HK1211345B - Methods of prognosis and diagnosis of cancer - Google Patents

Description

Methods for prognosis and diagnosis of cancer

Priority of U.S. provisional application No. 61/682,462, filed on 8/13/2012, the entire application of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to methods and immunoassay platforms for determining prognosis, diagnosis or risk identification of cancer in a patient by detecting biomarkers in the patient and determining the amount thereof. Biomarkers can be used to identify patients with cancer, identify patients as candidates for cancer treatment, classify the risk of a patient developing cancer, or classify the stage of cancer or risk of cancer progression in a patient, and determine a diagnosis, prognosis, or treatment regimen.

Background

Cancer remains a significant cause of morbidity and mortality in adults in developed countries. In some cases, improvements in cancer therapy have been able to increase patient survival from diagnosis to death. However, the overall success of cancer treatment often depends on early detection of the disease, which allows treatment to begin before the primary tumor expands and/or metastatic growth occurs. Accordingly, methods and assays that provide early and/or more accurate diagnosis of cancer are desirable because such methods and assays may allow early therapeutic intervention and may improve patient outcomes (e.g., quality of life, survival expectation, etc.).

laminin is a group of heterotrimeric proteins found in the basal lamina, and forms part of the basement membrane, these proteins are classified based on three different polypeptides that complex with each other to form laminin structures, these three polypeptides are identified as the alpha (α) chain, the beta (β) chain, and the gamma (γ) chain, and each have several molecular species (e.g., α 1- α 5, β 1- β 3, and γ 1- γ 2). laminin 5 (or LN5) is known to be present in the basal lamina, and is abundant in the basement membrane between epithelial cells and connective tissue supporting epithelial cells the structure of LN 2 is unique among known laminins in that it is the only laminin with a structure that includes the gamma-2 (γ 2) chain, which forms 5395 when complexed with the α 3 and β 3 chains. physiologically LN5 is known to be produced by epithelial cells, and can promote cell adhesion, proliferation, MT and/or migration from cells, e.g. 5 when it is treated with a metalloproteinases such as EGF-degrading enzymes (N-r) to promote cell adhesion, proliferation, MT 5) production from epithelial cells by a number of extracellular matrix proteins (e.g. EGF-2) and/or a number of peptides) which are known to promote cell growth and are treated with a number of extracellular matrix proteinMigratory and invasive) fragment [ Koshikawa, et al,J. Cell Biol., (2000)148:615-624]。

existing methods for detecting LN5 (including processed forms thereof) focus primarily on histological methods (e.g., immunostaining of tissue), urinalysis, or detection of proteolytic fragments (e.g., N-terminal fragments), and do not provide convenient and/or sensitive methods for detecting laminin gamma-2 monomers.

Summary of The Invention

In one aspect, the present disclosure provides a method for providing a diagnosis, prognosis or risk classification for a subject having or at risk of having cancer, the method comprising the steps of: obtaining a biological sample comprising blood from the subject; determining the concentration of laminin gamma-2 monomer in a biological sample from the subject; comparing the laminin gamma-2 monomer concentration from the sample to a reference laminin gamma-2 monomer concentration value, wherein a laminin gamma-2 monomer concentration in the sample that is greater than the reference laminin gamma-2 monomer concentration value identifies the subject as having cancer or as having an increased risk of developing cancer.

In one aspect, the present disclosure relates to a method for providing a diagnosis, prognosis or risk classification to a subject having or at risk of having cancer, the method comprising the steps of: obtaining a biological sample comprising blood from the subject; determining the concentration of laminin gamma-2 monomer in a biological sample from the subject; and providing a concentration of laminin gamma-2 monomer to identify the subject as having cancer or having an increased risk of developing cancer when compared to a reference laminin gamma-2 monomer concentration value.

In a further aspect, the present disclosure relates to a method for providing a diagnosis, prognosis or risk classification to a subject having or at risk of having cancer, the method comprising the steps of: obtaining a biological sample comprising blood from the subject; determining the concentration of laminin gamma-2 monomer in a biological sample from the subject; comparing the laminin gamma-2 monomer concentration from the sample to a reference laminin gamma-2 monomer concentration value; and providing a comparison, wherein when the comparison comprises a laminin gamma-2 monomer concentration in the sample that is greater than a reference laminin gamma-2 monomer concentration value, the comparison identifies the subject as having cancer or as having an increased risk of developing cancer.

In another aspect, the present disclosure provides a method for detecting, diagnosing, or prognosing a cancer in a subject, comprising determining the concentration of laminin gamma-2 monomer in a sample comprising blood from the subject, wherein the concentration of laminin gamma-2 monomer is detected by contacting an antibody that specifically binds laminin gamma-2 monomer with the sample, and detecting antibody binding, and wherein the cancer is detected, diagnosed, or prognosticated in the subject when the concentration of laminin gamma-2 monomer in the sample from the subject is higher relative to a reference laminin gamma-2 monomer concentration.

In yet another aspect, the present disclosure provides a method for detecting, diagnosing, or prognosing cancer in a subject, comprising determining a concentration of laminin gamma-2 monomer in a sample comprising blood from the subject, wherein the concentration of laminin gamma-2 monomer is determined by contacting an antibody that specifically binds laminin gamma-2 monomer with the sample and detecting antibody binding; and comparing the concentration of the laminin gamma-2 monomer in the sample from the subject to a reference laminin gamma-2 monomer concentration, wherein when the concentration of the laminin gamma-2 monomer in the sample from the subject is higher relative to the reference laminin gamma-2 monomer concentration, the cancer is detected, diagnosed, or prognosed in the subject.

In embodiments, the methods of the above aspects may further comprise detecting at least one additional cancer biomarker in the sample. In embodiments of the method, providing a diagnosis may be providing a diagnosis of cancer (such as, for example, bladder cancer or colorectal cancer). In other embodiments of the methods, providing a prognosis may be determining the disease stage of the cancer, or may be determining the likelihood or risk that a subject will develop an aggressive or aggressive form of cancer such as, for example, bladder or colorectal cancer.

The method may further comprise assessing at least one additional biomarker for cancer selected from the group consisting of: laminin gamma-2 fragment (e.g., EGF like fragment), carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (also known as cancer antigen 19-9, or CA19-9), and the like. The assessment of the additional biomarker may include, for example, measuring the concentration of the biomarker in a biological sample from the subject, or may include a clinical assessment of the subject. For additional biomarkers that are assessed by measuring the concentration of a biomarker in a biological sample from the subject, this method may further comprise comparing the measured concentration of at least one additional biomarker to a reference value for the biomarker. The reference value for any other biomarker used in the methods disclosed herein may relate to the biomarker concentration of a control sample, a biomarker cut-off value, or the median concentration of a plurality of control samples from a control subject group, or the like.

In one aspect, the present disclosure provides a method for identifying a subject as a candidate for a bladder cancer or colorectal cancer treatment regimen, the method comprising determining the concentration of laminin gamma-2 monomer in a biological sample comprising serum from the subject, and comparing the laminin gamma-2 monomer concentration in the sample to a reference laminin gamma-2 monomer concentration value, wherein when the laminin gamma-2 monomer concentration in the sample is greater than the reference laminin gamma-2 monomer concentration value, the subject is identified as a candidate for a cancer treatment regimen. In embodiments, the method may further comprise detecting at least one additional cancer biomarker in the sample.

In another embodiment, the present disclosure provides a method for diagnosing, prognosing and/or risk classifying a subject having or at risk of having a cancer, such as, for example, bladder cancer or colorectal cancer, wherein the method comprises detecting an increased laminin gamma-2 monomer concentration in the subject relative to a control subject that does not have cancer.

In any of the methods described herein, the reference laminin gamma-2 monomer value may be the laminin gamma-2 monomer concentration of the control sample, or a laminin gamma-2 monomer cutoff value. The laminin gamma-2 monomer concentration may be, for example, a reference value for laminin gamma-2 monomer concentration in blood (e.g., plasma or serum). The control sample can be a biological sample of a control subject, or a laminin gamma-2 monomer standard. The laminin gamma-2 monomer concentration of the control sample may be, for example, the median laminin gamma-2 monomer concentration of a plurality of control samples from a group of control subjects. Alternatively, the laminin gamma-2 monomer cutoff value may be determined by Receiver Operating Curve (ROC) analysis of biological samples from a patient group. Alternatively, the laminin gamma-2 monomer cutoff can be determined by a quartile analysis (quartile analysis) of biological samples of a patient group. Still further alternatively, the laminin gamma-2 monomer cutoff value may be determined by an average of biological samples of a patient group plus two standard deviation analyses. For example, the laminin gamma-2 monomer cutoff value may be determined by selecting a value corresponding to the median value of a patient group consisting of patients with cancer (such as, for example, bladder cancer or colorectal cancer), which may be from about 900-to about 1000 pg/ml serum. Alternatively, the laminin gamma-2 monomer cutoff value may be determined by selecting a value corresponding to the 75 th percentile of patient groups consisting of patients with bladder cancer or colorectal cancer, which may be, for example, about 1,100 to about 1,400 pg/mL serum. In other embodiments, a suitable cutoff value may be about 70 pg/mL to about 2,500 pg/mL serum. For example, a cut-off of about 1,000 pg/mL serum can be used to distinguish bladder cancer or colorectal cancer samples from normal samples. Similar cut-off values can be used for plasma and whole blood samples.

In any method, the method can be performed via an immunoassay. An example of an antibody employed in such immunoassays is monoclonal antibody 2H 2.

In any of the methods, the subject may be a human subject, and the biological sample of the subject and/or the control sample may be taken from the human subject. In any of the methods, the biological sample may be from a tissue or body fluid, including, for example, whole blood, plasma, or serum, or any of the cell culture suspensions or fractions thereof. In some embodiments of the methods described herein, the sample is whole blood, plasma or serum, suitably plasma or serum. A clotting inhibitor can be added to any peripheral blood sample. In the method, the determination of the concentration of laminin gamma-2 monomer and optionally of the at least one additional biomarker may be performed by an immunoassay in which an agent capable of specifically binding laminin gamma-2 monomer, and optionally an agent capable of specifically binding the additional biomarker, is used.

In another aspect, the present disclosure provides kits for performing any of the methods and assays disclosed herein, wherein the kits comprise at least one reagent capable of specifically binding laminin gamma-2 monomer, allowing for quantification of laminin gamma-2 monomer concentration in a biological sample from a subject; and a reference standard indicative of a reference concentration of laminin gamma-2 monomer. In a kit for performing a method for providing a diagnosis, prognosis or risk stratification of a subject having or at risk of having cancer (e.g., bladder cancer or colorectal cancer), the kit may further comprise at least one reagent capable of specifically binding to at least one additional biomarker of cancer in a biological sample, allowing for quantifying the concentration of the at least one additional biomarker in the biological sample; and a reference standard indicative of a reference concentration of the at least one additional biomarker for cancer in the biological sample. In any of the kits, the at least one agent capable of specifically binding laminin gamma-2 monomer may comprise at least one antibody capable of specifically binding laminin gamma-2 monomer. In some embodiments, the kit is suitable for use with an ELISA assay.

Brief Description of Drawings

FIGS. 1A and 1B depict serum concentrations (pg/mL) of laminin gamma-2 monomer in various samples (bladder, pancreatic, ovarian, colon, gastric, esophageal; and control samples). FIGS. 1A and 1B depict the same data, with FIG. 1B magnifying the y-axis of the concentration range from 0pg/mL to 2000 pg/mL. The mean concentration (solid line) and one standard deviation (dashed line) are indicated.

FIGS. 2A and 2B are graphs of the Receiver Operating Characteristics (ROC) of the biomarkers carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and laminin gamma-2 monomer (laminin gamma-2). Fig. 2A depicts biomarkers in bladder cancer. Fig. 2B depicts biomarkers in colorectal cancer.

FIG. 3 depicts the results of Western blot analysis of monoclonal antibodies D4B5 and 2H2 (each 1 μ g/mL). The data show that monoclonal antibody 2H2 binds specifically to laminin gamma-2 monomer and does not bind laminin gamma-2 monomer when it forms a laminin 5 complex.

FIG. 4 is a graphical representation of data sets from dilution assay experiments using various dilutions of the 2H2 monoclonal antibody and laminin gamma-2 monomer ("g-2") and assay substrate from normal samples (ABS 001).

FIGS. 5A and 5B illustrate laminin gamma-2 monomer ELISA. Fig. 5A depicts a schematic of a general ELISA assay used in embodiments described herein. FIG. 5B depicts a standard laminin gamma-2 monomer ELISA curve between the concentration range of 0-4,000 pg/mL. The assay sensitivity was determined to be 3.7 pg/mL.

Figure 6 illustrates the analytical sensitivity of the ARCHITECT assay using a sample dilution incorporating recombinant laminin gamma-2 monomer.

Figure 7 illustrates the results of further evaluation of dilution linearity using normal samples.

FIG. 8 illustrates measurement of laminin gamma-2 monomer levels in normal samples.

Detailed Description

The present disclosure is based on several unexpected developments and discoveries. In one general sense, the present disclosure relates to the use of laminin gamma-2 monomers as biomarkers for the diagnosis, prognosis and risk classification of certain cancers (such as, for example, bladder cancer and colorectal cancer). In another general sense, the present disclosure relates to the surprising development of methods, assays, and kits that provide for the detection of laminin gamma-2 monomer in a biological sample comprising blood (e.g., whole blood, plasma, or serum). In summary, these unexpected findings provide methods and assays for the general measurement and quantification of laminin gamma-2 monomer and fragments thereof with significant advantages over existing methods and assays. Accordingly, the present disclosure identifies novel associations between increased (i.e., higher) laminin gamma-2 monomer serum levels in patients with certain cancers (including bladder cancer and colorectal cancer) (e.g., subjects with cancer, subjects with increased risk of developing cancer, subjects identified as candidates for cancer therapy). As generally disclosed herein by way of example of several non-limiting embodiments, the presence of increased concentrations or levels of laminin gamma-2 monomer in a biological sample comprising blood may be associated with cancer (e.g., colorectal cancer and/or bladder cancer). The association between increased blood levels of laminin gamma-2 monomer and cancer is robust, predicting the disease stage, disease onset, clinical progression and/or disease severity of cancer. In contrast to existing methods and assays (e.g., which rely on measuring laminin gamma-2 monomer based on patient urine output and/or proteolytic processing of laminin gamma-2 monomer into its EGF-like fragment), embodiments of the methods and assays provided herein include simple and convenient steps that can be readily obtained from any subject. The assessed level of laminin gamma 2 in the blood may therefore improve existing methods and assays for diagnosing cancer, providing a prognosis for cancer treatment or cancer severity, and/or stratifying (stratify) or identifying a patient risk of developing cancer, thereby significantly benefiting patients having or at risk of developing cancer. Furthermore, the use of laminin gamma 2 monomer in combination with additional biomarkers may provide additional advantages.

Accordingly, the present disclosure provides methods of diagnosing, prognosing, or risk classifying/identifying a subject or group of subjects having or at risk of having a cancer, such as bladder cancer or colorectal cancer, using a laminin gamma-2 monomer as a clinical biomarker. Also provided are methods of identifying a candidate subject or group of candidate subjects for a cancer treatment regimen, such as treatment for bladder cancer or colorectal cancer, wherein the methods utilize laminin gamma 2 monomer as a biomarker. The disclosure also provides kits for practicing the disclosed methods.

The section headings used throughout this disclosure are for organizational purposes only and are not meant to be limiting.

A. Definition of

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For the numerical ranges set forth herein, each intervening value, to the same degree of accuracy between the stated ranges, is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are all expressly contemplated.

Unless otherwise indicated, the use of "or" means "and/or. Furthermore, the use of the terms "including" and "having" as well as other forms of those terms, such as "including", "having" and "having", is not limiting.

"component," "components," or "at least one component" generally refers to a capture antibody, a detection or conjugate calibrator (calibretor), a control, a group of sensitive subjects (sensory panel), a container, a buffer, a diluent, a salt, an enzyme, a cofactor for an enzyme, a detection reagent, a pretreatment reagent/solution, a substrate (e.g., as a solution), a stop solution, and the like, which may be included in a kit for the determination of a test sample, such as a patient urine, blood, serum, or plasma sample, according to the methods described herein and other methods known in the art. Some components may be in solution or lyophilized for reconstitution for use in the assay.

When referring to a composition, a "control," as used herein, can refer to a composition known to be free of an analyte of interest ("negative"), e.g., a laminin gamma-2 monomer (a laminin gamma-2 monomer or a variant of a gamma-2 monomer of laminin, or a combination thereof); or a composition containing an analyte of interest ("positive control"), e.g., a laminin gamma-2 monomer (such as a human laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof). The positive control may comprise a known concentration of laminin gamma 2 monomer. "control," "positive control," and "calibrator" are used interchangeably herein to refer to compositions comprising known concentrations of laminin gamma 2 monomer. A "positive control" can be used to establish an assay performance characteristic and is a useful indicator of the integrity of a reagent (e.g., an analyte). A "normal control" or "healthy control" may refer to a sample or specimen taken from a subject or actual subject who does not have cancer or is not at risk of developing cancer.

As used herein, the terms "laminin gamma-2 monomer", "laminin-5 gamma-2 monomer", "LN-5 gamma-2 monomer", "gamma-2", "g-2 monomer", or any of the foregoing terms in which the "gamma" symbol replaces the word "gamma" or the letter "g" are interchangeable and refer to one of the polypeptide chains that make up laminin-5 (also known as other synonyms such as "calnexin" and "nicein") and are identified as gamma (gamma) chains (as compared to α (α) and β (β) chains) of the gamma-2 molecular species (as opposed to the gamma-1 species)A laminin gamma-2 monomer may relate to any laminin gamma-2 monomer sequence, including amino acid sequences (e.g., proteins, polypeptides, peptides (precursor or mature), fusions, derivatives, variants, etc., or nucleic acid sequences encoding such amino acid sequences (e.g., DNA or RNA fragments, truncations, fusions, derivatives, SNPs, variants, etc.). laminin gamma-2 monomer may be from any organism, and in some embodiments, comprise an amino acid sequence from a higher eukaryote (including mammals.) in some non-limiting embodiments, laminin gamma-2 monomer may be selected from a human (including isoforms a and b, UniProtKB/Swiss-Prot: Q13753; RefSeq NP-005553.2), a mouse (little mouse: (little mouse): A and B)M. musculus) UniProt: E9Q7G3; RefSeq NP-032511.3), rat (Rattus norvegicus: (Rattus norvegicus) ((Rattus norvegicus))R. norvegicus) GenBank NP-001094110 (precursor protein); UniProtKB/TrEMBL F1LRH4) and chickens (G. gallusGenBank AAS92197; UniProtKB/TrEMBL Q6PVZ6 (partial sequence)), and any of flies and worms.

In some embodiments, a laminin gamma-2 monomer comprises a human laminin-5 gamma-2 monomer (encoded by GenBank accession No. NM _005562 (mRNA), or an amino acid sequence related to UniProtKB accession No. Q13753). In humans, the gene for the laminin gamma-2 monomer (or "LAMC 2") is located on the q-arm of chromosome 1 (1q 25.3). The human laminin gamma-2 monomer sequence may include a precursor protein sequence that includes a signal peptide (typically amino acids 1-21) that is cleaved off to produce the mature secreted protein (amino acids 22-1193). Laminin gamma-2 monomer may also encompass any fusion protein as well as any amino acid sequence variant. As shown above, the laminin gamma-2 monomer is unique to laminin 5, being located primarily in the basal layer and basement membrane.

As used herein, "label" and "detectable label" refer to moieties that bind to an antibody or analyte such that a reaction between the antibody and analyte is detectable, and the antibody or analyte so labeled is referred to as "detectably labeled". The marker may produce a signal that is detectable by visual or instrumental means. Various labels include signal-producing substances such as chromogens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that generate light, such as acridinium compounds, and moieties that generate fluorescence, such as fluorescein. Other labels are as described herein. In this regard, the moiety may not be detectable by itself, but may become detectable upon reaction with another moiety. The use of the term "detectably labeled" is intended to encompass such labels.

Any suitable detectable label known in the art may be used. For example, the detectable label may be a radioactive label (such as³H、¹²⁵I、³⁵S、¹⁴C、³²P, and³³p), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, glucose 6-phosphate dehydrogenase, etc.), chemiluminescent labels (such as acridinium esters (thioesters), thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters (phenanthridinium esters), and the like), fluorescent labels (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3' 6-carboxyfluorescein, 5(6) -carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., zinc sulfide capped (capped) cadmium selenide), thermometric labels, or immuno-polymerase chain reaction labels. Introduction to the labeling, labeling procedures and detection of the label is found in Polak and Van Noorden,Introduction to Immunocytochemistry,second edition, Springer Verlag, N.Y. (1997), and Haugland,Handbook of Fluorescent Probes and Research Chemicals(1996) which is a combination manual and catalog published by Molecular Probes, inc. Fluorescent labels can be used for FPIA (see, e.g., U.S. Pat. nos. 5,593,896,5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are incorporated herein by reference in their entirety). Acridinium compounds can be used as detectable labels in homogeneous chemiluminescence assays (see, e.g., Adamczyk et al, bioorg. Med. chem. Lett.16: 1324-1328 (2006); Adamczyk et al, bioorg. Med. chem. Lett.4: 2313-2317 (2004); Adamczyk et al, Bio rg. Med. chem. Lett. 14: 3917-3921(2004), and Adamczyk et al, org. Lett. 5: 3779-3782 (2003)).

In one aspect, the acridinium compound is acridinium-9-carboxamide. Methods for preparing acridinium-9-carboxamides are described In Mattingly, J.Biolumin. Chemimun.6: 107-114 (1991); Adamczyk et al, J.Org.Chem.63: 5636-5639 (1998); Adamczyk et al, Tetrahedron 55: 10899-10914(1999); Adamczyk et al, org. Lett.1: 779-781 (1999); Adamczyk et al, Bioconjugate Chem.11: 714-724 (2000); Mattingly et al, InLuminescence Biotechnology: Instruments and Applications;Dyke, K.V. Ed.; CRC Press: BocaRaton, pp. 77-105 (2002); Adamczyk et al, org. Lett. 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524, and 5,783,699 (each of which is incorporated herein in its entirety for its teachings).

Another example of an acridinium (acridinium) compound is an aryl acridinium-9-carboxylate. An example of an aryl acridinium-9-carboxylate of formula II is 10-methyl-9- (phenoxycarbonyl) acridinium fluorosulfonate (available from cayman chemical, Ann Arbor, MI). Methods for preparing arylacridinium-9-carboxylates are described in McCapra et al, Photochem. Photobiol.4: 1111-21 (1965), Razavi et al, Luminescence 15: 245-249(2000), Razavi et al, Luminescence 15: 239-244 (2000), and U.S. Pat. No. 5,241,070 (each of which is incorporated herein in its entirety for its teachings). Such aryl acridinium-9-carboxylates are effective chemiluminescent indicators of hydrogen peroxide in terms of signal intensity and/or rapidity of signal, which is generated in the oxidation of an analyte by at least one oxidase. The process of chemiluminescent emission of the arylacridinium-9-carboxylates is rapidly completed, i.e., in less than 1 second, while the chemiluminescent emission of the acridinium-9-carboxamides is extended by more than 2 seconds. However, the aryl acridinium-9-carboxylate loses its chemiluminescent properties in the presence of the protein. Thus, its use suitably includes the absence of protein during signal generation and detection. Methods for isolating or removing proteins from a sample are well known to those skilled in the art andand include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and/or digestion (see, e.g., Wells,High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategiesthe amount of protein removed or isolated from a test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, or at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Further details regarding aryl acridinium-9-carboxylates and their uses are described in U.S. patent application No. 11/697,835, filed on 9.4.2007. The aryl acridinium-9-carboxylate may be dissolved in any suitable solvent, such as degassed anhydrousN,N-Dimethylformamide (DMF) or aqueous sodium cholate.

"predetermined cutoff (cutoff)" and "predetermined level" generally refer to a measured cutoff value used to assess a diagnostic, prognostic, or therapeutic efficacy outcome by comparing the measured outcome to a predetermined cutoff/level, wherein the predetermined cutoff/level has been linked or correlated with various clinical parameters (e.g., presence of disease, disease stage, disease severity, disease progression, non-progression or improvement, etc.). The present disclosure provides exemplary predetermined levels. However, it is well known that the cut-off value may vary depending on the nature of the immunoassay (e.g., antibody employed, reaction conditions, sample purity, etc.). It is further well within the general ability of those skilled in the art to adjust the disclosure herein to other immunoassays based on the description provided by the disclosure to obtain immunoassay-specific cut-off values for those other immunoassays. Although the precise value of the predetermined cut-off/level may vary from assay to assay, the correlations described herein should be generally applicable.

As used in the diagnostic or prognostic assays described herein, a "pretreatment reagent," e.g., a lysis, precipitation and/or solubilization reagent, is a reagent that lyses any cells present in a test sample and/or solubilizes any analytes. As further described herein, not all samples require pretreatment. In addition, solubilizing the analyte (e.g., laminin gamma-2 monomer) causes the analyte to be released (e.g., dissociated or reduced in binding) from any endogenous binding protein present in the sample (such as a blood sample). The pretreatment reagents may be homogeneous (no separation step required) or heterogeneous (separation step required). When a heterogeneous pretreatment reagent is used, any precipitated analyte binding protein is removed from the test sample before proceeding to the next step of the assay. The pretreatment reagent optionally may comprise: (ii) one or more solvents and salts, (b) one or more solvents, salts and detergents, (C) detergents, (D) detergents and salts, or (e) any reagent or combination of reagents suitable for cell lysis and/or solubilization of an analyte.

In the context of the immunoassays and kits described herein, "quality control reagents" include, but are not limited to, calibrators, controls, and sensitivity test subject groups. To establish the concentration of a calibration (standard) curve interpolation (analyte), such as an antibody or analyte, a "calibrator" or "standard" (e.g., one or more, such as a plurality) is typically used. Alternatively, a single calibrator can be used that is close to a predetermined positive/negative cutoff. Multiple calibrators (i.e., more than one calibrator or different amounts of calibrators) may be used together to form a "sensitivity panel".

"sample", "test sample", "specimen", "sample from a subject" and "patient sample" are used interchangeably herein. Samples, such as samples of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes, may be used directly, as obtained from a patient, or may be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, in order to modify the characteristics of the sample as discussed herein or otherwise in some manner as is known in the art.

By "series of calibration compositions" is meant a plurality of compositions comprising a known concentration of laminin gamma-2 monomer, wherein each composition differs from the other compositions in the series by the concentration of laminin gamma-2 monomer.

A "specific binding partner" relates to a member of a specific binding pair. A specific binding pair comprises two different molecules that bind specifically to each other chemically or physically. Thus, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, and the like. In addition, a specific binding pair may include a member that is an analog of the initial specific binding member, e.g., an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments and antibodies, including monoclonal and polyclonal antibodies and complexes thereof and fragments thereof, whether isolated or recombinantly produced.

As used herein, "tracer" refers to an analyte or analyte fragment conjugated to a label, such as a laminin gamma-2 monomer conjugated to a fluorescein moiety, wherein the analyte conjugated to the label can effectively compete with the analyte for sites on an antibody specific for the analyte.

The term "cancer" as used herein refers to any malignant disease (e.g., angiogenesis, tumors, or tumorigenic cell growth) associated with dysregulated cell proliferation, growth, invasion, and metastasis or quality. In some embodiments, the cancer may comprise bladder cancer or colorectal cancer.

Bladder cancer, in general, is derived from cells lining the bladder (called transitional cells) and is classified based on the manner in which their tumors grow. The papillomas are wart-like in appearance and attach to the shaft. Non-papillary (sessile) tumors are rare, but more invasive and often associated with poor outcome. Many risk factors are associated with an increased likelihood of developing bladder cancer, including smoking, exposure to chemicals, long-term bladder infections, and chemotherapeutic drugs, cyclophosphamide, and radiation therapy.

Bladder cancer is often associated with a number of symptoms, which may include abdominal pain, hematuria, bone pain, fatigue, pain during urination, urinary frequency and/or urgency, urinary incontinence, and weight loss. Existing tests for detecting bladder cancer include abdominal CT scans, pelvic CT scans, bladder biopsy, cystoscopy, intravenous pyelography, urinalysis, and urine cytology. Bladder cancer is usually staged on a scale of 0 to IV, with stage 0 involving non-invasive tumors only lining the bladder; stage I involves penetration through the bladder lining, but not to the muscle layer of the bladder; stage II involves the tumor reaching the muscle layer; stage III involves tumor penetration through muscle into the tissues surrounding the bladder; and stage IV involves metastatic disease (e.g., to adjacent lymph nodes or remote sites). When bladder cancer spreads, it is often seen first in organs and tissues (including prostate, rectum, ureter, uterus, and vagina). Metastatic bladder cancer often involves bone, liver and/or lung.

Treatment is typically based on different stages of disease, with treatment of stage 0 and I disease including surgical removal of the tumor (e.g., partial resection) and chemotherapy and/or immunotherapy directed specifically to the bladder. In stages II and III, treatment may involve partial or complete removal of the bladder, followed by radiation and chemotherapy, preoperative chemotherapy to attempt to shrink the tumor before surgery, or a combination of radiation and chemotherapy for patients who are not eligible for surgery. In stage IV, bladder cancer is generally considered to be terminal, and the course of treatment typically includes chemotherapy.

Patients with stage 0 or stage I bladder cancer have a reasonably good prognosis. Although there is a high risk that the cancer will recover, most bladder cancers that recover can be surgically resected and cured. The cure rate for persons with stage III tumors is less than 50%. Patients with stage IV bladder cancer are rarely cured.

In some embodiments, the cancer may comprise a colorectal (or colon) cancer, which is typically a cancer derived from the large intestine (colon) or rectum (distal end of the colon). Colorectal cancer is often cited as one of the leading causes of cancer-related death in the united states. Early diagnosis is often associated with complete cure of the disease. Although there is no single cause for colon cancer, almost all colon cancer is of origin as polyps, which are initially benign and slowly progress to malignant cancer. Risk factors associated with colorectal cancer include age (greater than 60 years), smoking, drinking, red and/or meat-high diets, colorectal polyps, inflammatory bowel disease (e.g., ulcerative colitis or crohn's disease), family history of colorectal cancer, genetic predisposition including Lynch syndrome and Familial Adenomatous Polyposis (FAP).

In many cases, colorectal cancer may appear to be asymptomatic. However, some cases also present with abdominal pain and tenderness, bloody stool, diarrhea, constipation, stool stenosis and weight loss of unknown origin. As shown above, early detection of colorectal cancer often leads to excellent prognosis (cure). Current tests and screens for colorectal cancer include abdominal physical examination, Fecal Occult Blood Test (FOBT), colonoscopy, sigmoidoscopy, and blood tests for anemia and proper liver function. The various stages of colon cancer (0-IV) are generally characterized as follows: stage 0, cancer on the innermost layer of the intestine; stage I, cancer in several inner layers of the colon; stage II, the cancer has spread to the muscle wall of the colon; stage III, the cancer has spread to lymph nodes; in stage IV, the cancer has spread to other organs.

Treatment of colorectal cancer may include any one or combination of surgery (e.g., colectomy), chemotherapy, and radiation therapy, depending generally on the stage of the disease. Generally, patients who detect and treat early (e.g., stages 0-III) colorectal cancer can survive 5 years after diagnosis and are considered to be cured of the disease. Stage IV colorectal cancer is generally considered curable.

Diagnosis of cancer is typically performed by any one or more of the clinical or diagnostic tests shown above, and may include any one or combination of physical examination, imaging tests, radiographic (X-ray) and laboratory diagnosis as described herein or as known in the art. Several biomarkers, including carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9), were used in the diagnosis of colorectal cancer.

CEA is a glycoprotein involved in cell adhesion and is usually produced during fetal development and stops prenatally. CEA was first detected in tissue extracts from human colon cancer extracts and increased levels in serum have been associated with colorectal cancer as well as carcinomas of the stomach organs, pancreas, lungs, breast and medullary thyroid. The normal level of CEA is about 2.5 ng/mL. Nonetheless, CEA markers are not completely reliable for diagnosing cancer or as a screening test for early detection of cancer.

CA19-9, although related and identified as a marker for colon and pancreatic cancer, has been associated with high occurrence of both false negative results as well as false positive results. Furthermore, CA19-9 was not expressed in patients lacking lewis antigens, even when the patients had tumors. Nonetheless, since CA19-9 may be elevated in many types of gastrointestinal cancer, such as colorectal cancer, esophageal cancer, and hepatocellular carcinoma, it may be useful as a cancer biomarker (e.g., colorectal cancer).

Thus, both CEA and CA19-9 have been associated with certain types of cancer and used as biomarkers to diagnose disease (e.g., bladder cancer or colorectal cancer). However, these markers lack the desired sensitivity and specificity needed to accurately and/or early diagnose cancer in a patient.

As used herein, the term "risk assessment", "risk classification", "risk identification" or "risk stratification" of a subject (e.g., patient) refers to the assessment of factors, including biomarkers, to predict the risk of occurrence of a future event, including the onset of a disease or progression of a condition, so that a treatment decision for the subject can be made on a more informed basis.

The term "cancer risk" or "risk of developing cancer" of a subject as used herein refers to an assessment factor, including biomarkers, to predict the risk of cancer development (including cancer onset, increased likelihood of cancer progression, occurrence/severity of clinical symptoms associated with cancer). In addition to laminin gamma-2 monomer levels, other factors that may indicate an adverse disease prognosis include tumor size, disease stage, serum concentration of CA19-9 and/or CEA, family and/or personal history of cancer, and any increased clinical severity presenting symptoms. Thus, in some embodiments, the methods relate to providing a prognosis of cancer onset or cancer progression, comprising detecting/determining the level of laminin gamma-2 monomer and any one or more prognostic factors described herein or known in the art in a sample from a patient.

The term "specifically binds" or "specifically binds" as used herein refers to the interaction of an antibody, protein or peptide with a second chemical species, wherein the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies recognize and bind to specific protein structures but not to all proteins (protein genes). If the antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in the reaction comprising labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

The term "antibody" as used herein refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab')₂Fragments which may be produced by treating an antibody with an enzyme (e.g. pepsin). Examples of antibodies useful in the present disclosure include, but are not limited to, antisera, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, recombinant antibodies, single chain Fvs ("scFv"), affinity matured antibodies, single chain antibodies, single domain antibodies, F (ab) fragments, F (ab') fragments, disulfide linked Fvs ("sdFv") and anti-idiotypic ("anti-Id") antibodies, and functionally active epitope-binding fragments of any of the above.

The terms "subject" and "patient" as used herein are used interchangeably, whether the subject has or is currently undergoing any form of treatment. The term "subject" and "subjects" as used herein may refer to any vertebrate animal, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as cynomolgus or macaque, chimpanzees, etc.) and humans). In some embodiments, the subject may be a human or a non-human. In some embodiments, the subject may be a human patient at risk of developing cancer (such as, for example, bladder cancer or colorectal cancer) or already having cancer (such as, for example, bladder cancer or colorectal cancer).

The terms "sample" and "biological sample" as used herein generally refer to a biological material that is tested for and/or suspected of containing an analyte of interest (such as a laminin gamma-2 monomer). The sample may be any tissue sample taken or derived from a subject. In some embodiments, the sample from the subject may comprise a protein. In some embodiments, a sample from a subject can comprise nucleic acids (e.g., polynucleotides, mRNA, etc.).

Any cell type, tissue or body fluid may be used to obtain the sample. Such cell types, tissues and fluids may include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood (such as whole blood), plasma, serum, sputum, stool, tears, mucus, saliva, bronchoalveolar lavage (BAL) fluid, hair, skin, red blood cells, platelets, interstitial fluid, ocular lens fluid, cerebrospinal fluid, sweat, nasal fluid, synovial fluid, menstrual fluid, amniotic fluid, semen, and the like. Cell types and tissues may also include lymph, ascites, gynecological fluid, urine, peritoneal fluid, cerebrospinal fluid, fluid collected by vaginal washing, or fluid collected by vaginal rinsing. The tissue or cell type may be provided by removing a cell sample from the animal, but may also be accomplished by using cells previously isolated (e.g., isolated by another person at another time and/or for another purpose). Archival tissues, such as those with a history of treatment or outcome, may also be used. Protein or nucleotide isolation and/or purification may not be necessary.

In the practice of the present disclosure, methods for collecting, processing and processing urine, blood, serum and plasma, and other bodily fluids, well known in the art, are used, for example, when the antibodies provided herein are used as immunodiagnostic reagents and/or in laminin gamma-2 monomer immunoassay kits. The test sample may comprise other moieties in addition to the analyte of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides or polynucleotides. For example, the sample may be a whole blood sample obtained from a subject. It may be necessary or desirable to process the test sample, particularly whole blood, prior to the immunoassay as described herein (e.g., with a pretreatment reagent). Even where pretreatment is unnecessary (e.g., most urine samples, pretreated archival samples, etc.), pretreatment of the sample is an option that can be done for convenience only purposes (e.g., as part of a protocol on a commercial platform). The sample may be used directly as obtained from the subject or after pretreatment to modify the characteristics of the sample. Pre-treatment may include extraction, concentration, inactivation of interfering components, and/or addition of reagents.

The pretreatment reagent can be any reagent suitable for use with the assays (e.g., immunoassays) and kits described herein. The pretreatment optionally comprises: (a) one or more solvents (e.g., methanol and ethylene glycol), and a salt, (b) one or more solvents, salts, and detergents, (c) detergents, or (d) detergents and salts. Pretreatment reagents are known in the art, and such pretreatments can be employed, for example, for determination on Abbott TDx, AxSYM, and ARCHITECT ® analyzers (Abbott laboratories, Abbott Park, IL), as described in the literature (see, e.g., Yatscoff et al, Abbott TDx Monoclonal Antibody Evaluation for Measuring Cyclosporine Blood, Clin. chem. 36: 1969-1973 (1990), and Wallmacq et al, Evaluation of the New AxSYM Cyclosporine Assay: composite with TDxMonochromatic and EMCycline Assays, Clin. chem. 45: 432-1999), and/or commercially available. Further, pretreatment can be accomplished as described in Abbott's U.S. patent No. 5,135,875, european patent application No. 0471293, and U.S. patent application publication No. 2008/0020401 (the teachings of which regarding pretreatment are incorporated by reference in their entirety). The pretreatment reagent may be a heterogeneous reagent or a homogeneous reagent.

When a heterogeneous pretreatment reagent is used, the pretreatment reagent precipitates analyte binding proteins (e.g., proteins that can bind laminin gamma-2 monomer) present in the sample. Such a pretreatment step includes removing any analyte binding protein by adding a pretreatment reagent to the sample to separate the resulting mixture supernatant from the precipitated analyte binding protein. In such assays, the supernatant of the mixture, in the absence of any binding protein, is used in the assay, proceeding directly to the antibody capture step.

When a homogeneous pretreatment reagent is used, there is no such separation step. The entire mixture of test sample and pretreatment reagent is contacted with a labeled binding partner specific for a laminin gamma-2 monomer, or variant of a laminin gamma-2 monomer, such as a labeled anti-laminin gamma-2 monomer monoclonal antibody (or antigen-reactive fragment thereof). The pretreatment reagents employed in such assays are typically diluted in the pretreated test sample mixture prior to or during capture by the first specific binding partner. Despite such dilution, a specific amount of pretreatment reagent (e.g., 5M methanol and/or 0.6M ethylene glycol) remains present (or retained) in the test sample mixture during capture.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. For example, any of the terms and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The meaning and scope of the terms should be clear; however, in the case of any potential ambiguity, the definitions provided herein are superior to any dictionary or external definition. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

B. Method of producing a composite material

The method encompasses providing a diagnosis or prognosis of the subject, comprising, with respect to cancer, any one or more of: determining that the subject has cancer, determining the severity of cancer, determining the subject's risk of developing cancer (i.e., the likelihood of disease onset), determining the efficacy of a cancer treatment regimen, identifying the subject as a candidate for cancer treatment, and assessing risk for cancer progression in a subject with disease. The methods are based, in part, on the unexpected discovery that the concentration of laminin gamma-2 monomer in a biological sample (e.g., blood, serum, or plasma) from a subject predicts or diagnoses cancer (e.g., bladder cancer or colorectal cancer) in the subject, and thus laminin gamma-2 monomer can be used as a prognostic or diagnostic biomarker for cancer.

The method involves providing or obtaining a biological sample from a subject, which may be obtained by any known means, including needle punching, needle biopsy, swab, and the like. In an embodiment of the method, the biological sample is a blood sample, preferably a plasma or serum sample, which may be obtained by standard techniques such as, for example, by venipuncture. The biological samples used in the methods can be stored or preserved under suitable tissue storage conditions, or can be obtained from samples that have been previously stored or preserved under suitable conditions. In some embodiments, the methods comprise assaying a biological sample from the subject for previously determined or analyzed data (e.g., measuring laminin gamma-2 monomer and/or another cancer biomarker, such as, for example, any one or more of CEA and CA 19-9).

The methods encompass methods for the diagnosis, prognosis and/or risk stratification of cancer in a subject having cancer or suspected of having cancer by determining the laminin gamma-2 monomer concentration in the subject. Providing a diagnosis may be, for example, providing a diagnosis of cancer in a subject, wherein the subject may not have been previously diagnosed as having cancer (or not identified as at risk of having cancer), is suspected of having cancer, or not. Alternatively, or in addition, providing a prognosis may be, for example, determining the severity or stage of cancer, or may be a risk assessment, i.e., determining the likelihood that a subject will develop cancer. The method further comprises identifying one or more patients or subpopulations of patients having an increased risk of developing cancer. A feature common to all methods is the determination of laminin gamma-2 monomer concentration in a biological sample as described herein, wherein an increase in laminin gamma-2 monomer concentration in the sample relative to a reference value for laminin gamma-2 monomer concentration indicates cancer, or an increased risk of developing cancer.

Laminin gamma-2 monomer concentration is considered to be an increase as compared to a reference value or predetermined level, i.e., a reference laminin gamma-2 monomer concentration value described herein. For example, a laminin gamma-2 monomer serum concentration that can be used as a reference concentration value is about 500 pg/ml, but can also be higher or lower, such as about 200 pg/ml or about 1000 pg/ml (e.g., about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pg/ml or more) in serum. Laminin gamma-2 monomer concentration, when detectably higher (e.g., about 1% to about 10% higher), or significantly higher, such as at least 20% higher (1.2 times higher), at least 30% higher (1.3 times higher), at least 40% higher (1.4 times higher), at least 50% higher (1.5 times higher), at least 60% higher (1.6 times higher), at least 70% higher (1.7 times higher), at least 80% higher (1.8 times higher), at least 100% higher (2.0 times or twice higher), at least 150% higher (2.5 times higher), or at least 200% higher (3.0 times or three times higher), can be considered an increase as compared to a reference value.

The presence, concentration, or amount of laminin gamma-2 monomer in the biological sample can be readily determined using any suitable assay known in the art. Examples include, but are not limited to, immunoassays, such as sandwich immunoassays (e.g., monoclonal-polyclonal sandwich immunoassays, including radioisotope assays (radioimmunoassays (RIA)) and enzyme assays (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA) (e.g., Quantikine ELISA assays, R & D Systems, Minneapolis, MN)), competitive inhibition immunoassays (e.g., forward and reverse), Fluorescence Polarization Immunoassays (FPIA), Enzyme Multiplex Immunoassay Techniques (EMIT), Bioluminescence Resonance Energy Transfer (BRET), and homogeneous luminescence assays, etc. in SELDI-based immunoassays, capture reagents that specifically bind to the laminin γ -2 monomer of interest (or a portion thereof) are attached to the surface of a mass spectrometry probe, such as a pre-activated protein chip array On the chip, and detecting the captured laminin gamma-2 monomer by mass spectrometry. Alternatively, laminin gamma-2 monomer may be eluted from the capture reagent and detected by conventional MALDI (matrix assisted laser desorption/ionization) or by SELDI. Chemiluminescent microparticle immunoassays, particularly those using ARCHITECT automatic analyzers (Abbott Laboratories, Abbott Park, IL), are examples of preferred immunoassays. Other methods include, for example, mass spectrometry and immunohistochemistry (e.g., with sections from tissue biopsies) using antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) or fragments thereof that specifically bind laminin gamma-2 monomer. Anti-laminin gamma-2 monomeric antibodies and fragments thereof may be produced according to methods known in the art as described herein. Alternatively, commercially available anti-laminin gamma-2 monomeric antibodies may be used as described herein. Other detection methods include those described in, for example, U.S. patent nos. 6,143,576; 6,113,855; 6,019,944, respectively; 5,985,579, respectively; 5,947,124, respectively; 5,939,272, respectively; 5,922,615, respectively; 5,885,527, respectively; 5,851,776, respectively; 5,824,799, respectively; 5,679,526, respectively; 5,525,524, respectively; and 5,480,792, each of which is incorporated herein by reference in its entirety.

Laminin gamma-2 monomer or its variants or any combination can be analyzed using an immunoassay. The presence or amount of laminin gamma-2 monomer may be determined by using an antibody and detecting specific binding to laminin gamma-2 monomer. If desired, one or more of the antibodies described herein can be used in combination with one or more commercially available monoclonal/polyclonal antibodies. Such antibodies are available from companies such as Life span Biosciences, Inc. (Seattle, WA), AcrisAntibodies, Inc. (San Diego, CA), Raybiotech, Inc. (Norcross, GA), atlas antibodies (Stockholm, Sweden), Sigma-Aldrich (St. Louis, MO), IMGENEX (SanDiego, CA), GeneTex (Irvine, CA), Abcam (Cambri, MA), Novus Biologicals (Littleton, CO), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Sciences (Scien, MA), US Biological (Abpsamcott, MA), Abd Setec (Raigigh, NC), R & DSysic (Heat applied, Inc.), Pinewortz electronics, Inc., Cell, Inc., and Pluronic (Cell, Inc., Cell).

Any immunoassay may be utilized. The immunoassay may be an enzyme-linked immunoassay (ELISA), a Radioimmunoassay (RIA), a competitive inhibition assay, such as a forward or reverse competitive inhibition assay, a fluorescence polarization assay or a competitive binding assay (for example). The ELISA may be a sandwich ELISA.

Heterogeneous formats may be used. For example, after obtaining a sample from a subject, a first mixture is prepared. The mixture contains a test sample evaluated for a laminin gamma-2 monomer (including a variant of laminin gamma-2 monomer, or any combination thereof) and a first specific binding partner, wherein the first specific binding partner and any laminin gamma-2 monomer contained in the test sample form a first specific binding partner-laminin gamma-2 monomer complex. Preferably, the first specific binding partner is an anti-laminin gamma-2 monomeric antibody or fragment thereof. The order in which the test sample and the first specific binding partner are added to form the mixture is not critical. Preferably, the first specific binding partner is immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific binding partner, and optionally the second specific binding partner) may be any solid phase known in the art, such as, but not limited to, magnetic particles, beads, test tubes, microtiter plates, cups, membranes, scaffold molecules, thin films, filter papers, disks (disks), and chips.

After forming a mixture containing the first specific binding partner-laminin gamma-2 monomer complex, any unbound laminin gamma-2 monomer is removed from the complex using any technique known in the art. For example, unbound laminin gamma-2 monomer may be removed by washing. Suitably, however, the first specific binding partner is present in an amount greater than any laminin gamma-2 monomer present in the test sample, such that all laminin gamma-2 monomers present in the test sample are bound by the first specific binding partner.

After removal of any unbound laminin gamma-2 monomer, a second specific binding partner is added to the mixture to form a first specific binding partner-laminin gamma-2 monomer-second specific binding partner complex. The second specific binding partner is preferably an anti-laminin gamma-2 monomeric antibody that binds to an epitope on laminin gamma-2 monomer that is different from the epitope on laminin gamma-2 monomer to which the first specific binding partner binds. Furthermore, it is also preferred that the second specific binding partner is labelled with or comprises a detectable label as described above.

As indicated above, the use of immobilized antibodies or fragments thereof can be incorporated into immunoassays. The antibodies can be immobilized on various supports such as magnetic or chromatographic matrix particles, surfaces of assay plates (such as microtiter wells), components of solid base materials, and the like. The assay strip may be prepared by coating one or more antibodies in an array on a solid support. The strip can then be immersed in a test biological sample and then rapidly processed through washing and detection steps to generate a measurable signal, such as a colored dot.

Sandwich ELISAs measure the amount of antigen between two layers of antibody (i.e., a capture antibody (i.e., at least one capture antibody) and a detection antibody (i.e., at least one detection antibody.) the capture and detection antibodies bind to different epitopes on the antigen (e.g., laminin gamma-2 monomer).

Typically, at least two antibodies are used to isolate and quantify laminin gamma-2 monomer (including variants of laminin gamma-2 monomer or any combination thereof) in a test sample. More specifically, at least two antibodies bind to certain epitopes of the laminin gamma-2 monomer or portions of the laminin gamma-2 monomer that form an immune complex known as a "sandwich". One or more antibodies can be used to capture laminin gamma-2 monomers (e.g., laminin gamma-2 monomers or variants of laminin gamma-2 monomers, or any combination thereof) in a test sample (these antibodies are often referred to as "capture" antibody (s)), and one or more antibodies are used to bind a detectable (i.e., quantitative) label to the sandwich (these antibodies are often referred to as "detection" antibody (s)). In a sandwich assay, the binding of an antibody to its epitope is expected not to be diminished by the binding of any other antibody in the assay to its respective epitope. In other words, the antibodies are selected such that one or more first antibodies contacted with a test sample containing or suspected of containing a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer or a variant of a laminin gamma-2 monomer, or any combination thereof) do not bind to all or a portion of the epitope recognized by a second or subsequent antibody, thereby not interfering with the ability of one or more second detection antibodies to bind to a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof).

The antibody may be used as the primary antibody in the immunoassay. Preferably, the antibody is at 4.2x10^-11M to 7.4x10^-13K of M_DImmunospecifically binds to an epitope comprising at least three consecutive (3) amino acids of a laminin gamma-2 monomer. The immunoassay can beA second antibody comprising an epitope that immunospecifically binds to at least three consecutive (3) amino acids comprising a laminin gamma-2 monomer, wherein the consecutive (3) amino acids bound by the second antibody are different from the three (3) consecutive amino acids bound by the first antibody. In some embodiments, the antibody can preferentially bind laminin gamma-2 monomer over laminin-5 or over a fragment of laminin gamma-2 monomer (e.g., an EGF-like fragment of laminin gamma-2 monomer).

In one embodiment, a test sample suspected of containing a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) may be contacted with at least one or more capture antibodies and at least one detection antibody, either simultaneously or sequentially. In a sandwich assay format, a test sample suspected of containing a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) is first contacted with at least one capture antibody that specifically binds a particular epitope under conditions that allow for the formation of an antibody-laminin gamma-2 monomer complex. If more than one capture antibody is used, multiple capture antibody-laminin gamma-2 monomer complexes are formed. In a sandwich assay, the antibody, preferably at least one capture antibody, is used in an amount in molar excess of the maximum amount of laminin gamma-2 monomer or laminin gamma-2 monomer variant expected in the test sample. For example, about 5 μ g/mL to about 1 mg/mL antibody/mL microparticle coating buffer may be used.

Optionally, prior to contacting the test sample with the at least one first capture antibody, the at least one capture antibody can be bound to a solid support that facilitates isolation of the antibody-laminin gamma-2 monomer complex from the test sample. Any solid support known in the art may be used, including, but not limited to, solid supports made of polymeric material in the form of wells, tubes, or beads. One or more antibodies can be bound to the solid support by adsorption, by covalent bonding using a chemical coupling agent, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind laminin gamma-2 monomer or laminin gamma-2 monomer variant. In addition, the solid support may be derivatized, if desired, to allow reactivity with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, such as, but not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

After contacting a test sample suspected of containing a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) with at least one capture antibody, the test sample is incubated to allow formation of one or more capture antibody-laminin gamma-2 monomer complexes. The incubation can be carried out at a pH of about 4.5 to about 10.0, at a temperature of about 2 ℃ to about 45 ℃ for a period of time of at least about one (1) minute to about eighteen (18) hours, about 2-6 minutes, or about 3-4 minutes.

After formation of the one or more capture antibody-laminin gamma-2 monomer complexes, the complexes are then contacted with at least one detection antibody (under conditions that allow formation of the one or more capture antibody-laminin gamma-2 monomer-one or more detection antibody complexes). If the capture antibody-laminin gamma-2 monomer complex is contacted with at least one detection antibody, one or more capture antibody-laminin gamma-2 monomer-one or more detection antibody complexes are formed. As with the capture antibody, when contacting at least one detection (and subsequently) antibody with the capture antibody-laminin gamma-2 monomer complex, an incubation period of time under conditions similar to those described above is required to form one or more capture antibody-laminin gamma-2 monomer-one or more detection antibody complexes. Preferably, at least one detection antibody contains a detectable label. The detectable label may be bound to at least one detection antibody prior to, simultaneously with, or after formation of one or more capture antibody-laminin gamma-2 monomer-one or more detection antibody complexes. Any detectable label known in the art can be used as discussed herein and known in the art.

Chemiluminescence assays can be performed according to the methods described in Adamczyk et al, anal. Chim. Acta 579(1): 61-67 (2006). Although any suitable assay format may be used, a microplate chemiluminescence analyzer (Mithras LB-940, Berthold Technologies u.s.a., LLC, Oak Ridge, TN) enables rapid assay of small volumes of multiple samples. The chemiluminescence analyzer can be equipped with multiple reagent syringes using 96-well black polystyrene microtiter plates (Costar # 3792). Each sample may be added to a separate well followed by the simultaneous/sequential addition of other reagents as determined by the type of assay employed. Desirably, the formation of spurious bases in neutral or alkaline solutions using acridinium aryl esters (such as by acidification) is avoided. The chemiluminescent response was then recorded well by well. In this regard, the time taken to record the chemiluminescent response will depend in part on the delay between the addition of the reagent and the particular acridinium employed.

The order of addition of the test sample and the one or more specific binding partners to form a mixture of the chemiluminescent assay is not critical. If the first specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-laminin gamma-2 monomer complex is formed. Alternatively, if a second specific binding partner is used and the second specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-laminin gamma-2 monomer-second specific binding partner complex is formed. Any unbound specific binding partner, whether labeled or not, may be removed from the mixture using any technique known in the art, such as washing.

The hydrogen peroxide can be generated in situ in the mixture, or can be supplied to or supplemented with the mixture, before, simultaneously with, or after addition of the acridinium compound described above. The hydrogen peroxide may be generated in situ in a number of ways, such as those apparent to those skilled in the art.

Alternatively, the source of hydrogen peroxide may simply be added to the mixture. For example, the source of hydrogen peroxide may be one or more buffers or other solutions known to contain hydrogen peroxide. In this regard, the hydrogen peroxide solution may simply be added.

Upon simultaneous or subsequent addition of at least one basic solution to the sample, a detectable signal, i.e. a chemiluminescent signal, is generated indicating the presence of laminin gamma-2 monomer or variant thereof. The alkaline solution contains at least one base and has a pH greater than or equal to 10, preferably greater than or equal to 12. Examples of alkaline solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of alkaline solution added to the sample depends on the concentration of the alkaline solution. The amount of alkaline solution added to the sample can be readily determined by one skilled in the art based on the concentration of alkaline solution used.

The generated chemiluminescent signal may be detected using conventional techniques known to those skilled in the art. Based on the intensity of the signal generated, the amount of laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) in the sample can be quantified. Specifically, the amount of laminin gamma-2 monomer in the sample is directly proportional to the intensity of the signal generated. The amount of laminin gamma-2 monomer present may be quantified by comparing the amount of light generated to a standard curve for laminin gamma-2 monomer or by comparison to a reference standard. Standard curves can be generated by mass spectrometry, gravimetric methods, and other techniques known in the art using serial dilutions of the laminin gamma-2 monomer or solutions of known concentration.

In the chemiluminescent particle assay using the ARCHITECT @ (or successor thereof) analyzer, the conjugate diluent pH should be about 5.8 +/-0.2, the particle coating buffer should be maintained at room temperature (i.e., about 17 to about 27 ℃), the particle coating buffer pH should be about 5.5 +/-0.2, and the particle diluent pH should be about 6.0 +/-0.2. The solids are preferably less than about 0.2%, such as less than about 0.15%, less than about 0.14%, less than about 0.13%, less than about 0.12%, less than about 0.11%, less than about 0.10%, less than about 0.09%, less than about 0.08%, less than about 0.07%, less than about 0.06%, less than about 0.05%, less than about 0.04%, or less than about 0.03%, such as about 0.025%.

In the forward competition format, an aliquot of a known concentration of labeled laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) is used to compete with laminin gamma-2 monomer in the test sample for laminin gamma-2 monomer antibodies (such as immobilized laminin gamma-2 monomer antibodies).

In a forward competition assay, an immobilized antibody (such as a laminin gamma-2 monomeric antibody) may be contacted with a test sample and a labeled laminin gamma-2 monomer or laminin gamma-2 monomer variant, either sequentially or simultaneously. Laminin gamma-2 monomeric protein or laminin gamma-2 monomeric variant can be labeled with any detectable label (including those detectable labels discussed above as well as anti-laminin gamma-2 monomeric antibodies). In this assay, the antibody may be immobilized to a solid support. Alternatively, the antibody may be coupled to an antibody, such as an anti-species antibody, which has been immobilized on a solid support, such as a microparticle.

The labeled laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof), test sample, and antibody are incubated under conditions similar to those described above with respect to the sandwich assay format. Two different species of antibody-laminin gamma-2 monomer complexes may then be generated. Specifically, one of the resulting antibody-laminin gamma-2 monomer complexes contains a detectable label, while the other antibody-laminin gamma-2 monomer complex does not contain a detectable label. The antibody-laminin gamma-2 monomer complex may, but need not, be separated from the remainder of the test sample prior to quantifying the detectable label. Whether or not the antibody-laminin gamma-2 monomer complex is separated from the remainder of the test sample, the amount of detectable label of the antibody-laminin gamma-2 monomer complex is then quantified. The concentration of laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) in the test sample may then be determined by comparing the amount of detectable label in the antibody-laminin gamma-2 monomer to a standard curve. The standard curve can be generated by mass spectrometry, gravimetric analysis, and by other techniques known in the art using serial dilutions of known concentrations of laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, variants of laminin gamma-2 monomer, or any combination thereof).

The antibody-laminin gamma-2 monomer complex may be separated from the test sample by: the antibody is bound to a solid support, such as those discussed above with respect to the sandwich assay format, and the remaining portion of the test sample is then removed from contact with the solid support.

In a reverse competition assay, an immobilized laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) may be contacted with a test sample and at least one labeled antibody, either sequentially or simultaneously. Preferably, the antibody specifically binds to an epitope comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, at least 25, or at least 30 amino acids of a laminin gamma-2 monomer.

Laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) may be bound to a solid support, such as the solid support discussed above with respect to the sandwich assay format.

The immobilized laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof), the test sample, and the at least one labeled antibody are incubated under conditions similar to those described above with respect to the sandwich assay format. Two different kinds of laminin gamma-2 monomer-antibody complexes were then generated. Specifically, one of the resulting laminin γ -2 monomer-antibody complexes is immobilized and contains a detectable label, while the other laminin γ -2 monomer-antibody complex is not immobilized and contains a detectable label. The non-immobilized laminin gamma-2 monomer-antibody complex and the remainder of the test sample are removed from the immobilized laminin gamma-2 monomer-antibody complex present by techniques known in the art, such as washing. Once the non-immobilized laminin gamma-2 monomer antibody complex is removed, the amount of detectable label in the immobilized laminin gamma-2 monomer-antibody complex is then quantified. The concentration of laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) in the test sample may then be determined by comparing the amount of detectable label in the laminin gamma-2 monomer-complex to a standard curve. Standard curves can be generated by mass spectrometry, gravimetric analysis, and by other techniques known in the art using serial dilutions of a known concentration of laminin gamma-2 monomer or laminin gamma-2 monomer variant.

In a fluorescence polarization assay, an antibody or functionally active fragment thereof can be first contacted with a monomer suspected of containing laminin gamma-2 (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) to form an unlabeled laminin gamma-2 monomer-antibody complex. The unlabeled laminin gamma-2 monomer-antibody complex is then contacted with a fluorescently labeled laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof). The labeled laminin gamma-2 monomer competes with any unlabeled laminin gamma-2 monomer in the test sample for binding to an antibody or functionally active fragment thereof. The amount of labeled laminin gamma-2 monomer-antibody complex formed is determined, and the amount of laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) in the test sample is determined via use of a standard curve.

The antibody used in the fluorescence polarization assay specifically binds to an epitope comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or at least 30 amino acids of a laminin gamma-2 monomer.

The antibody, labeled laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, variant of laminin gamma-2 monomer, or any combination thereof), test sample, and at least one labeled antibody may be incubated under conditions similar to those described above with respect to the sandwich immunoassay.

Alternatively, the antibody or functionally active fragment thereof can be contacted simultaneously with a fluorescently labeled laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, variant of laminin gamma-2 monomer, or any combination thereof) and an unlabeled test sample suspected of containing a laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, variant of laminin gamma-2 monomer, or any combination thereof) to form both a labeled laminin gamma-2 monomer-antibody complex and an unlabeled laminin gamma-2 monomer-antibody complex. The amount of labeled laminin gamma-2 monomer-antibody complex formed is determined, and the amount of laminin gamma-2 monomer in the test sample is determined via the use of a standard curve. The antibody used in the immunoassay may specifically bind to an epitope comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or at least 30 amino acids of a laminin gamma-2 monomer.

Alternatively, the antibody or functionally active fragment thereof is first contacted with a fluorescently labeled laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) to form a labeled laminin gamma-2 monomer-antibody complex. The labeled laminin gamma-2 monomer-antibody complex is then contacted with an unlabeled test sample suspected of containing a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof). Any unlabeled laminin gamma-2 monomer in the test sample competes with a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) for binding to an antibody or functionally active fragment thereof. The amount of labeled laminin gamma-2 monomer-antibody complex formed was used to determine the amount of laminin gamma-2 monomer in the test sample via the use of a standard curve. The antibody used in the immunoassay specifically binds to an epitope comprising at least three 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or at least 30 amino acids of a laminin gamma-2 monomer.

Mass Spectrometry (MS) analysis can be used alone or in combination with other methods. Other methods include immunoassays and those described above for the detection of specific polynucleotides. Mass spectrometry methods can be used to determine the presence and/or amount of one or more biomarkers. The MS analysis may comprise matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS analysis, such as, for example, guided-spot MALDI-TOF or liquid chromatography MALDI-TOF analysis. In some embodiments, the MS analysis comprises electrospray ionization (ESI) MS, such as Liquid Chromatography (LC) ESI-MS. Mass spectrometry can be done using a commercially available spectrometer. Methods for analysis using MS, including MALDI-TOF MS and ESI-MS, can be used to detect the presence and amount of biomarker peptides in biological samples. See, for example, U.S. patent nos. 6,925,389; 6,989,100, respectively; and 6,890,763, each of which is incorporated herein by reference for guidance.

It may be desirable to include a control sample or calibrator, such as a series of calibrators. The control sample may be analyzed simultaneously with the sample from the subject. The results obtained from the subject sample can be compared to the results obtained from the control sample. A standard curve can be provided against which the results of the measurement of the biological sample can be compared. Such standard curves present levels as a function of assay units, i.e., fluorescence signal intensity if a fluorescent label is used. Using samples taken from multiple donors, a standard curve can be provided for a control level of laminin gamma-2 monomer in normal tissue and an "at risk" level of laminin gamma-2 monomer in tissue taken from a donor that may have one or more of the above characteristics.

Thus, in view of the foregoing, methods are provided for determining the presence, amount, or concentration of a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) in a test sample. The method comprises assaying a test sample for laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) by an immunoassay, e.g., using at least one antibody and at least one detectable label, and comprising comparing a signal generated by the detectable label as a direct or indirect indication of the presence, amount, or concentration of laminin gamma-2 monomer in the test sample to a signal generated as a direct or indirect indication of the presence, amount, or concentration of laminin gamma-2 monomer in a calibrator. The calibrant is optionally and preferably part of a series of calibrants, wherein each calibrator differs from the other calibrants in the series by the laminin gamma-2 monomer concentration. One of the at least one antibody is an isolated antibody that specifically binds a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof), wherein the antibody has a domain or region selected from the group consisting of (i) a variable heavy chain domain region, or (ii) a variable heavy chain domain region and a variable light domain region. Alternatively, one of the at least one antibody is an isolated antibody that specifically binds a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof), wherein the antibody has (i) a variable heavy chain comprising Complementarity Determining Regions (CDR)1, CDR2, and CDR3 and (ii) a variable light chain comprising CDR1, CDR2, and CDR 3. Examples of at least one antibody that may be used are Antibodies that specifically bind to laminin gamma-2 monomer, such as Antibodies commercially available from companies such as Life span Biosciences, Inc. (Seattle, WA), Acris Antibodies, Inc. (San Diego, CA), Raybiotech, Inc. (Norcross, GA), Atlas Antibodies (Stockholm, Sweden), Sigma-Aldrich (St. Louis, MO), IMGENENEX (San Diego, CA), GeneTex (Irvine, CA), Abcam (Cambridge, MA), Novus Biologicals (Little, CO), Santa Cruzz biotechnology, Inc. (Cell Cruzz, CA), Sciences (Canton, MA), US Biological (Swamyl, Abamet), Seinterstitial (elongated, thermal), Inc. (Inc, and laser Inc. (Inc, Cell, Inc, and laser, Inc, and pipeline, Inc, laser, and pipeline, Inc, and pipeline, Inc, pipeline, and pipeline, Inc, and pipeline, pipeline, PA).

The method may include: (i) contacting the test sample with at least one capture antibody that binds to an epitope on a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) so as to form a capture antibody/laminin gamma-2 monomer complex, (ii) contacting the capture antibody/laminin gamma-2 monomer complex with at least one detection antibody that comprises a detectable label and binds to an epitope on a laminin gamma-2 monomer that is not bound by the capture antibody (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) to form a capture antibody/laminin gamma-2 monomer/detection antibody complex, and (iii) determining the amount of laminin gamma-2 monomer in the test sample based on the signal generated by the detectable label in the capture antibody/laminin gamma-2 monomer/detection antibody complex formed in (ii).

Alternatively, the method may comprise: (i) contacting the test sample with at least one capture antibody that binds to an epitope of a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) so as to form a capture antibody/laminin gamma-2 monomer complex, and simultaneously or sequentially, in any order, contacting the test sample with a detectably labeled laminin gamma-2 monomer that can compete for binding of the at least one capture antibody with any laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) in the test sample. Any laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, variant of laminin gamma-2 monomer, or any combination thereof) and detectably labeled laminin gamma-2 monomer present in the test sample compete with one another to form a capture antibody/laminin gamma-2 monomer complex and a capture antibody/detectably labeled laminin gamma-2 monomer complex, respectively. The method further comprises (ii) determining the presence, amount or concentration of laminin gamma-2 monomer in the test sample based on the signal generated by the detectable label in the capture antibody/detectably labeled laminin gamma-2 monomer complex formed in (ii). The signal generated by the detectable label in the capture antibody/detectably labeled laminin gamma-2 monomer complex is inversely proportional to the amount or concentration of laminin gamma-2 monomer complex in the test sample.

In some embodiments, the methods can include any technique and assay known in the art for measuring the amount of laminin gamma-2 monomer in a sample. For example, a polyclonal, monoclonal, chimeric, humanized or human anti-laminin gamma-2 monomeric antibody (Ab) may be attached directly or indirectly, e.g., via a sheep (or other species) anti-human Ab, to a solid support. Any laminin gamma-2 monomer present in the sample and in contact with the solid support is bound by polyclonal, monoclonal, chimeric humanized or human anti-laminin gamma-2 monomer Ab. Biotin-labeled mouse anti-laminin gamma-2 monomer Ab also binds laminin gamma-2 monomer. Streptavidin linked to horseradish peroxidase (HRPO) bound biotin on mouse anti-laminin gamma-2 monomer Ab. After contact with o-phenylenediamine, HRPO converts the o-phenylenediamine to 2, 3-diaminophenol oxazine, which is orange brown in color and can be measured spectrophotometrically at 492 nm.

The method may further comprise diagnosing, determining a prognosis, or assessing the efficacy of a treatment (therapeutic or prophylactic) for the patient from whom the test sample is obtained. If the method further comprises assessing the efficacy of a therapeutic/prophylactic treatment on the patient from whom the test sample is obtained, the method optionally further comprises adjusting the therapeutic/prophylactic treatment of the patient as needed to improve efficacy. The method may be adapted for use in an automated system or a semi-automated system.

In general, a predetermined level can be used as a benchmark against which to evaluate results obtained after determining a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) of a test sample. Typically, in making such comparisons, the predetermined level is obtained by running a particular assay under appropriate conditions a sufficient number of times such that a link or association between the presence, amount, or concentration of the analyte and a particular stage or endpoint of a disease, disorder, or condition (e.g., cancer), or to a particular clinical marker, can be obtained. Typically, the predetermined level is obtained using an assay of a reference subject (or population of subjects). The laminin gamma-2 monomer measured may include fragments thereof, degradation products thereof, and/or enzymatic cleavage products thereof.

In particular, the amount or concentration of a laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) may be "unchanged", "advantageous" (or "favorably altered"), or "unfavorable" (or "unfavorably altered") with respect to a predetermined level for monitoring disease progression and/or treatment. "elevated" or "increased" refers to an amount or concentration in a test sample that is above a normal or normal level or range (e.g., a predetermined level), or above another reference level or range (e.g., an earlier or baseline sample). The term "reduced" or "reduced" refers to an amount or concentration in a test sample that is below a normal or normal level or range (e.g., a predetermined level), or below another reference level or range (e.g., an earlier or baseline sample). The term "altered" refers to an alteration (increase or decrease) in the amount or concentration in a sample as compared to a normal or normal level or range (e.g., a predetermined level), or as compared to another reference level or range (e.g., an earlier or baseline sample).

The usual or normal levels or ranges for laminin gamma-2 monomer are defined according to standard practice. A so-called change level or change may be considered to have occurred when there is any net change from the usual or normal level or range, or reference level or range, that cannot be explained by experimental error or sample variation. Thus, the level measured in a particular sample will be compared to the level or range of levels determined in a similar sample from a so-called normal subject. In this context, for example, a "normal subject" is an individual without a detectable disease or disorder, while a "normal" (sometimes referred to as a "control") patient or population is those who do not exhibit a detectable disease or disorder, respectively. An "apparent normal subject" is a subject in which laminin gamma-2 monomer has not been evaluated or is being evaluated. An analyte level is said to be "elevated" when the analyte is normally undetectable (e.g., the normal level is zero, or in the range of about 25 to about 75 percentiles of the normal population), but detectable in the test sample, and when the analyte is present in the test sample at a higher than normal level. Thus, the present disclosure provides, among other things, methods of screening for subjects having or at risk of having cancer.

Assays also involve the determination of other labels, etc., as discussed herein and known in the art. For example, the assay method may also involve assaying (detecting) laminin gamma-2 monomer and/or laminin gamma-2 monomer fragment, CEA, and CA19-9 (for example).

Thus, the methods described herein can also be used to determine whether a subject has or is at risk of developing a cancer (e.g., bladder cancer or colon cancer), such as those discussed herein and known in the art. In particular, such a method may comprise the steps of:

(a) determining a concentration or amount of a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) in a test sample from a subject (using methods described herein or known in the art); and is

(b) Comparing the concentration or amount of the laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) determined in step (a) to a predetermined level, wherein, if the concentration or amount of laminin gamma-2 monomer determined in step (a) is favorable relative to the predetermined level, the subject is determined to not have or be at risk of a cancer discussed herein and known in the art. However, if the concentration or amount of laminin gamma-2 monomer determined in step (a) is unfavorable (such as, for example, increased) relative to a predetermined level, the subject is determined to have or be at risk of a cancer as discussed herein and known in the art.

Further, provided herein are methods of monitoring disease progression in a subject. In some embodiments, the method comprises the step of

(a) Determining a concentration or amount of a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) in a test sample from a subject;

(b) determining a concentration or amount of laminin gamma-2 monomer in a later test sample from the subject; and is

(c) Comparing the concentration or amount of laminin gamma-2 monomer determined in step (b) with the concentration or amount of laminin gamma-2 monomer determined in step (a), wherein if the concentration or amount determined in step (b) is unchanged or disadvantageous (such as, for example, increased) when compared to the concentration or amount of laminin gamma-2 monomer determined in step (a), it is determined that the disease in the subject has continued, progressed, or worsened. In contrast, if the concentration or amount of laminin gamma-2 monomer determined in step (b) is favorable when compared to the concentration or amount of laminin gamma-2 monomer determined in step (a), then it is determined that the disease in the subject has stopped, resolved, or ameliorated.

As described herein, in some embodiments, the various methods disclosed herein include providing any of a concentration, an amount, or a comparison of laminin gamma-2 monomer as determined in the various method steps. Once provided, the concentration, amount, or comparison of laminin gamma-2 monomer in the sample can be used to provide a diagnosis, prognosis, or risk assessment of a disease (e.g., an assessment of disease progression or an assessment of the likelihood of developing a disease), or to monitor the course of a disease in a subject (e.g., in a subject undergoing treatment or a subject who has been treated and monitored for disease recurrence). Optionally, the method further comprises comparing the concentration or amount of laminin gamma-2 monomer determined in step (b), for example, to a predetermined level. Further, optionally, if the comparison shows that the concentration or amount of laminin gamma-2 monomer determined in step (b) is, for example, detrimentally altered (such as, for example, increased) relative to a predetermined level, the method comprises treating the subject with one or more pharmaceutical compositions for a period of time.

In addition, the methods can be used to monitor treatment in a subject receiving treatment with one or more pharmaceutical compositions. In particular, such methods involve providing a first test sample from the subject prior to administering one or more pharmaceutical compositions (such as one or more chemotherapeutic or biological agents) to the subject. Next, the concentration or amount of laminin gamma-2 monomer in the first test sample from the subject is determined (e.g., using methods described herein or known in the art). After determining the concentration or amount of the laminin gamma-2 monomer, the concentration or amount of the laminin gamma-2 monomer is optionally then compared to a predetermined level. If the concentration or amount of laminin gamma-2 monomer determined in the first test sample is below a predetermined level, the subject is not treated with one or more pharmaceutical compositions. However, if the concentration or amount of laminin gamma-2 monomer determined in the first test sample is above a predetermined level, the subject is treated with one or more pharmaceutical compositions for a period of time. One skilled in the art can determine the period of time for which the subject is treated with one or more pharmaceutical compositions (e.g., the period of time can be from about (1) day to about thirty (30) days, at which point the success of the treatment can be assessed (e.g., using clinical criteria or determining the concentration or amount of laminin gamma-2 monomer after treatment has begun).

During the course of treatment with one or more pharmaceutical compositions, a second and subsequent test sample is then obtained from the subject. The number of test samples and the time to obtain the test sample from the subject is not critical. For example, a second test sample may be obtained seven (7) days after a first administration of one or more pharmaceutical compositions to a subject, a third test sample may be obtained two (2) weeks after a first administration of one or more pharmaceutical compositions to a subject, a fourth test sample may be obtained three (3) weeks after a first administration of one or more pharmaceutical compositions to a subject, a fifth test sample may be obtained four (4) weeks after a first administration of one or more pharmaceutical compositions to a subject, etc.

After each second or subsequent test sample is obtained from the subject, the concentration or amount of laminin gamma-2 monomer (e.g., laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof) in the second or subsequent test sample is determined (e.g., using methods described herein or known in the art). The concentration or amount of laminin gamma-2 monomer determined in each of the second and subsequent test samples is then compared to the concentration or amount of laminin gamma-2 monomer determined in the first test sample (e.g., the test sample that was initially optionally compared to a predetermined level). If the concentration or amount of laminin gamma-2 monomer determined in step (c) is favorable when compared to the concentration or amount of laminin gamma-2 monomer determined in step (a), then it is determined that the disease or infection in the subject has stopped, resolved, or ameliorated, and the subject should continue to be administered the one or more pharmaceutical compositions of step (b). However, if the concentration or amount determined in step (c) is unchanged or disadvantageous (such as, for example, increased) when compared to the concentration or amount of laminin gamma-2 monomer determined in step (a), it is determined that the disease has continued, progressed or worsened, and the subject should be treated with a higher concentration of the one or more pharmaceutical compositions administered to the subject in step (b), or should be treated with one or more pharmaceutical compositions that are different from the one or more pharmaceutical compositions administered to the subject in step (b). In particular, a subject may be treated with one or more pharmaceutical compositions that are different from one or more pharmaceutical compositions that the subject has previously received and the efficacy of the different compositions to reduce or reduce the level of laminin gamma-2 monomer in the subject and/or ameliorate the symptoms of the disease is evaluated.

Typically, for assays that can be tested repeatedly (e.g., to monitor disease progression and/or response to treatment), a second or subsequent test sample is obtained at a time after a first test sample has been obtained from the subject. In particular, the second test sample from the subject may be obtained minutes, hours, days, weeks or years after the first test sample has been obtained from the subject. For example, a second test sample may be obtained from the subject at the following time period after the first test sample is obtained from the subject: about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 22 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 22 weeks, About 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5.0 years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 10.0 years, or more, or at least one of the foregoing time periods. When used to monitor disease progression, the above assays can be used to monitor disease progression in a subject having cancer and/or any condition associated with cancer. Such conditions may be generally chronic when the cancer is not cured, or such conditions may be acute (also referred to as critical care conditions). Acute conditions are often life-threatening diseases or other critical medical conditions involving, for example, the cardiovascular, nervous or excretory systems. Generally, critical care conditions refer to those conditions that require acute medical intervention in a hospital-based institution (including but not limited to an emergency room, an intensive care unit, a trauma center, or other emergency care institution) or are managed by a paramedic or other on-site medical personnel. For critical care conditions, repeated monitoring is typically performed over a short time frame of minutes, hours, or days (e.g., every about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, 4 about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, or at least about one of the foregoing time frames), and the initial measurements are also typically performed over a short time frame, such as about minutes, hours, or days from the onset of the disease or condition.

Suitably, the assay may also be used to monitor disease progression in subjects suffering from chronic or non-acute conditions. Non-critical care or non-acute conditions refer to conditions other than acute, life-threatening diseases or other critical medical conditions. Generally, non-acute conditions include those having a long or chronic duration. For non-acute conditions, repeated monitoring is typically performed over an extended period of time, such as hours, days, weeks, months, or years (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 9 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 9.0 years, or more years, and the duration of the onset of the disease is typically measured within the same or longer time period, e.0, e.g., as the onset of the initial disease, and is typically measured, or more typically measured within the same age of the subject, Days, months or years.

In addition, the above assays may be performed using a first test sample obtained from a subject, wherein the first test sample is obtained from a source, such as blood, urine, serum, or plasma. Optionally, the above assay can then be repeated using a second test sample obtained from the subject, wherein the second test sample is obtained from the same or another source. For example, if the first test sample is obtained from urine, the second test sample may be obtained from serum or plasma. Results obtained from assays using the first test sample and the second test sample may be compared. The comparison can be used to assess the disease or condition state in the subject.

In addition, the disclosure also relates to methods of determining whether a subject predisposed to or suffering from cancer would benefit from treatment. In particular, the present disclosure relates to laminin gamma-2 monomer conjugate diagnostic methods and products. Thus, the method of "monitoring disease treatment in a subject" as described herein further may also best encompass selecting or identifying candidates for treatment, such as treatment with chemotherapeutic drugs, biologies, radiation, palliative treatment, hormonal treatment, and/or surgery.

Thus, in some embodiments, the present disclosure also provides methods of determining whether a subject having cancer or at risk of having cancer (as discussed herein and known in the art) is a candidate for a particular cancer treatment. Typically, a subject is one that has experienced some disease symptoms or has actually been diagnosed as having or at risk of cancer and/or is indicative of an adverse concentration or amount of a laminin gamma-2 monomer or variant thereof as described herein (such as, for example, an increased concentration of laminin gamma-2 monomer when compared to a predetermined level).

The method optionally comprises an assay as described herein, wherein the analyte is assessed before and after treatment of the subject with one or more pharmaceutical compositions, or wherein the analyte is assessed after such treatment and the concentration or amount of the analyte is compared to a predetermined level. An observed adverse analyte concentration (such as, for example, an increased concentration when compared to a predetermined level) or amount after treatment confirms that the subject will not benefit from receiving further or continued treatment, while an observed favorable analyte concentration or amount after treatment confirms that the subject will benefit from receiving further or continued treatment. This demonstration helps manage clinical studies and provide improved patient care.

While certain embodiments herein are useful in assessing cancer or the risk of cancer onset, the assays and kits are also optionally useful in assessing laminin gamma-2 monomer in other diseases, disorders, and conditions, as appropriate.

In general, any method that can detect or quantify a biomarker in a sample can be used in the methods described herein. These methods include physical and molecular biological methods in addition to immunological methods. For example, suitable physical methods include mass spectrometry methods, Fluorescence Resonance Energy Transfer (FRET) assays, chromatography, and dye-detection methods. Suitable molecular biological methods that may be used include, but are not limited to, Northern or Southern blot hybridization, nucleic acid dot or strip blot hybridization, in situ hybridization, nucleic acid chip assay, PCR, reverse transcriptase PCR (RT-PCR) or real-time PCR (taq-manPCR). Other methods of detecting biomarkers include, for example, Nuclear Magnetic Resonance (NMR), fluorometry, colorimetry, radiometry, luminescence (luminometry) or other spectroscopy, plasmon resonance (e.g., BIACORE), and one-or two-dimensional gel electrophoresis.

Once measured, the laminin gamma-2 monomer concentration and the concentration of any other additional biomarkers evaluated are compared to predetermined reference values for the specific biomarkers. A measured (i.e., determined) laminin gamma-2 monomer concentration that exceeds the reference value for laminin gamma-2 monomer indicates that the subject has cancer, or an increased risk of cancer. The reference value may be determined in one of a number of ways. For example, the reference value for laminin gamma-2 monomer may be the concentration of laminin gamma-2 monomer measured in a sample taken from a control subject, or may be the median value of laminin gamma-2 monomer concentration calculated from the concentrations measured in a plurality of control samples taken from a group of control subjects. The median laminin gamma-2 monomer concentration is preferably obtained from a group of at least 20 control subjects, at least 30 control subjects, or at least 40 control subjects. The predetermined reference value for the biomarker may be a predetermined cutoff value.

A "control subject" is a healthy subject, i.e., a subject without clinical signs or symptoms of cancer. Preferably, the control subjects are clinically evaluated for other undetected signs or symptoms of cancer, which evaluation may include routine physical examination and/or laboratory testing.

Alternatively, the laminin gamma-2 monomer cutoff value (or predetermined cutoff value) may be determined from a biological sample of a patient group by Receiver Operating Curve (ROC) analysis. As is commonly known in the biological fieldIt is well known that ROC analysis is the ability of a test to distinguish one condition from another (e.g., diseased versus normal), or to compare the diagnostic performance of two or more laboratory or diagnostic tests. A description of ROC analysis applied according to the present disclosure is by p.j. heiagrty et al,Time-dependent ROC curves for censored survival data and a diagnostic markerbiometrics 56:337-44 (2000), the disclosure of which is incorporated by reference herein in its entirety. Alternatively, the laminin gamma-2 monomer cutoff value may be determined by a quartile analysis of biological samples of a patient group. For example, the laminin gamma-2 monomer cutoff value may be determined by selecting a value corresponding to any value in the 25 th to 75 th percentile range (preferably a value corresponding to the 25 th percentile, the 50 th percentile, or the 75 th percentile, and most preferably the 75 th percentile). Still further alternatively, the laminin gamma-2 monomer cutoff value may be determined by an average of biological samples of a patient group plus two standard deviation analyses. An exemplary reference laminin gamma-2 monomer value obtained from the median value of the relevant patient group is about 950 pg/ml in serum. The reference value for the exemplary laminin gamma-2 monomer obtained from the quartile analysis at the 75 th percentile is about 1200 pg/ml in serum. Such statistical analysis may be performed using any method known in the art, and may be performed by any number of commercially available Software packages (e.g., from analysis-it Software ltd., Leeds, UK; StataCorp LP, College Station, TX; SAS Institute inc., Cary, NC.). An exemplary laminin gamma-2 monomer reference obtained from the mean plus two standard deviation analyses is about 1050pg/ml in serum.

In some embodiments, the method comprises the following laminin gamma-2 monomer cutoff values: about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 780, about 790, about 840, about 860, about 850, about 910, about 830, about 910, about 880, about 700, about 900, about 750, about 780, about 790, about 780, about 700, about 930, about 940, about 950, about 960, about 970, about 980, about 990, or about 1,000 pg/mL.

The method may further comprise assessing at least one other biomarker of cancer, for example, by measuring the concentration of the at least one other biomarker in the biological sample and comparing the measured concentration to a reference value for each other biomarker assessed. One, two, three, four or more additional biomarkers can be assessed. Additional such biomarkers for cancer include, but are not limited to, fragments of CEA, CA19-9, and laminin gamma-2 monomer (e.g., EGF-like fragments generated by MT 1-MMP). As described herein for determining reference values for laminin gamma-2 monomer, reference values (or predetermined levels) for any other biomarker of cancer may be similarly determined. Typically, a measured (i.e., determined) concentration of any other biomarker in the biological sample that exceeds the reference value for that biomarker also indicates that the subject has cancer or an increased risk of cancer onset. Nevertheless, an example of a true opposite biomarker is also possible, i.e. a biomarker whose concentration in the biological sample is inversely proportional to the situation where the risk of cancer or cancer onset is increased, such that a measured biomarker concentration below the reference value for the biomarker indicates that the subject has cancer or an increased risk of cancer onset.

For example, elevated levels of CEA in the blood have been used as diagnostic biomarkers for cancer, such as colorectal cancer. CEA is often evaluated in patients suspected of having some form of cancer, even though a positive result may be due to other causes, while a negative result does not exclude disease. Nonetheless, in combination with signs and symptoms, CEA can play a role in both diagnosis and disease prognosis, and is part of the usual disease diagnostic criteria for some cancer types. Increased levels of CEA may occur in cancer. Generally, higher levels of CEA in a sample correlate with a greater probability of the presence or onset of cancer. Accordingly, embodiments of the methods described herein include determining the concentration of CEA (and/or CA19-9, fragments of laminin gamma-2 monomer, etc.) and laminin gamma-2 monomer in the sample.

Thus, in some embodiments, the methods comprise detecting a laminin gamma-2 monomer and at least one marker selected from the group consisting of CEA, CA19-9, and a fragment of a laminin gamma-2 monomer. In some embodiments, the methods encompass detecting a laminin gamma-2 monomer and CEA, and optionally, at least one marker selected from the group consisting of CA19-9 and fragments of laminin gamma-2 monomer. In some embodiments, the methods encompass detecting laminin gamma-2 monomer and CA19-9, and optionally, at least one marker selected from fragments of CEA and laminin gamma-2 monomer. In some embodiments, the methods encompass detecting laminin gamma-2 monomer and fragments of laminin gamma-2 monomer, and optionally, at least one marker selected from CEA and CA 19-9. In some embodiments, the methods encompass detecting fragments of laminin gamma-2 monomer, CEA, CA19-9, and laminin gamma-2 monomer.

C. Reagent kit

Provided herein are kits that can be used to treat a subject having, or at increased risk of, cancer, or to diagnose a subject with cancer as previously described herein.

The kit to be used for treating a patient will contain an antibody specific for the laminin gamma-2 monomer. The kit preferably includes instructions for treating a subject with the antibodies described herein. The instructions included in the kit may be affixed to packaging material or may be included as a package insert. Although the description is generally of written or printed material, they are not so limited. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tape, cartridges, chips), optical media (e.g., CD ROM), and the like. The term "specification" as used herein may include an internet address that provides the specification.

Also provided are kits for determining a laminin gamma-2 monomer (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) of a test sample. The kit comprises at least one component for determining a laminin gamma-2 monomer of a test sample and instructions for determining a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof of a test sample. The at least one component includes at least one composition comprising an isolated antibody that specifically binds (e.g., a laminin gamma-2 monomer, a variant of laminin gamma-2 monomer, or any combination thereof). The antibody has a variable heavy domain region and a variable light domain region. The antibody is optionally detectably labeled.

For example, the kit can comprise instructions for assaying a test sample (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof) by an immunoassay, such as a chemiluminescent microparticle immunoassay. The instructions may be in a paper form or a computer readable form such as a compact disc, CD, DVD, or the like. The antibody may be a laminin gamma-2 monomer capture antibody and/or a laminin gamma-2 monomer detection antibody. Alternatively or additionally, the kit may comprise a calibrator or control, e.g., purified, and optionally lyophilized, (e.g., a laminin gamma-2 monomer, a variant of a laminin gamma-2 monomer, or any combination thereof), and/or at least one container for conducting an assay (e.g., a tube, microtiter plate, or strip that has been coated with an anti-laminin gamma-2 monomer monoclonal antibody), and/or a buffer, such as an assay buffer or wash buffer, any of which may be provided as a concentrated solution, a substrate solution for a detectable label (e.g., an enzymatic label), or a stop solution. Preferably, the kit comprises all components necessary to perform the assay, i.e. reagents, standards, buffers, diluents, etc. The instructions may also include instructions for generating a standard curve or reference standard for the purpose of quantifying laminin gamma-2 monomer.

Any antibody provided in the kit, such as a recombinant antibody specific for a laminin gamma-2 monomer, may incorporate a detectable label, such as a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, and the like, or the kit may include a reagent for labeling the antibody or a reagent for detecting the antibody (e.g., a detection antibody) and/or a reagent for labeling the analyte or a reagent for detecting the analyte. The antibodies, calibrators, and/or controls may be provided in separate containers or pre-dispensed into a suitable assay format, for example, into a microtiter plate.

Optionally, the kit includes quality control components (e.g., sensitivity panels, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described in the insert for various immunodiagnostic products. Members of the subject panel are optionally used to establish assay performance characteristics, and further optionally useful indicators of immunoassay kit reagent integrity and assay standardization.

The kit may also optionally include other reagents required to perform diagnostic assays or to facilitate quality control assessments, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, and the like. Other components, such as buffers and solutions (e.g., pre-treatment reagents) for separating and/or processing the test sample may also be included in the kit. The kit may additionally include one or more other controls. One or more components of the kit may be lyophilized, in which case the kit may further comprise reagents suitable for reconstituting the lyophilized components.

The various components of the kit are optionally provided in suitable containers (e.g., microtiter plates) as desired. The kit may further comprise a container for holding or storing the sample (e.g., a container for a blood sample or a cartridge). The kit optionally may also contain reaction vessels, mixing vessels, and other components to facilitate preparation of reagents or test samples, as appropriate. The kit may also include one or more instruments that assist in obtaining the test sample, such as syringes, pipettes, forceps, measuring spoons (measuredspoons), and the like.

If the detectable label is at least one acridinium compound, the kit can comprise at least one acridinium-9-carboxamide, at least one aryl acridinium-9-carboxylate, or any combination thereof. If the detectable label is at least one acridinium compound, the kit can also comprise a source of hydrogen peroxide, such as a buffer, a solution, and/or at least one basic solution.

If desired, the kit may contain a solid phase such as magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, membranes, filter paper, quartz crystals, disks or chips. The kit may also include a detectable label, which may be an antibody or conjugated to an antibody, such as an antibody that functions to detect antibodies. The detectable label may, for example, be a direct label, which may be an enzyme, an oligonucleotide, a nanoparticle chemiluminescent, a fluorophore, a fluorescence quencher, a chemiluminescent quencher, or biotin. The kit may optionally include any other reagents required to detect the label.

If desired, the kit may further comprise one or more components, alone or in further combination with instructions, for determining another analyte of the test sample, which may be a biomarker, such as a biomarker for cancer. Examples of analytes include, but are not limited to, laminin gamma-2 monomer, CEA, CA19-9, and fragments of laminin gamma-2 monomer, as well as other analytes and biomarkers discussed herein or otherwise known in the art. In some embodiments, the one or more components used to determine the laminin gamma-2 monomer of the test sample enable the determination of the presence, amount, or concentration of laminin gamma-2 monomer. Samples, such as serum samples, can also be assayed for laminin gamma-2 monomer using TOF-MS and internal standards.

Kits (or components thereof) and methods for determining the concentration of laminin gamma-2 monomer in a test sample by immunoassay as described herein may be adapted for use in a variety of automated and semi-automated systems, including those in which the solid phase comprises microparticles, as described, for example, in U.S. patent nos. 5,089,424 and 5,006,309, and commercially marketed, for example, by Abbott laboratories (Abbott Park, IL) as ARCHITECT.

Some differences between automated or semi-automated systems as compared to non-automated systems (e.g., ELISA) include the substrate to which the first specific binding partner (e.g., analyte antibody or capture antibody) binds (which may affect sandwich formation and analyte reactivity) and the length and timing of the capture, detection, and/or any optional washing steps. While non-automated formats (such as ELISA) may require relatively long incubation times (e.g., about 2 hours) with the sample and capture reagents, automated or semi-automated formats (e.g., ARCHITECT @andany subsequent platform, Abbott Laboratories) may have relatively short incubation times (e.g., about 18 minutes for ARCHITECT @). Similarly, while a non-automated format (such as ELISA) may incubate the detection antibodies (such as the conjugate reagents) for a relatively long incubation time (e.g., about 2 hours), an automated or semi-automated format (e.g., ARCHITECT @, and any subsequent platforms) may have a relatively short incubation time (e.g., about 4 minutes for ARCHITECT @, and any subsequent platforms).

Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM @, IMx @ (see, e.g., U.S. Pat. No. 5,294,404, which is incorporated herein by reference in its entirety), PRISM @, EIA (beads), and Quantum @ II, among others. In addition, the assays, kits, and kit components can be employed in other formats, such as in electrochemical or other portable or point-of-care assay systems. The present disclosure is applicable, for example, to a commercial Abbott Point of Care (i-STAT Laboratories) electrochemical immunoassay system that performs a sandwich immunoassay. Immunosensors in single-use testing devices and methods of making and operating the same are described, for example, in U.S. patent No. 5,063,081, U.S. patent application publication No. 2003/0170881, U.S. patent application publication No. 2004/0018577, U.S. patent application publication No. 2005/0054078, and U.S. patent application publication No. 2006/0160164, which are incorporated by reference in their entirety for their teachings.

Specifically, the following configuration is preferable for the measurement of the adjustment to the I-STAT systems. Microfabricated silicon chips were fabricated using a pair of gold amperometric working electrodes and a silver-silver chloride reference electrode. On one of the working electrodes, polystyrene beads (0.2 mm diameter) with immobilized capture antibody were attached to a polymer coating of patterned polyvinyl alcohol on the electrode. The chip is assembled into I-STAT drug cartridges in a fluidics form suitable for immunoassay. A layer comprising a detection antibody labeled with alkaline phosphatase (or other label) is present on a portion of the wall of the sample-holding chamber of the cartridge. Within the fluid pouch of the cartridge is an aqueous reagent comprising a phosphate ester of para-aminophenol.

In operation, a sample suspected of containing laminin gamma-2 monomer was added to the holding chamber of the test cartridge and the cartridge was inserted into an I-STAT reader. After the secondary antibody (detection antibody) is dissolved into the sample, a pump element in the cartridge forces the sample into the tube containing the chip. Where it is shaken to facilitate the formation of a sandwich between the first capture antibody, laminin gamma-2 monomer and labeled second detection antibody. In the penultimate step of the assay, fluid is forced out of the bag and into the tubing to wash the sample off the chip and into the waste chamber. In the final step of the assay, the alkaline phosphatase label reacts with the p-aminophenol phosphate to cleave the phosphate group and allow the released p-aminophenol to be electrochemically oxidized at the working electrode. Based on the measured current, the reader can calculate the amount of laminin gamma-2 monomer in the sample through an embedded algorithm and a factory-determined calibration curve.

It will be understood that the methods and kits described herein necessarily include other reagents and methods for performing immunoassays. For example, the present disclosure encompasses various buffers, such as are known in the art and/or are readily prepared or optimized for use, e.g., for washing, as conjugate diluents, and/or as calibrator diluents. An exemplary conjugate diluent is ARCHITECT conjugate diluent used in certain kits (Abbott Laboratories, Abbott Park, IL) and containing 2- (N-morpholino) ethanesulfonic acid (MES), salts, protein blockers, antimicrobials, and detergents. An exemplary calibrator diluent is ARCHITECT human calibrator diluent used in certain kits (Abbott Laboratories, Abbott Park, IL) and containing buffers comprising MES, other salts, protein blocking agents, and antimicrobial agents. In addition, improved signal generation can be obtained, for example, in the I-STAT cartridge format, using nucleic acid sequences linked to signal antibodies as signal amplifiers, as described in U.S. patent application No. 61/142,048, filed 12/31/2008.

If desired, various antibodies can be included in the kit at various concentrations to facilitate generation of a standard curve against which the signal detected in the test sample can be compared. Alternatively, a standard curve can be generated by preparing a dilution of a single antibody solution provided in the kit.

It will be apparent to those skilled in the art that other suitable variations and modifications of the disclosed methods described herein are readily applicable and perceivable, and may be practiced using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, it will be more clearly understood by reference to the following examples, which are intended merely to illustrate some aspects and embodiments of the present disclosure, and are not to be taken as limiting the scope of the disclosure. The disclosures of all journal references, U.S. patents and publications cited herein are hereby incorporated by reference in their entirety.

Examples

Materials and methods

The rabbit polyclonal antibodies used in the examples were established by immunizing rabbits with purified domain III of the human laminin γ -2 chain, which was recombinantly expressed in e.coli (domain III (383-608 aa) expressed as a GST fusion protein using Gateway technology (Invitrogen, Grand Island, NY)). Mouse monoclonal antibody 2H2 was supplied by doctor Koshikawa and doctor Seiki, university of tokyo (monoclonal antibody 2H2 is described in Koshikawa N, et al.Cancer Res.2008, (68) (2). 2008, 1 month, 15 days). Monoclonal antibodies D4B5 and 1H3 (D4B5 is commercially available from Millipore, Billerica, Massachusetts. 1H3 is described in Koshikawa N, et al.Cancer Res.2008 (68) (2). 2008/1/15/2008. samples from bladder cancer patients were purchased from ProMedDx (Norton, MA). Samples from colorectal cancer patients were purchased from ProMedDx (Norton, MA) and KAC co., Ltd. (Kyoto, Japan). Samples from pancreatic cancer patients were purchased from proMedDx (Norton, MA) bioreduction LLC (Long Island, NY) and KAC Co., Ltd. (Kyoto, Japan). Samples of ovarian cancer patients were purchased from bioreduction LLC (Long Island, NY) and KAC co., ltd. (Kyoto, Japan). Samples from gastric cancer patients were purchased from ProMedDx (Norton, MA) and KAC co., ltd. (Kyoto, Japan). Samples from patients with esophageal cancer were purchased from Biorecamation LLC (Long Island, NY). A total of 109 normal samples were purchased from several suppliers including KAC co., ltd. (Kyoto, Japan), bioreduction LLC (Long Island, NY), sera carelifer sciences Inc. (Milford, MA), and Complex Antibodies, Inc (Fort Lauderdale, FL).

Example 1: validation of antibodies and prototype ELISA

A series of experiments were performed to characterize the binding properties of various antibodies used in an exemplary ELISA for the establishment of laminin gamma-2 monomers.

Binding specificity of monoclonal antibody 2H2 to gamma-2 monomer

Western blot assays were performed to assess the specificity of monoclonal antibody 2H2 for the γ -2 monomer and its degree of cross-reactivity with laminin 5. As illustrated in FIG. 3, antibody monoclonal D4B5 (purchased from Millipore, Billerica, Massachusetts) showed binding activity to both laminin 5 complex (at 20 ng; lane 1 of FIG. 3) and gamma-2 monomer (at 4 ng; lane 2 of FIG. 3). In contrast, monoclonal antibody 2H2 showed binding specificity to the γ -2 monomer without any binding activity to the laminin 5 complex. Each monoclonal antibody was used at a concentration of 1 μ g/mL.

Spike recovery and dilution linearity

To evaluate antibody performance in dilution alignment, monoclonal antibodies D4B5 (5 μ g/mL), 1H3 (10 μ g/mL), and 2H2 (10 μ g/mL) (source D4B5 was purchased from D4B5, marketed by Millipore, Billerica, Massachusetts. 2H2 and 1H3 were provided by doctor Koshikawa and doctor Seiki (university of Tokyo) and described in Koshikawa N, et al.Cancer Res.2008, (2). 2008/15/1) were coated in wells of a 96-well microtiter plate (Costar) using standard procedures. Briefly, wells were coated with various concentrations of antibody in Phosphate Buffered Saline (PBS) overnight at 4 ℃. Each well was blocked with 3% Bovine Serum Albumin (BSA) for 1 hour at 37 ℃. Recombinant laminin gamma-2 monomer was added to dilutions (400x, 2000x, 10000x and no additions/admixtures) and to two different dilutions (3x and 10x) of normal serum sample samples (Complex Antibodies, Inc.). Each antibody showed good linearity of response with dilutions of laminin gamma-2 monomer as well as dilutions of matrix (i.e., normal serum samples). An illustrative example of the data from this experiment is shown in figure 4 for the 2H2 monoclonal antibody.

Monoclonal antibody 2H2 was coated at 10 μ g/mL in a 96-well microtiter plate and further evaluated using the spike recovery assay. Recombinant laminin gamma-2 monomer was spiked into diluent (PBS) and normal serum sample (ProMedDx). The target concentration ranges for laminin gamma-2 monomer for this experiment span 0-20 ng/mL (0.00, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00, and 20.00 ng/mL). Once the concentration in the normal sample was adjusted for the endogenous signal (i.e. no spike), the average spike recovery of laminin gamma-2 monomer was calculated to be 86.3%.

Sensitivity of ELISA established Using 2H2 as capture antibody

ELISA assays were performed using 2H2 monoclonal antibody as capture antibody, rabbit polyclonal antibody against domain III of human laminin gamma-2 as detection antibody, and secondary anti-rabbit antibody conjugated to horseradish peroxidase in order to evaluate the analytical sensitivity and reliable concentration range of laminin gamma-2 monomer ELISA. Briefly, each well in a 96-well plate was coated with 50 μ L of 2H2 antibody (10 μ g/mL) overnight at 4 ℃. Each well was washed three times with 200 μ L/well of PBS. Each well was blocked with 200 μ L of a solution containing BSA in PBS (3%) for 1 hour at 37 ℃. After three washes with 0.1% Tween-20 in 200 μ L PBS, the plates were stored at-20 ℃ until use.

For ELISA, the following solutions were prepared:

the sample solution was prepared as a 10x sample dilution in PBS containing 1% BSA, 10mM EDTA, 2% Tween 20, 0.2mg/mL HBR.

The washing solution was PBS containing 0.1% Tween-20.

The detection antibody solution (or "first" antibody solution) contained polyclonal rabbit antibodies in PBS containing 1% BSA, 10mM EDTA, 2% Tween-20, 0.2mg/mL HBR. The solution was added to each well (50 μ L) at room temperature and incubated for 1 hour.

The secondary antibody solution (or "secondary" antibody solution) contained 5000x excess of donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) or F (ab') 2 fragment of goat anti-rabbit IgG in PBS containing 1% BSA, 10mM EDTA, 2% Tween-20, 0.2 mg/mLHBR.

ELISA protocol: to each well 50 μ L of sample solution was added and allowed to incubate for 2 hours at 37 ℃. Each well was washed three times with 200. mu.L/wash. After washing, the detection antibody solution was added to each well (50 μ L/well) at room temperature and incubated for 1 hour. Each well was washed three times again, 200 μ L washing solution/wash. A secondary antibody solution (50 μ L/well) was added to each well at room temperature and incubated for 1 hour. After four rounds of washing (200 μ L/wash), 100 μ L of Tetramethylbenzidine (TMB) was added to each well. The reaction was stopped by adding 100. mu.l/well of 0.6% sulfuric acid solution and incubated for 20 min. OD readings were taken at 450 and 630 nm (Bio-Rad).

As shown in fig. 5B, the established standard curve indicates that the assay sensitivity is about 3.7 pg/mL, such that the reliable detection limit for the ELISA is about 4 pg/mL and the linear detection range is at least 0-4,000 pg/mL.

Dilution line analysis of cancer and normal samples using laminin gamma-2 monomer spiking (spiked)

Further evaluation of the dilution linearity was performed using two cancer samples (one bladder cancer sample, one pancreatic cancer sample) and two normal samples. Normal samples were spiked with recombinant laminin gamma-2 monomer. For dilution factors ranging from 1:8 to 1:1 (i.e., 8x to 1x dilutions), the results indicate 95-116% (compared to no spiked) recovery in cancer samples and 93-113% in normal samples.

Example 2: measurement of laminin gamma-2 monomer

Serum samples from ten patients with bladder cancer and twenty-five patients with colorectal cancer were prepared along with normal samples (109) for determination of various biomarker concentrations. The measurement of samples was performed using the ARCHITECT system for CEA and CA19-9, while laminin gamma-2 monomer concentration was determined using the ELISA assay detailed above. All commercial kits were used according to the manufacturer's instructions.

Results

Fig. 1A and 1B depict a dot plot of the levels of laminin gamma-2 monomer in serum samples from patients with various cancers, including bladder cancer (n = 10) and colorectal cancer (n = 25), as well as serum samples from healthy control patients. [ median level of laminin gamma-2 monomer in patients with bladder cancer was 992 pg/mL, 929 pg/mL for colorectal cancer patients, and 485 pg/mL for healthy controls. Using Student's t-test: bladder cancer was p =0.000041 compared to healthy controls and colorectal cancer was p =0.00031 compared to healthy controls. Serum samples from bladder and colorectal cancer samples had significantly higher concentrations of laminin gamma-2 monomer when compared to the concentrations of laminin gamma-2 monomer in normal samples or other cancer samples (e.g., pancreas, ovary, stomach, and esophagus).

Sensitive and specific serum markers for LN gamma-2 monomer-bladder and colorectal cancer

Receiver Operating Characteristic (ROC) plots were generated from the observed true positive rates for the levels of the three measured biomarkers (laminin gamma-2 monomer, CEA, CA19-9) in bladder cancer and colorectal cancer samples versus the observed false positive rates for the levels of the three biomarkers in normal samples (fig. 2A and 2B). A diagonal line from the lower left axis through the graph to the upper right corner (e.g., (1,1) coordinates) would indicate that the graph has the worst possible prediction method, where the marker levels would be completely indistinguishable from cancer samples and normal samples. The best possible prediction method is expected to yield a point at the top left corner or coordinate (0,1) of the ROC space, representing 100% sensitivity (no false negatives) and 100% specificity (no false positives). Thus, the area under the curve (AUC) derived from the actual data plot of values close to 1.0 represents the best possible prediction method. As shown in fig. 2A and 2B, ROC graph curves demonstrate the sensitivity and specificity of laminin gamma-2 monomeric marker for both bladder cancer and colorectal cancer when compared to other biomarkers (CEA and CA19-9) associated with bladder and colorectal cancer diagnosis. From this data, laminin gamma-2 monomer demonstrates excellent sensitivity and specificity as a biomarker for bladder cancer and colorectal cancer relative to existing biomarkers for those diseases.

TABLE 1 ROC summary (bladder cancer vs (v.) Normal)。

Marker substance	Area of	95% CI	SE
				Laminin gamma 2 monomer	0.91	0.83-0.99	0.039
CEA	0.55	0.29-0.61	0.083
				CA19-9	0.66	0.48-0.84	0.091

TABLE 2 ROC overview (colorectal versus (v.) Normal)。

Marker substance	Area of	95% CI	SE
				Laminin gamma 2 monomer	0.90	0.85-0.96	0.028
CEA	0.55	0.37-0.73	0.092
				CA19-9	0.58	0.43-0.74	0.077

This data establishes for the first time the unexpected association of a) the ability to detect laminin gamma-2 monomer in serum, and b) an increased concentration of laminin gamma-2 monomer in serum (compared to concentrations in normal (healthy) controls and samples of other cancer types) with the occurrence of bladder and colorectal cancer in patient samples. Furthermore, the data also establish that laminin gamma-2 monomer shows excellent diagnostic accuracy for both bladder cancer and colorectal cancer when compared to other existing and clinically relevant biomarkers. The present disclosure also establishes for the first time a diagnostic test that can be used to detect laminin gamma-2 monomers in serum with increased sensitivity and specificity (i.e., it does not detect laminin 5), and does not require proteolytic treatment of laminin gamma-2 monomers for detection (i.e., the assay is not specific for the laminin gamma-2N-terminal fragment produced by MT 1-MMP).

Thus, laminin gamma-2 monomers may be used to diagnose cancer, such as bladder cancer and colorectal cancer, in patient samples, or to provide a prognosis of the risk or progression of cancer, such as bladder cancer and colorectal cancer, in a subject or patient. Similarly, elevated levels of laminin gamma-2 monomer may be used to identify a patient as a candidate for treatment comprising one or more cancer treatments.

Example 3: establishment of prototype reagents for use in an automated immunoassay Instrument ARCHITECT

A series of experiments were performed to transfer ELISA reagents to automated immunoassay reagents for the detection of laminin gamma-2 monomers.

Monoclonal antibody 2H2 was coated on paramagnetic microspheres (Varian Medical Systems, Palo Alto, Calif.). The carboxyl-modified microparticles were then washed with MES buffer (ph5.5), and then MES buffer containing N- (3-dimethylaminopropyl) -N' -ethyl-carbodiimide hydrochloride (SIGMA-ALDRICH, St Louis, MO) and N-hydroxysuccinimide (SIGMA-ALDRICH, St Louis, MO) was added to the microparticles. After incubation at room temperature for 30 min, the reagents were washed out and the antibody diluted with MES buffer was added to the microparticles. The final concentration of antibody in this reaction was 0.3 mg/mL. After 2 hours incubation at room temperature, microparticles were washed with TBS with 1% Tween-20 and stored at 2-8 degrees Celsius.

A rabbit polyclonal antibody directed against domain III of human laminin gamma-2 (used as detection antibody in ELISA) was conjugated with acridinium in PBS buffer containing 0.5% CHAPS. Excess acridinium was removed by a Zeba Micro desalting spin column (Thermo Fisher Scientific, Waltham, MA). Assay sample dilutions were prepared based on phosphate buffer with 1% BSA and 0.1% Tween-20.

ARCHITECT ™ assay is carried out in an i2000 ARCHITECT ™ analyzer. Recombinant laminin gamma-2 monomer was incorporated into sample dilutions (PBS with 1% BSA and 0.1% Tween-20). The calibrant range for the laminin gamma-2 monomer used in this experiment spans 0-20 ng/mL (0.00, 0.01, 0.02, 0.10, 1.00, 10.00, and 20.00 ng/mL).

As shown in fig. 6, the preliminary standard curve indicates that the assay sensitivity is about or near 10 pg/mL. Thus, the assay is quite sensitive.

Dilute linear analysis of cancer and normal samples using laminin gamma-2 monomer spiking

Further evaluation of dilution linearity was performed using five normal samples. Normal samples were spiked with recombinant laminin gamma-2 monomer. Spiked samples were diluted with sample diluent to produce a three-fold dilution series. As shown on fig. 7, the dilution factor ranges from 1:81 to 1:3 (i.e., 81x to 3x dilution). The results show an average recovery of 100-113% in 5 normal samples (compared to no spiked). ARCHITECT;, shows good performance in the dilution linearity test.

Evaluation of commercially available Normal samples

A total of 66 normal samples were purchased from several suppliers including ProMedDx (Norton, MA), KAC co., ltd. (Kyoto, Japan), SeraCare life sciences Inc. (Milford, MA), and complexantifoods, Inc (Fort Lauderdale, FL). Laminin gamma-2 monomer levels in normal samples were measured by the ARCHITECT ™ cell. The results are shown in fig. 8. The median value of the normal sample was 46.0 pg/mL and the 95 percentile was 95.0 pg/mL. The mean value (64 normal samples: excluding 2 samples, 2496 pg/mL and 302 pg/mL) was 50.2 pg/mL. From the standard deviation analysis (excluding 2 samples, 2496 pg/mL and 302 pg/mL), the standard deviation value was 17.8 pg/mL, and the mean plus two standard deviations (appropriate cut-offs) were 85.8 pg/mL.

Claims (18)

1. Use of a polyclonal antibody that specifically binds to domain III of a laminin gamma-2 monomer and does not bind to an N-terminal fragment of a laminin gamma-2 monomer in the preparation of a kit for providing an immunoassay for diagnosis, prognosis or risk classification of a subject having or at risk of having colorectal cancer or bladder cancer, the immunoassay comprising the steps of:

a. obtaining a blood sample from the subject;

b. determining the concentration of laminin gamma-2 monomer in the blood sample by contacting the polyclonal antibody with the blood sample and detecting antibody binding; and

c. comparing the laminin gamma-2 monomer concentration from the blood sample to a reference laminin gamma-2 monomer concentration value, wherein the laminin gamma-2 monomer concentration in the blood sample that is greater than the reference laminin gamma-2 monomer concentration value identifies the subject as having, or having an increased risk of developing, colorectal cancer or bladder cancer.

2. The use of claim 1, wherein the immunoassay further comprises determining the concentration of at least one additional biomarker of colorectal cancer or bladder cancer in the blood sample; and comparing the concentration of the at least one additional biomarker to a reference concentration value for the at least one biomarker.

3. The use of claim 2, wherein the additional biomarker is a laminin gamma-2 fragment, carcinoembryonic antigen (CEA), or carbohydrate antigen 19-9 (CA 19-9).

4. The use of any one of claims 1-3, wherein the reference laminin gamma-2 monomer concentration value is the laminin gamma-2 monomer concentration value of a control sample, or a laminin gamma-2 monomer cutoff value.

5. The use of claim 4, wherein the reference laminin gamma-2 monomer concentration value is the laminin gamma-2 monomer concentration of a control sample.

6. The use of claim 5, wherein the control sample is a blood sample from a human subject.

7. The use of claim 5 or claim 6, wherein the reference laminin gamma-2 monomer concentration value is a laminin gamma-2 monomer concentration standard, a median laminin gamma-2 monomer concentration of a plurality of control samples from a group of control subjects, or an average laminin gamma-2 monomer concentration of a plurality of control samples from a group of control subjects.

8. The use of claim 4, wherein the reference laminin gamma-2 monomer concentration value is a laminin gamma-2 monomer cutoff value.

9. The use of claim 8, wherein the laminin gamma-2 monomer cutoff value is determined from a biological sample of a patient group by Receiver Operating Curve (ROC) analysis.

10. The use of claim 8, wherein the laminin gamma-2 monomer cutoff value is determined by an average plus 2 standard deviation analysis of biological samples of a patient group.

11. The use of claim 8, wherein the reference laminin gamma-2 monomer cutoff is about 1000 pg/mL.

12. The use of any one of claims 1-3, wherein the laminin gamma-2 monomer concentration in the blood sample is at least two times the reference laminin gamma-2 monomer concentration value.

13. The use of any one of claims 1-3, wherein the subject is a human.

14. The use of any one of claims 1-3, wherein the prognosis comprises determining the severity or stage of colorectal cancer or bladder cancer in the subject and/or the likelihood that the subject will develop colorectal cancer or bladder cancer.

15. The use of any one of claims 1-3, wherein the immunoassay further comprises a capture antibody.

16. The use of claim 15, wherein the capture antibody is monoclonal antibody 2H 2.

17. A kit for performing an immunoassay for determining the concentration of laminin gamma-2 monomer in a biological sample, the kit comprising:

a. a polyclonal antibody capable of specifically binding to domain III of a laminin gamma-2 monomer, which allows for quantification of laminin gamma-2 monomer concentration in a biological sample, wherein said polyclonal antibody does not bind to an N-terminal fragment of laminin gamma-2;

b. a reference standard indicative of a reference laminin gamma-2 monomer concentration; and

c. instructions for performing an immunoassay.

18. The kit of claim 17, further comprising at least one additional reagent capable of binding to at least one additional biomarker of colorectal cancer or bladder cancer in the biological sample, said additional reagent allowing for quantification of the concentration of the at least one additional biomarker in the biological sample, and a reference standard indicative of a reference concentration of the at least one additional biomarker of colorectal cancer or bladder cancer in the biological sample.