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WO2015184144A1 - Amplification de signal de molécule rare - Google Patents

Amplification de signal de molécule rare Download PDF

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Publication number
WO2015184144A1
WO2015184144A1 PCT/US2015/032985 US2015032985W WO2015184144A1 WO 2015184144 A1 WO2015184144 A1 WO 2015184144A1 US 2015032985 W US2015032985 W US 2015032985W WO 2015184144 A1 WO2015184144 A1 WO 2015184144A1
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Prior art keywords
rare
cells
rare cells
tyramide
signal
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Inventor
Michael Pugia
Julia PHILIP
Karen Marfurt
Mary FOLTZ
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Siemens Healthcare Diagnostics Inc
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Siemens Healthcare Diagnostics Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the present invention relates to cellular assays, and more particularly to signal amplification of rare molecules in cellular assays utilizing tyramide signal amplification (TSA) without a corresponding amplification of non-rare molecules.
  • TSA tyramide signal amplification
  • Rare molecule analysis is important in medical applications, such as for diagnosis of many diseases including cancers, cardiovascular disease, neurological diseases, immune disease, infectious disease and others. These applications typically require isolation of certain rare molecules of interest which represent only a small fraction of the analyzed sample.
  • rare cells such as circulating tumor cells (“CTC") are of particular interest in the diagnosis of metastatic cancers.
  • CTC are isolated from whole blood by first removing red blood cells ("RBC") by lyses.
  • RBC red blood cells
  • WBC white blood cells
  • Other exemplary rare cells include circulating endothelial cells, rare immune cells, and fetal cells.
  • normal cells e.g., WBC
  • WBC normal cells
  • sample volume must be reduced from 10 ml to a few hundred microliters or less.
  • This method allows for automation of membrane filtration with high recovery of rare cells. For example, the above method allows for a > 99% reduction of WBC and removes all RBC. For a whole blood sample containing 1000 cancer cells, the ratio of WBC to cancer cells may be decreased from 50,000-100,000/1 to ⁇ 200/1 . Rare cells may be measured using an immunocytochemistry (ICC) or in situ hybridization (ISH) method.
  • ICC immunocytochemistry
  • ISH in situ hybridization
  • WO2012/159820 a multiplexing assay of markers on the membrane is described.
  • the labels employed are fluorescent labels and a different fluorescent label is employed for each antibody such that multiple fluorescent-labeled antibodies are employed in any one assay conducted on an isolated rare cell preparation.
  • the sensitivity of traditional fluorescent labels for detecting biomarkers is generally poor for rare cell analysis.
  • Excess affinity molecules have been used to increase the number of probes associated with the cell or tissue. However, this approach has had limited success in significantly enhancing rare cell detection.
  • Another approach that has been developed is to utilize enzymes to catalytically increase the number of fluorescent labels.
  • a Tyramide Signal Amplification (TSA) method uses peroxidase enzymes to catalyze the deposition of multiple fluorophores on tyrosines of proteins by formation of reactive tyramide molecules with phenolic-labeled fluorophores.
  • the enzyme may be associated with an affinity label to direct which cells are reacted (see, for example, Bobrow et al., 1993).
  • TSA is thus presently impractical for rare cell analysis by filtration as WBC remain on the membrane after filtration.
  • FIG. 1 shows the improvement in background reduction in a TSA assay as a result of the addition of novel blocking agents in accordance with an aspect of the present invention.
  • aspects of the present invention are directed to TSA cellular assays that provide for signal amplification of rare cells without corresponding signal amplification of non-rare cells (aka WBC).
  • aspects of the present invention improve rare cell detection methods, reduce background noise, substantially reduce or eliminate false positive results for rare cells caused by WBC detection - all without requiring physical separation of rare cells from non-rare cells.
  • novel blocking agents that are effective to reduce tyramide binding to WBC without reducing tyramide binding to rare cells.
  • a method for signal enhancement in a tyramide signal amplification (TSA) assay for detection of rare cells in a sample includes rare cells and non-rare cells (such as white blood cells).
  • contact of HRP with the tyramide in an unreactive state results in the generation of reactive tyramide molecules in the presence of hydrogen peroxide.
  • the reactive tyramide binds to rare cells in the vicinity of the affinity partner to amplify a detection signal for the rare cells.
  • the blocking agent is effective to reduce an amount of reactive tyramide binding to non-rare cells, and thus reduces background noise for rare cell detection caused by undesired non-rare cell signal detection.
  • the method further includes detecting the presence of the rare cells in the sample.
  • a system for signal enhancement in a tyramide signal amplification (TSA) assay for detection of rare cells in a sample comprising rare cells and non-rare cells.
  • the system comprises effective amounts of: (1 ) hydrogen peroxide; (2) an affinity partner for the rare cells, the affinity partner directly or indirectly linked to horse radish peroxidase (HRP); (3) a conjugate comprising tyramide in an unreactive state and an imaging agent; and (4) a blocking agent comprising a compound of the formula:
  • R aryl, alkyl, polyalkylene oxide, or polymer
  • aryl may comprise phenyl, substituted phenyl, naphyl and substituted naphyl.
  • alkyl alkyl may comprise alkyl chains of 1 to 16, linked via a C-C, C-O , C-N, or C-S bond.
  • contact of HRP with the tyramide in an unreactive state results in the generation of reactive tyramide in the presence of hydrogen peroxide.
  • the reactive tyramide binds to rare cells in the vicinity of the affinity partner to amplify a detection signal for the rare cells.
  • the blocking agent is effective to reduce an amount of reactive tyramide binding to non-rare cells, and thus reduces background noise for rare cell detection caused by undesired non-rare cell signal detection.
  • Standard techniques are used for recombinant DNA, oligonucleotide synthesis, cellular assays, and tissue culture and transformation (e.g., electroporation, Iipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Coligan et al.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, systems, and methods of this presently disclosed and claimed inventive concept(s) may be described in terms of aspects or embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, systems, and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the presently disclosed and claimed inventive concept(s). All such similar substitutes and
  • the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
  • the phrases “amount effective,” “effective amount,” or the like refer to amounts at concentrations and for periods of time necessary to achieve the desired result.
  • alkyl or “alkylene” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; for example, "C1 -C10 alkyl” denotes alkyl having 1 to 10 carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.
  • aryl refers to aromatic monocyclic or multicyclic groups containing carbon atoms.
  • Aryl groups include, but are not limited to groups such as unsubstituted or substituted phenyl, and unsubstituted or substituted naphthyl.
  • the term "at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc.
  • the term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results.
  • the terms “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • the phrase "or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • affinity partner refers to any type of molecule that binds to a specific biomarker as described herein.
  • probes include, but are not limited to, antibodies (or binding fragments or derivatives thereof), receptors, organic molecules, inorganic molecules, ligands, nucleic acids (including but not limited to, DNA, RNA, microRNA, mRNA, siRNA, etc.), peptides, polypeptides, proteins, epitopes, antigens, ligands, receptors, complexes, lipids, glycoproteins, glycolipids, glycosaminoglycans,
  • antibody is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
  • monoclonal antibodies including full length monoclonal antibodies
  • polyclonal antibodies include full length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
  • multispecific antibodies e.g., bispecific antibodies
  • antibody or “antibody peptide(s)” refer to a full length immunoglobulin molecule (i.e., an intact antibody), or a binding fragment thereof that competes with the intact antibody for specific antigen binding. Binding fragments may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single- chain antibodies, single domain antibodies (such as but not limited to, NANOBODIES®) and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (Nature Med., 9:129-134 (2003).
  • the terms "antigen binding fragment” or “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to bind to an antigen.
  • the antigen-binding function of an antibody can be performed by fragments of an intact antibody.
  • binding fragments encompassed within the term "antigen-binding fragment” of an antibody include but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single- chain antibodies, single domain antibodies (such as but not limited to,
  • NANOBODIES® isolated CD H3
  • other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments are obtained using conventional recombinant and/or enzymatic techniques and are screened for antigen binding in the same manner as intact antibodies.
  • biomarker as used herein will be understood to refer to any target site on the surface of or inside of a cell, e.g., a rare cell, that an affinity partner can have affinity therefor and thus can bind to said moiety.
  • biomarker may be, for example but not by way of limitation, a nucleic acid, peptide, polypeptide, protein, epitope, antigen, ligand, receptor, complex (i.e., an MHC-peptide complex), lipid, glycoprotein, glycolipid, glycosaminoglycan, carbohydrate,
  • polycarbohydrate polycarbohydrate, glycoconjugate, and any combination or derivative thereof.
  • blocking agent refers to a molecule which interacts with or binds to a binding site on a cell to a degree sufficient to prevent a different intended molecule (e.g., an affinity agent) from binding to the same binding site.
  • label or "imaging agent” refers to a functional group or molecule that produces or can be induced to produce a detectable signal.
  • the terms "linked,” “conjugated,” and the like along with their noun forms refers to all possible ways by chemical interaction or bonding, whereby at least one first attachment site and the at least one second attachment site are connected. Linking thus includes bonding, directly or indirectly (through a spacer or additional moiety or molecule), one molecule to another.
  • polypeptide refers to a polymer of amino acid residues.
  • polypeptide is a generic term that refers to native protein, protein fragments, or analogs of a polypeptide sequence. Hence, native protein, protein fragments, and analogs are species of the polypeptide genus.
  • polymer refers to molecule composed of at least 2 repeating structural units typically connected by covalent chemical bonds.
  • the repeating structural unit may be one type of monomer, and, in such case, the resulting polymer is a homopolymer.
  • a polymer may include two or more different types of monomers.
  • Tyramide Signal Amplification (TSA) [0036]
  • TSA Tyramide Signal Amplification
  • Tyramide is a phenolic compound that, when activated by the enzyme horseradish peroxidase (HRP) in the presence of hydrogen peroxide, covalently binds to electron rich moieties on a surface (e.g., predominantly to tyrosine residues in proteins in tissue or cell preparations).
  • HRP horseradish peroxidase
  • HRP converts tyramides into short-lived, extremely reactive tyramide intermediates.
  • the activated molecules then rapidly react with and covalently bind to electron rich regions (typically tyrosine in the form of polypeptides chains of proteins) of adjacent proteins as described.
  • This binding of activated tyramide molecules typically occurs immediately adjacent to the sites at which the affinity partner and activating HRP enzyme is bound.
  • Multiple deposition of the labeled tyramide occurs in a very short time (generally within 3-10 minutes).
  • Subsequent detection of the label which may be linked to the affinity partner and/or the reactive tyramide molecules, yields effectively large signal amplification for rare cells.
  • the blocking agent prevents this reaction with non-rare cells by preventing tyramide from being in the proximity of the proteins on the non-rare cells.
  • the blocking agents, compositions, systems, and processes described herein are particularly useful to reduce background noise associated with undesired reactive tyramide binding to non-rare cells (e.g., white blood cells) in the sample. While it is desirable to increase rare cell response utilizing TSA, known TSA methods are characterized by false positives provided by reactive tyramide binding to white blood cells in particular. Thus, a sample having no rare cells may actually be characterized as positive for rare cells, although primarily white blood cells and no rare cells are present.
  • TSA methods involve using an enzyme to catalyze a chemical reaction that amplifies an associated signal. See US 20140038.
  • a primary affinity partner such as an antibody, having an affinity for a biomarker on a rare cell is used, which may or may not have an imaging agent or label linked thereto.
  • the primary affinity partner is conjugated to an enzyme horse radish peroxidase (HRP).
  • HRP horse radish peroxidase
  • One or more mediums comprising a label such as fluorophore dye, hydrogen peroxide, and a tyramide mixture in a non-reactive form are added to the sample. It is appreciated that subsequent washing and/or incubation steps may follow each addition.
  • HRP converts the non-reactive tyramide into a short-lived free-radical tyramide form that deposits in the neighborhood of the site of the bound affinity partners, through binding to nucleophilic protein tyrosine side chains.
  • This free-radical mechanism allows multiple reactive labeled tyramides to be deposited, thereby amplifying the signal.
  • the short-lived nature of the converted labeled tyramides means they will not diffuse, and will stay locally close to the affinity partners that were linked to HRP.
  • a TSA method is utilized in the context of an immunocytochemistry (ICC) and/or an in situ hybridization (ISH) method that provide for at least filtration of the sample to be examined for the presence of rare cells and for attachment of one more affinity partners specific for one or more respective biomarkers on the rare cells.
  • ICC and ISH methods are based on the binding of specific affinity agents to detect biomolecule target positive cells.
  • Immunocytochemistry (ICC) methods use antibodies as the affinity agent.
  • In-situ hybridization (ISH) methods use nucleic acid probes as the affinity partner. These methods can be "direct affinity assays" that conjugate a label, such as a fluorescent probe, to an affinity partner.
  • the ICC and ISH methods may also be performed as an "indirect assay.”
  • An “indirect assay” may comprise a secondary affinity agent conjugated to a label where the secondary affinity agent binds the primary affinity partner as described above.
  • HRP can be directly linked to the affinity partner.
  • HRP can be bound to a secondary antibody or probe which can bind to the affinity partner as an "indirect method.”
  • the HRP may also be bound to an affinity partner through a streptavdin binding interaction to biotin where either streptavdin or biotin is directly attached to the affinity agent (e.g., "conjugated").
  • the labeled target cell may be detected using suitable methods known in the art.
  • suitable methods known in the art.
  • Fluorescence microscope with excitation, emission, and cut-off filters specific for each probe is utilized.
  • Multiple fluorescent probes can be used in a cocktail to detect multiple biomolecules of a target cells.
  • Cells may be then characterized, for example, by the positive (fluorescent) or negative (non- fluorescent) response using a scanning fluorescent microscope.
  • fluorescence substrates procedures may also be utilized for rare cell identification.
  • reaction of HRP with these substrates occurs when isolated materials are reacted with the substrates.
  • the amount of signal generated is read by spectrophotometer, western blot, or luminometer.
  • Chromogenic dye staining methods are often used as an additional test to determine if cells detected are rare or non-rare cells. Chromogenic dye counterstaining procedures are based on Hematoxylin & Eosin (H&E) methods widely used by pathologists. The procedure produces visible spectra that viewed under a microscope with color camera. Hematoxylin & Eosin (H&E) are chromogenic dyes.
  • Hematoxylin stains nuclei of cells blue. Eosin Y stains the eosinophilic structures of cells in various shades of red, pink and orange.
  • the use of chromogenic dyes for counterstaining or indirect immunoassay for is not compatible with fluorescence methods. The signal, e.g., fluorescence, of an immunoassay is thus read prior to chromogenic dyes counter staining. Therefore, chromogenic dye staining can be done as a sequential step after the fluorescent signal is read.
  • sample processing and steps of the methods described herein may be automated by robot.
  • a robot or automated instrumentation may be provided that is specially equipped with a filtration device and software to integrate the robotics for cell isolation, reagent additions and steps required for the detection methods.
  • cells may be fixed with
  • the methods described herein may also include series of wash steps to wash away unbound antibody and probe, blocking steps to reduce non-specific binding, and incubation steps for multiple step assays as would be appreciated by those skilled in the art.
  • the methods described herein also contemplate the use of DAPI (4',6- diamidino-2-phenylindole) or Hoechst fluorescent DNA stains to stain the nuclei of the cells and application cover media to help preserve the signaling, e.g., fluorescent intensity, of the imaging agents, if desired.
  • the samples to undergo analysis as described herein may be from any suitable source and be prepared according to known methods in the art.
  • the whole blood sample is first processed so as to remove the majority of red blood cells, platelets and the like, to isolate rare cells (if present).
  • the remaining sample having rare cells typically also comprises a quantity of white blood cells.
  • whole blood samples may be mixed with buffer and separated through filtration onto a porous microscope slide to isolate rare cells (if present) with some non-rare cells (white blood cells) and no red blood cells.
  • Rare cells are those cells that are present in a sample in relatively small quantities when compared to the amount of non-rare cells in a sample. In some examples, rare cells are present in an amount of about 10 "8 % to about 10 ⁇ 2 % by weight of a total cell population in a sample suspected of containing the rare cells. CEC are included in the category of rare cells.
  • Non-rare cells are those cells that are present in relatively large amounts when compared to the amount of rare cells such as CEC in a sample.
  • non-rare cells are at least about 10 times, or at least about 102 times, or at least about 103 times, or at least about 104 times, or at least about 105 times, or at least about 106 times, or at least about 107 times, or at least about 108 times greater than the amount of the rare cells in the total cell population in a sample suspected of containing non-rare cells and rare cells.
  • the non-rare cells may be, but are not limited to, white blood cells (WBC), platelets, and red blood cells (RBC), for example.
  • WBC white blood cells
  • RBC red blood cells
  • Enhancement of the concentration of rare cells in a sample from a subject may be achieved by filtration using one or more of the following forces: centrifugation, pressure, vacuum, capillary hydrostatic forces, ultracentrifugation, differential thermal gradient and electrophoretic forces, for example.
  • Filtration involves contacting the sample with a porous matrix usually under vacuum pressure. Filtration techniques include, but are not limited to, microfiltration, ultrafiltration, or cross-flow filtration, for example.
  • the porous matrix may be part of a filtration module where the porous matrix is part of an assembly for convenient use during filtration ("porous matrix filtration assembly").
  • the porous matrix can be a membrane, film, or microfluidic structure that has pores or obstacles through which liquid with cells flows across or through.
  • a porous matrix filtration assembly by way of illustration and not limitation, is described by Friedrich, et al., in U.S. Patent Application Publication No. 2012/0315664, the relevant disclosure of which is incorporated herein by reference. 1 .5 Affinity Agents
  • one or more affinity agents are selected to one or more respective target rare cell biomarkers and are added to a cellular sample in effective amounts that would be appreciated by the skilled artisan.
  • the affinity agent comprises an antibody specific for a biomarker on a rare cell.
  • Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, lgG1 , lgG2a, lgG2b and lgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, and Fab', for example. In addition, aggregates, polymers, and conjugates of
  • immunoglobulins or their fragments may be used where appropriate so long as binding affinity for a particular molecule is maintained.
  • the affinity agent is comprises a nucleic acid specific for a marker on a rare cell.
  • the nucleic acid may be any polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • isolated nucleic acid and “isolated polynucleotide” are used interchangeably; a nucleic acid or polynucleotide is considered “isolated” if it: (1 ) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide” is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
  • rare cell type biomarkers include cancer cell type biomarkers, oncoproteins and oncogenes, chemo resistance biomarkers, metastatic potential biomarkers, endothelial cell typing markers and others. See Pugia
  • Cancer cell type biomarker include but are not limited to cytokeratins (CK) (CK1 , CK2, CK3, CK4, CK5, CK6, CK7, CK8 and CK9, CK10, CK12, CK 13, CK14, CK16, CK17, CK18, CK19 and CK2, Epithelial cell adhesion molecule (EpCAM), N- cadherin, E-cadherin and vimentin.
  • Oncoproteins and oncogenes with likely therapeutic relevance due to mutations include but are not limited to WAF, BAX-1 , PDGF, JAGGED
  • Endothelial Cell typing markers include, by way of illustration and not limitation, CD136, CD105/Endoglin, CD144A/E- cadherin, N-cadherin, E-cadherin CD145, CD34, Cd41 CD136, CD34, CD90,
  • the rare cells may be endothelial cells which are detected using markers, by way of illustration and not limitation, CD136,
  • the rare molecules may be biomarkers that detect rare immune cells.
  • monocytes are indicated by CD45+, CD14+; T lymphocytes are indicated by CD45+, CD3+; T helper cells are indicated by
  • CD45+,CD3+, CD4+; cytotoxic T cells are indicated by CD45+,CD3+, CDS+; ⁇ - lymphocytes are indicated by CD45+, CD19+ or CD45+, CD20+; thrombocytes are indicated by CD45+, CD61 +; and natural killer cells are indicated by CD16+, CD56+, and CD3-.
  • CD4 and CD8 are, in general, used as markers for helper and cytotoxic T cells, respectively. These molecules are defined in combination with CD3+, as some other leukocytes also express these CD molecules (some macrophages express low levels of CD4; dendritic cells express high levels of CDS).
  • the rare molecules detected may be biomarkers for detection of cancer, cardiac damage, cardiovascular disease, neurological disease, hemostasis/hemastasis, fetal maternal assessment, fertility, bone status, hormone levels, vitamins, allergies, autoimmune diseases, hypertension, kidney disease, diabetes, liver diseases, infectious diseases and other biomolecules useful in medical diagnosis of
  • the sample to be tested may be a whole blood, urine, plasma, serum, salvia or other natural sample from a mammal such as, but not limited to, a human subject, for example.
  • HRP is linked to the affinity agent. This may be accomplished by suitable incubation and washing steps as are known, such as by incubation with effective amounts of an HRP source, such as Goat-anti-rabbit HRP.
  • an HRP source such as Goat-anti-rabbit HRP.
  • labels may be similarly linked to the affinity agent by suitable reaction of functional groups, for example, to provide for enhanced signaling in the respective assay.
  • the labels used herein may be any molecule that produces or can be induced to produce a signal, and may be, for example, a fluorescer, a radiolabel, an enzyme, a chemiluminescer or a photosensitizer.
  • the signal may be detected and/or measured by detecting enzyme activity, luminescence, light absorbance or radioactivity, depending on the nature of the label.
  • the label can directly produce a signal and, therefore, additional components are not required to produce a signal.
  • Numerous organic molecules, for example fluorescers are able to absorb ultraviolet and visible light, where the light absorption transfers energy to these molecules and elevates them to an excited energy state. This absorbed energy is then dissipated by emission of light at a second wavelength.
  • Other labels that directly produce a signal include radioactive isotopes and dyes, for example.
  • the label is part of a signal producing system, which may include components other than the label for generating a signal in conjunction with the label.
  • the label is a fluorescent label.
  • fluorescent labels Various types, depending on the application and purpose, may be employed in accordance with aspects of the present invention.
  • a different fluorescent label may be associated with multiple different affinity agents such that multiple fluorescent-labeled antibodies, for example, may be employed in any one assay conducted on an isolated rare cell preparation.
  • fluorescent labels are commercially available and sold under the trademarks BD HorizonTM V450 Pacific BlueTM AmCyan BD HorizonTM V500 (Em-Max 500 nm) , Alexa Fluor®, FITC, R-phycoerythrin (R-PE), Texas Red®, APC, CyTM PerCP and DyLightTM .
  • suitable fluorophores are described herein below. For example, but not by way of limitation, a comprehensive catalogue exists online at http://www.fluorophores.org (the entire contents of which are expressly incorporated herein by reference).
  • the label may require components to produce a signal, and the signal producing system would then include all the components required to produce a measurable signal as would be appreciated by the skilled artisan.
  • Such other components may include substrates, coenzymes, enhancers, additional enzymes, substances that react with enzymatic products, catalysts, activators, cofactors, inhibitors, scavengers, metal ions, and a specific binding substance required for binding of signal generating substances.
  • the label and other signal producing system members may be bound to the affinity partner, tyramide molecules, a support, and/or become bound to a cell that is disposed on a support.
  • the binding may be direct or indirect, covalent or non-covalent and may be accomplished by well known techniques, commonly available in the literature.
  • Cells may be bound to a solid support in any manner known in the art, provided only that the binding does not substantially interfere with the ability of a biomarker on the cell to bind with an affinity agent.
  • the cells may be coated or covalently bound directly to the solid support. Linking groups may also be used to covalently couple the solid support and the cells. Other methods of binding the cells are also possible.
  • a solid support may have a coating of a binder for a small molecule such as, for example, avidin or an antibody, and a small molecule such as, e.g., biotin, hapten, etc., can be bound to the cells or vice versa.
  • a small molecule such as, for example, avidin or an antibody
  • a small molecule such as, e.g., biotin, hapten, etc.
  • the support may be comprised of an organic or inorganic, solid or fluid, water insoluble material, which may be transparent or partially transparent.
  • the support can have any of a number of shapes, such as particle, including bead, film, membrane, tube, well, strip, rod, planar surfaces (such as, e.g., sheet, plate and slide), and fiber, for example.
  • the support may or may not be suspendable in the medium in which it is employed.
  • suspendable supports are polymeric materials such as latex; lipid bilayers or liposomes; oil droplets, and metallic supports such as, e.g., magnetic particles; for example.
  • support compositions include polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4 methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, and polyvinyl butyrate), either used by themselves or in conjunction with other materials.
  • polymers such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4 methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, and polyvinyl butyrate), either used by themselves or in conjunction with other materials.
  • the label and/or other signal producing system member may be bound to a member of a specific binding pair.
  • the label can be linked to a member of a specific binding pair such as an affinity partner as described herein.
  • two signal producing system members such as a fluorescer and quencher can each be bound to different antibodies that form a specific complex. Formation of the complex brings the fluorescer and quencher in close proximity, thus permitting the quencher to interact with the fluorescer to produce a signal.
  • the blocking agents are added to the cellular sample, affinity agent, and tyramide, which may be labeled, in amounts effective to reduce at least an amount of a non-rare cell signal upon detection of the same in a TSA method.
  • the novel blocking agents will bind to non-rare cells, e.g., white blood cells (WBC), at least preferentially as compared to rare cells.
  • WBC white blood cells
  • the blocking agents will bind to WBC to a greater degree than to the rare cells.
  • the non-rare cells e.g., white blood cells
  • the blocking agents will prevent subsequent binding of labeled tyramide species to the non- rare cells.
  • one or more of the novel blocking agents are effective to reduce background noise by at least 10%, 30%, 50%, 70%, 90%, or 100%, or more.
  • the signal to noise ratio for the associated detection method may be increased by a corresponding amount.
  • the amount of blocking agents utilized for a particular can be readily established by monitoring the effect of adding the blocking agent.
  • the amount of blocking agent is typically 1 to 5 % in buffer.
  • the amount of blocking agent may be deemed too little if too much background noise is seen
  • affinity agent binds non target
  • signal amplification is not optimal.
  • the amounts of hydrogen peroxide and tyramide (labeled or unlabeled) to be included in the assay may be readily established.
  • one or more known blocking agents and/or components effective to reduce background noise caused by the presence of non-rare cells may be utilized.
  • additional (second) blocking agents known in the art may be utilized, such as casein, albumin, serum, gelatin and other known to those skilled in the art.
  • the novel blocking agents may be added to a casein blocking buffer as is known in the art.
  • effective amount of surfactants may be added to the medium comprising the blocking agents, or in a separate medium, and combined with the sample. The surfactants also aid in reducing background noise caused by non-rare cells in a rare cell detection method.
  • Suitable surfactants include, but are not limited to, Tween 80, Tween 20, Triton X100, CHAPS and other known to those skilled in the art.
  • the novel blocking agents may be utilized with any other composition or process designed to reduce background noise in a rare cell detection assay, such as with ICC and ISH.
  • the assays and compositions described herein may be carried out/disposed in an aqueous buffered medium at a moderate pH, generally that which provides optimum assay sensitivity.
  • the pH for the assay medium will usually be in the range of about 4 to about 1 1 , or in the range of about 5 to about 10, or in the range of about 6.5 to about 9.5.
  • the pH will usually be a compromise between optimum binding of the binding members of any specific binding pairs, and the pH optimum for other reagents of the assay such as members of a signal producing system, for example.
  • Various buffers may be used to achieve the desired pH and maintain the pH during the incubation period.
  • Illustrative buffers include borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, BICINE, and the like.
  • Various ancillary materials may be employed in the assay methods.
  • the medium may comprise stabilizers for the medium and for the reagents employed.
  • proteins may be included, such as albumins; quaternary ammonium salts; polyanions such as dextran sulfate; and binding enhancers, for example. All of the above materials are present in a concentration or amount sufficient to achieve the desired effect or function.
  • One or more incubation periods may be applied to the medium at one or more intervals including any intervals between additions of various reagents mentioned above.
  • the medium is usually incubated at a temperature and for a time sufficient for binding of various components of the reagents to occur.
  • Moderate temperatures are normally employed for carrying out the method and usually constant temperature, preferably, room temperature, during the period of the measurement.
  • Incubation temperatures normally range from about 5°C to about 99°C or from about 15°C to about 70°C, or about 20°C to about 45°C, for example.
  • the time period for the incubation is about 0.2 seconds to about 24 hours, or about 1 second to about 6 hours, or about 2 seconds to about 1 hour, or about 1 to about 15 minutes, for example.
  • the time period depends on the temperature of the medium and the rate of binding of the various reagents. Temperatures during measurements will generally range from about 10°C to about 50°C or from about 15°C to about 40°C.
  • the concentrations for the various reagents in the assay medium will generally be determined by factors such as the concentration range of interest for the particular biomarker, the nature of the assay, affinity level of the affinity agent, avidity and antibody fragmentation, for example.
  • the final concentration of each of the reagents may be determined empirically to optimize the sensitivity of the assay over a range. Considerations such as the nature of a signal producing system and the nature of the coagulation factor normally determine the concentrations of the various reagents.
  • the order of addition may be varied widely, there will be certain preferences depending on the nature of the assay.
  • the simplest order of addition is to add all the materials simultaneously and determine the effect that the assay medium has on the signal as in a homogeneous assay.
  • the reagents can be combined sequentially. Washing and incubation steps may be involved preceding and/or subsequent to each addition as would be appreciated by one skilled in the art. The length of the incubation period is that which is sufficient to accomplish the desired function.
  • Blood samples of -10 ml_ were collected from patients following an IRB approved protocol using blood collection tubes and tube holders containing K3EDTA & 0.45 ml Transfix (Vacutest Kima TVT-09-50-45). Tubes were inverted 10 times after collection and were used directly after 1 h from collection. The samples were stored at room temperature for up to 5 days before isolation. If sample was shipped, it was kept between 2-25° C during shipping. Blood samples were delivered to a 50 ml_ centrifuge tube and QS to 20 ml_ with PBS solution. The H226 or H2228 lung cancer cell line (ATCC) was used to test the response of the assay. These cells were added at 50, 200, 400, and 800 cell counts per blood tube.
  • ATCC H226 or H2228 lung cancer cell line
  • the blood samples were filtered through a membrane having an average pore size of 8 ⁇ according to a method disclosed in U.S. Patent Application Publication No. 2012/0315664, the relevant portions of which are incorporated herein by reference.
  • the sample on the membrane was subjected to a negative mBar, that is, a decrease greater than about -30 mBar from atmospheric pressure.
  • the vacuum ap plied varies from 1 to -30 mBar as the volume of the sample reduces from during filtration. High pressure drops are allowable dependent on reservoir and sample volume and filtration rate.
  • the membrane was washed with PBS, and the sample was fixed with formaldehyde, washed with PBS, subjected to permeabilization using of 0.2% TRITON® X100 in PBS for 1 min and washed 5 times with 1 ml_ of PBS. Hydrogen peroxide (3%) incubation for 30 min can optionally be used for removal of endogenous peroxidase activity, which can help the specificity of when HRP is used as the catalyst.
  • lung cancer cells H226 ATCC captured on the membrane were detected with an affinity agent that was a nucleic acid for EGFR.
  • the rare cells were detected with 10 ⁇ _ fluorochrome-labeled (Texas Red, Rhodamine) probe (HER FISH probe mix, Dako, Ca).
  • the probe was pipetted onto the area to be hybridized, and the slides were heated to 82 °C for 5 min.
  • the ISH reagent was provided in liquid form in hybridization solution containing 45% formamide, 10% dextransulphate, 0.21 % N-methyl-2-pyrrolidone, 300 mmol/L NaCI, 5 mmol/L phosphate, nucleic acid blocking sequence and then incubated overnight at 60 °C. Post-hybridization washes were done at RT with 1 ml_ SSC wash (2X saline-sodium citrate buffer (SSC) and 0.3% NP-40 nonyl phenoxypolyethoxylethanol) to remove unbound probe.
  • SSC saline-sodium citrate buffer
  • NP-40 nonyl phenoxypolyethoxylethanol
  • lung cancer cells H2228 ATCC captured on the membrane were detected with multiple affinity agents that were antibodies for multiple rare cancer target molecules.
  • a blocking buffer of 10% casein in PBS was dispensed on the membrane to block non-specific binding of antibody to the membrane.
  • the membrane was washed with 1 ml_ PBS to wash extra blocking agent.
  • an antibody-conjugate mix was dispensed to the membrane followed by an incubation period of 20 min at RT.
  • the multiplexed antibody conjugates (in 10% casein in PBS) at 5 g /ml_ used in this example included: 260 ⁇ _ of monoclonal mouse antibodies for CK8/18/19 rare cell type biomarkers were labeled with Dyl_ight550 (Siemens Elkhart, IN) which were used to detect the cancer cells; and anti- CD45 antibodies conjugated to DyLight 650 which were used to detect the non-rare white blood cells; and a rabbit monoclonal antibody (Cell Signaling Technologies Inc, clone D5F3) for activin receptor-like kinase 1 (ALK1 ) which was used for oncoprotein detection of this low abundance rare cell type biomarker.
  • the N2228 (ATCC) non-small lung cancer cells were used to test the response of the assay.
  • the signal for ALK was measured by using an "indirect method" with goat-anti-rabbit-HRP and fluorescence TSA amplification.
  • the TSA reaction is done by 25 min incubation at RT of 260 ⁇ of tyramide-Alexa488 in 0.0015% hydrogen peroxide (25min) (Amplification step 3) and washing 5 times with 1 ml_ of 0.2% TritonX in PBS to wash away unbound tyramide-Alexa488. The reacted slide is then incubated with 500 ⁇ _ of DPAI and washed twice with 1 ml_ of PBS-T to wash extra DAPI.
  • the fluorescence amplification steps noted above were demonstrated using the Tyramide Signal Amplification Kit (Molecular Probes, Nr.: T-20916, T-20936 and T-30954).
  • This kit is based on HRP-conjugate (Goat-anti-mouse-HRP) and tyramide-Alexa488.
  • This kit uses a quenching buffer PBS-T (Phosphate Buffered Saline with 0.05% Tween 20) with 2% H 2 O 2 to destroy background HRP.
  • a blocking solution is made by diluting at 10mg/ml blocking reagent in PBS-T.
  • Tyramide-Alexa488 stock solution is made by dissolve the solid material in 150 ⁇ DMSO.
  • the tyramide-Alexa488 working solution is made diluting the tyramide stock solution 1 :100 in amplification buffer/0.0015% H 2 O 2 .
  • Modifications to this procedure were made and the results are shown in Table 1 .
  • Reagents were either removed or replaced from the method shown above for this example to generate the data shown in Table 1 . None of these reagents were able to cause any improvement in nonspecific signal from WBC.
  • Chromogenic dye staining can be done as regressive method with Harris hematoylin (Sigma Aldrich) or progressive method with Mayer's or Gill's solutions (ScyTeck Laboratories HAE-1 -1 FU or Diff-Quick Stain method) as known to those skilled in the art.
  • Harris hematoylin Sigma Aldrich
  • Mayer's or Gill's solutions ScyTeck Laboratories HAE-1 -1 FU or Diff-Quick Stain method
  • the more complex regressive H&E is done by pre-washing the slide 2 times with 1 mL water. Next, 500 ⁇ of hematoxylin (Sigma Aldrich HHS 32) was incubated for 5 mins followed by washing the slide 3 times with 1 mL water.
  • the slide can be stained after ICC and ISH results are read.
  • the H&E method allows identification of non-rare WBC cells from rare cancer cells and was used as an additional test to determine if false WBC cell results were measured by the standard TSA method.
  • the measurement of an amount of signal obtained in any of the above assay methods for rare comprises of measuring two or more biomarker may correlated with the level of disease in the subject. This measurement is explained more fully as follows: To first determine if the cell of is of interest using a rare cell marker with separate distinct signal and by comparison to a non-rare cell marker in a second separate distinct signal. The rare cells are counted when the rare cell signal is above a threshold and the non-rare signal is below a threshold. The threshold is defined as the point at which the signal is detectable over the noise. The noise in this case is the background signal on the filtration matrix. An additional separate distinct signal for other rare cell features may be measured for each rare cell.
  • the rare cells with additional rare cell features signal above the threshold are counted as a rare cell with this feature.
  • the filtration process allows all signals to be measured simultaneously.
  • a separate signal threshold is selected for each determination of whether a cell is a rare cell, a non- rare cell or whether a rare cell has additional features.
  • the non-rare signal threshold is established using a negative control with non-rare cells.
  • the rare cell and additional feature signal thresholds are determined using a positive control with non-rare cells and cultured rare cells that are fixed with the additional feature. Signals below the threshold are set to zero. An unknown sample may be run and only the additional feature signals above the threshold are measured on cells that are rare cell positive and non-rare negative cells.
  • the above methods may be employed to determine a potential of a mammalian subject for exhibiting disease.
  • the signal obtained in any of the above assay methods for rare cell comprising one or more additional features is correlated to the potential of a mammalian subject for exhibiting disease by measuring the rare cell and non-rare cell signals identifying the cells above the rare cell threshold and below the non-rare cell threshold.
  • the additional feature signal on the rare cell is then measured if above the positive threshold.
  • the numbers of rare cell are counted for each subject or patient. If the signal for the feature is above the threshold for the feature, the cell is considered to have this feature.
  • the higher the increase in the feature signal above the threshold the greater is the feature for the rare cell.
  • the TSA amplification method increased the sensitivity for cells which are bound by the affinity partner.
  • the H2228 single cells could be detected with TSA, but not without. No H2228 cells could be detected using Dyl_ight488 labeled ALK antibody.
  • TSA method has a background problem for white blood WBC. Over 30%of WBC were clearly positive (false) for ALK when TSA was used. These WBC were detected without any goat-anti-rabbit-HRP indicating the tyramide-Alexa488 alone caused the positive response.
  • the amine blocker was added at 50 to 100 ⁇ into the casien blocking solution and the experiment was run as described above. Slides were then placed on a slide holder of a fluorescent microscope (Leica DM5000 (Leica Microsystems GmbH, Wetzlar, Germany) and images were captured during the scanning of the membrane for each of the fluorescent using the filter sets for respective fluorophore labels used.
  • the TSA signal for Alexa488 was measured with an excitation band pass filter at 460 - 500 nm and an em ission band pass filter at 51 2 -543 nm . At leastI O cancer cells and 10 white blood cells after staining were compared to 10 white blood cells which were not exposed to any Tyramide-Alexa488.
  • the cancer and white blood cells not exposed to any Tyramide-Alexa488 did exhibit the expected autofluoresence noise which was removed by normalization.
  • the improvement in WBC background due to TSA binding was quantified by analyzing the background signal strength.
  • the normalized background (WBC background - WBC autofluorescence)/WBC autofluoresence shown in Figure 1 .
  • the brightness and contrast of the image was adjusted to define the area represented by the WBC background, and then the average signal strength of this area was measured.
  • the WBC signal strength seen in different experimental conditions was used to determine which blocking agents provided the greatest background reduction. All images were taken with the same exposure times and camera gain so that comparison of the signal strength from different images would accurately reflect differences in the underlying signal on the slides.

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Abstract

L'invention concerne un système et un procédé qui permettent d'amplifier un signal dans un essai d'amplification de signal de tyramide (TSA) pour la détection de cellules rares dans un échantillon comportant des cellules rares et des cellules non rares. Le système et le procédé comprennent l'utilisation de nouveaux agents de blocage qui sont efficaces pour réduire une quantité de tyramide réactive se liant à des cellules non rares, ce qui réduit le bruit de fond provoqué par une détection indésirable de signal de cellules non rares.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018017604A1 (fr) * 2016-07-18 2018-01-25 Cell Idx, Inc. Composés réactifs, compositions, kits et procédés pour dosages amplifiés
JP2018155601A (ja) * 2017-03-17 2018-10-04 東洋インキＳｃホールディングス株式会社 生化学分析用ブロッキング剤。
CN111830256A (zh) * 2020-07-01 2020-10-27 北京基谱生物科技有限公司 一种石蜡切片免疫荧光多重染色试剂盒及使用方法
US11834703B2 (en) 2008-05-27 2023-12-05 Agilent Technologies, Inc. Hybridization compositions and methods
US11867696B2 (en) 2015-02-06 2024-01-09 Cell Idx, Inc. Antigen-coupled immunoreagents
US12404543B2 (en) 2019-04-25 2025-09-02 Agilent Technologies, Inc. Hybridization compositions and methods for making and using compositions

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030073149A1 (en) * 2001-10-12 2003-04-17 Archer Robert A. Antibody complexes and methods for immunolabeling
US20120071338A1 (en) * 2010-07-15 2012-03-22 Christopher Bowman Polymerization-based amplification for immunostaining and biodetection
US20120270242A1 (en) * 2009-10-20 2012-10-25 Jesper Lohse Immunochemical detection of single target entities
US20130260379A1 (en) * 2012-03-27 2013-10-03 Ventana Medical Systems, Inc. Signaling conjugates and methods of use
WO2015028382A1 (fr) * 2013-08-28 2015-03-05 Ventana Medical Systems, Inc. Essai immunohistochimique pour la détection de cd3 et cd16

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030073149A1 (en) * 2001-10-12 2003-04-17 Archer Robert A. Antibody complexes and methods for immunolabeling
US20120270242A1 (en) * 2009-10-20 2012-10-25 Jesper Lohse Immunochemical detection of single target entities
US20120071338A1 (en) * 2010-07-15 2012-03-22 Christopher Bowman Polymerization-based amplification for immunostaining and biodetection
US20130260379A1 (en) * 2012-03-27 2013-10-03 Ventana Medical Systems, Inc. Signaling conjugates and methods of use
WO2015028382A1 (fr) * 2013-08-28 2015-03-05 Ventana Medical Systems, Inc. Essai immunohistochimique pour la détection de cd3 et cd16

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11834703B2 (en) 2008-05-27 2023-12-05 Agilent Technologies, Inc. Hybridization compositions and methods
US12209276B2 (en) 2008-05-27 2025-01-28 Agilent Technologies, Inc. Hybridization compositions and methods
US11867696B2 (en) 2015-02-06 2024-01-09 Cell Idx, Inc. Antigen-coupled immunoreagents
WO2018017604A1 (fr) * 2016-07-18 2018-01-25 Cell Idx, Inc. Composés réactifs, compositions, kits et procédés pour dosages amplifiés
JP2018155601A (ja) * 2017-03-17 2018-10-04 東洋インキＳｃホールディングス株式会社 生化学分析用ブロッキング剤。
US12404543B2 (en) 2019-04-25 2025-09-02 Agilent Technologies, Inc. Hybridization compositions and methods for making and using compositions
CN111830256A (zh) * 2020-07-01 2020-10-27 北京基谱生物科技有限公司 一种石蜡切片免疫荧光多重染色试剂盒及使用方法

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