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WO2020033539A1 - Dosage à écoulement latéral pour évaluer une conjugaison cible - Google Patents

Dosage à écoulement latéral pour évaluer une conjugaison cible Download PDF

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Publication number
WO2020033539A1
WO2020033539A1 PCT/US2019/045497 US2019045497W WO2020033539A1 WO 2020033539 A1 WO2020033539 A1 WO 2020033539A1 US 2019045497 W US2019045497 W US 2019045497W WO 2020033539 A1 WO2020033539 A1 WO 2020033539A1
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Prior art keywords
target
test device
test
reagent
antibody
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Hong Qi
Hong Xu
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Qoolabs Inc
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Qoolabs 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow

Definitions

  • the present disclosure relates to lateral flow test devices, and uses thereof, for quantitatively assessing target conjugation.
  • the present disclosure relates to the use of lateral flow immunoassay to quantitatively assess biotinylation level, enzyme conjugation level or drug conjugation level of a target polypeptide or target antibody.
  • Bioconjugation is widely used in the field of research, diagnostics and therapeutics.
  • Antibody biotinylation is one of the most commonly used bioconjugation, which is the process of attaching biotin molecules to antibodies.
  • the resulted biotin-antibody can bind to streptavidin and avidin with an extremely high affinity and high specificity.
  • Multiple biotin molecules can be conjugated to each antibody molecule, which allows binding of multiple streptavidin, avidin or Neutravidin protein molecules and increases the sensitivity of detection of the protein of interest.
  • conjugation efficiency For every bioconjugation reaction, it is important to quantitate the actual conjugation efficiency, i.e. , the ratio of biomolecule to the modified molecule in the final product. It is often challenging to quantify the efficiency of the conjugation reaction.
  • Several common methods for quantitation of conjugation efficiency use the physical/chemical properties of the biomolecule, the linker or the modified molecule, such as optical spectral property, gel migration property, fluorescent property, hydrophobicity, molecular weight, etc.
  • fluorescent dye conjugation can be quantified by absorption or fluorescent spectra. Unfortunately, many conjugations do not have these distinct properties for easy detection, oftentimes the detection devices for some properties are very expensive, like mass spectroscopy.
  • biotinylation for a biomolecule is critical for downstream applications. Low biotin density may result in reduced interaction with (strept)avidin while overlabeling may affect the bioaffinity of labelled biomolecules, both of which may lead to decreased sensitivity of a detection assay. Quantification of biotin enables optimized biotinylation protocols, and more importantly, quality control of biotinylation process in production.
  • HABA 2-(4’ hydroxyazobenzene) benzoic acid
  • avidin which has a higher affinity to biotin than to HABA, causing a shift of the absorption peak.
  • biotin quantification assay is based on FRET principle ⁇ ].
  • avidin tagged with a quencher dye is briefly incubated with biotin.
  • fluorescent biotin is added to the assay mixture. The fluorescent signal (Ex490nm/Em520nm) is directly proportional to the amount of biotin in the sample.
  • HABA assay requires at least 0.3 nmole biotin. Assuming the biotin: Ab ratio is 1:1, 45 pg of Ab is used in one assay. Even for the most sensitive commercially available FRET-based kit (sensitivity of 2 pmole), minimum of 0.3 pg Ab is used in one assay with microtiter plate (100-200 pl). In many research applications where only a few microgram of antibodies are used, the high amount of antibodies required for quantitation of biotinylation become a significant cost of antibody and often is prohibitive for performing the assay.
  • Quant*Tag biotin kit An alternative to avidin-based assays is the Quant*Tag biotin kit from vector laboratories [4]. This kit contains reagents which chemically react with biotin, yielding a visible color change quantifiable by spectrophotometry. Because steric interference is not an issue, accurate biotin quantification is possible without digestion. However, besides its rather low sensitivity (50 pmoles), this Quant*Tag kit does not distinguish between free biotin and conjugated biotin, which is also true for avidin-based assays.
  • MS mass spectrometry
  • conjugation quantitation methods e.g., biotinylating quantitation methods
  • biotinylating quantitation methods are time consuming, with low sensitivity and/or difficult to perform.
  • improved assays for quantitatively assessing target conjugation e.g., biotinylation level, enzyme conjugation level or drug conjugation level of a target polypeptide or target antibody.
  • target conjugation e.g., biotinylation level, enzyme conjugation level or drug conjugation level of a target polypeptide or target antibody.
  • the present disclosure provides a lateral flow test device for quantitatively assessing a conjugated target comprising a target portion and a conjugate portion, which device comprises a porous matrix that comprises a test location on said porous matrix, said test location comprising: 1) a test reagent 1 that binds to said conjugated target to form a sandwich comprising said test reagent l-said conjugated target-a labeled binding reagent, e.g., a fluorescently labeled binding reagent, for said conjugate portion of said conjugated target;
  • a labeled binding reagent e.g., a fluorescently labeled binding reagent
  • test reagent 2 that competes with said conjugated target for binding to a labeled binding reagent, e.g., fluorescently labeled binding reagent, for said conjugate portion of said conjugated target, wherein a liquid sample flows laterally along said test device and passes said test location to form a detectable signal from said sandwich and/or a complex between said test reagent 2 and said labeled or fluorescently labeled binding reagent, said detectable signal being measured for quantitatively assessing said conjugated target.
  • a labeled binding reagent e.g., fluorescently labeled binding reagent
  • kits for quantitatively assessing a conjugated target which kit comprises 1) a lateral flow test device as described above; and 2) a control sample, a diluting buffer and/or an instruction for using said kit.
  • the present disclosure provides a system for quantitatively assessing a conjugated target, which system comprises: 1) a lateral flow test device or a kit as described above; and 2) a reader, e.g., a fluorescence reader, configured for detecting a detectable signal at the test location.
  • a reader e.g., a fluorescence reader
  • the present disclosure provides a method for quantitatively assessing a conjugated target, which method comprises: a) contacting a liquid sample containing a conjugated target comprising a target portion and a conjugate portion with a lateral flow test device as described above, wherein said liquid sample is applied to a site of said test device upstream of said test location; b) transporting said conjugated target and/or a labeled or fluorescently labeled binding reagent for said conjugate portion of said conjugated target to said test location; and c) assessing a detectable signal at said test location for quantitatively assessing said conjugated target.
  • Figure 1 illustrates an exemplary lateral flow assay test strip with its components.
  • Figure 2 illustrates an exemplary streptavidin-protein A lateral flow assay quantitating bioti Ab ratio below 8:1.
  • different ratio of Biotin Ab samples range from 0.1:1 to 8:1 were tested, the ratios were plotted against adjusted signal.
  • Figure 3 illustrates an exemplary competition lateral flow assays for quantitation of biotin conjugation at higher ratio.
  • different ratio of Biotin Ab samples range from 4: 1 to 100:1 were tested, the ratios were plotted against adjusted signal.
  • FIG 4 illustrates an exemplary combination of Streptavidin/Protein A and competition lateral flow assay.
  • different ratio of Biotin Ab samples range from 0.1:1 to 200:1 were tested, the ratios were plotted against adjusted signal.
  • Figure 5 illustrates exemplary pictures of test strips after running samples with various bioti Ab ratio. Left, QNowTM strips; right, Expedeon test strips. For both sets, Strip #1 was run with blank buffer, Strip #2 to 13 were run with samples of BiotimAb ratios at: 0.1, 0.2, 0.5, 1, 2, 4, 8, 20, 40, 60, 100, and 200.
  • composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab') 2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g. , sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rlgG recombinant IgG
  • scFv single chain variable fragments
  • single domain antibodies e.g. , sdAb, sdFv, nanobody
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • the term“antibody” should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • The“class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
  • CDR complementarity determining region
  • HVR hypervariable region
  • FR-H1, FR-H2, FR-H3, and FR-H4 there are four FRs in each full- length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
  • immunoglobulin variable domains an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme).
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Rabat scheme is based structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Rabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions
  • the two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • a“CDR” or“complementary determining region,” or individual specified CDRs (e.g.,“CDR-H1, CDR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes.
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given V H or VL amino acid sequence
  • such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes.
  • FR or individual specified FR(s) e.g., FR- Hl, FR-H2
  • FR- Hl, FR-H2 FR- H2
  • FR-H2 FR- H2
  • the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Rabat, Chothia, or Contact method.
  • variable region or“variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et ak, J. Immunol. 150:880-887 (1993); Clarkson et al, Nature 352:624-628 (1991).
  • Fc region herein is used to define a C-terminal region of an
  • immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Rabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • an“antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a camelid single-domain antibody.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
  • the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody.
  • A“humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs.
  • the term“chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • monoclonal antibodies including monoclonal antibody fragments.
  • the term“monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different epitopes
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen.
  • the term is not to be construed as requiring production of the antibody by any particular method.
  • a monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.
  • polypeptide and“protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length.
  • Polypeptides including the provided antibodies and antibody chains and other peptides, may include amino acid residues including natural and/or non-natural amino acid residues.
  • the terms also include post expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • Bind refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
  • An“affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • HVRs hypervariable regions
  • the term“specific binding” refers to the specificity of a binder, e.g., an antibody, such that it preferentially binds to a target, such as a polypeptide antigen.
  • a binding partner e.g., protein, nucleic acid, antibody or other affinity capture agent, etc.
  • “specific binding” can include a binding reaction of two or more binding partners with high affinity and/or complementarity to ensure selective hybridization under designated assay conditions.
  • specific binding will be at least three times the standard deviation of the background signal.
  • the binding partner binds to its particular target molecule and does not bind in a significant amount to other molecules present in the sample.
  • binders, antibodies or antibody fragments that are specific for or bind specifically to a target bind to the target with higher affinity than binding to other non-target substances.
  • binders, antibodies or antibody fragments that are specific for or bind specifically to a target avoid binding to a significant percentage of non- target substances, e.g., non- target substances present in a testing sample. In some embodiments, binders, antibodies or antibody fragments of the present disclosure avoid binding greater than about 90% of non-target substances, although higher percentages are clearly contemplated and preferred.
  • binders, antibodies or antibody fragments of the present disclosure avoid binding about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99% or more of non-target substances. In other embodiments, binders, antibodies or antibody fragments of the present disclosure avoid binding greater than about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of non target substances.
  • An“individual” or“subject” includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats.
  • An“individual” or“subject” may include birds such as chickens, vertebrates such as fish and mammals such as mice, rats, rabbits, cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeys and other non-human primates.
  • the individual or subject is a human.
  • sample refers to anything which may contain an analyte for which an analyte assay is desired.
  • a“sample” can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • the sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.
  • Biological tissues are aggregate of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
  • the sample is a biological sample.
  • a biological sample of the present disclosure encompasses a sample in the form of a solution, a suspension, a liquid, a powder, a paste, an aqueous sample, or a non-aqueous sample.
  • a“biological sample” includes any sample obtained from a living or viral (or prion) source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid, protein and/or other macromolecule can be obtained.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom.
  • the sample can be derived from a tissue or a body fluid, for example, a connective, epithelium, muscle or nerve tissue; a tissue selected from the group consisting of brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, gland, and internal blood vessels; or a body fluid selected from the group consisting of blood, urine, saliva, bone marrow, sperm, an ascitic fluid, and subfractions thereof, e.g., serum or plasma.
  • An“isolated” antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • An“isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. _ B. Lateral flow test devices, kits and systems for quantitatively assessing target conjugation
  • the present disclosure provides a lateral flow test device for quantitatively assessing a conjugated target comprising a target portion and a conjugate portion, which device comprises a porous matrix that comprises a test location on said porous matrix, said test location comprising: 1) a test reagent 1 that binds to said conjugated target to form a sandwich comprising said test reagent l-said conjugated target-a labeled binding reagent, e.g., a fluorescently labeled binding reagent, for said conjugate portion of said conjugated target; and/or 2) a test reagent 2 that competes with said conjugated target for binding to a labeled binding reagent, e.g., a fluorescently labeled binding reagent, for said conjugate portion of said conjugated target, wherein a liquid sample flows laterally along said test device and passes said test location to form a detectable signal from said sandwich or a complex between said test reagent 2 and said labeled binding reagent,
  • the target portion of a conjugated target can comprise any suitable moiety.
  • the target portion can comprise an organic moiety.
  • Exemplary organic moiety can be an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid or a complex thereof.
  • the target portion can comprise a polypeptide moiety, e.g., an antibody moiety.
  • the conjugate portion of a conjugated target can comprise any suitable moiety.
  • the conjugate portion can comprise an organic moiety.
  • Exemplary' organic moiety can be an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid or a complex thereof.
  • the conjugate portion can comprise a water-soluble B vitamin moiety, e.g., a biotin moiety.
  • the conjugated target is a biotinylated target polypeptide, e.g., a biotinylated target antibody.
  • the conjugate portion can comprise an enzyme moiety .
  • the enzyme moiety can be any suitable enzyme moiety, e.g. , a horseradish peroxidase (HRP), alkaline phosphatase or luciferase.
  • the conjugated target is a target polypeptide-enzyme conjugate, e.g., a target polypeptide-HRP conjugate, a target polypeptide- alkaline phosphatase conjugate, or a target polypeptide-Iuciferase conjugate.
  • the conjugate portion can comprise a drug moiety.
  • the conjugated target can be a target polypeptide-drug conjugate, e.g., a target antibody-drug conjugate (ADC).
  • ADC target antibody-drug conjugate
  • the target portion of a conjugated target can comprise a water-soluble B vitamin moiety, e.g., a biotin moiety, and the conjugate portion can comprise a polypeptide moiety, e.g. , an antibody moiety.
  • a labeled binding reagent e.g., a fluorescently labeled binding reagent
  • the labeled or fluorescently labeled binding reagent can comprise protein A and/or protein G beads.
  • the matrix can have any suitable structure.
  • the matrix can have a porous structure.
  • the matrix can comprise any suitable material(s).
  • porous plastics material such as polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and polytetrafluoro- ethylene can be used. See e.g., U.S. patent No. 6,187,598. It can be advantageous to pre-treat the membrane with a surface-active agent during manufacture, as this can reduce any inherent hydrophobicity in the membrane and therefore enhance its ability to take up and deliver a moist sample rapidly and efficiently.
  • the matrix can also be made from paper or other cellulosic materials.
  • the matrix comprises or is made of nitrocellulose or glass fiber.
  • the test location can comprise any suitable test reagent 1.
  • the test location can comprise a test reagent 1 that binds, e.g., specifically binds, to the conjugated target to form a sandwich comprising the test reagent l-the conjugated target- a labeled binding reagent, e.g., a fluorescently labeled binding reagent, for the conjugate portion of the conjugated target.
  • the test reagent 1 can bind to the target and/or conjugate portion of a conjugated target.
  • the target portion comprises a polypeptide moiety and the test reagent 1 binds to the polypeptide moiety.
  • the target polypeptide moiety comprises a target antibody moiety
  • the test reagent 1 binds to the target antibody moiety.
  • test reagent 1 comprises protein A and/or protein G.
  • test reagent 1 comprises an antibody that binds to the target antibody moiety in a conjugated antibody.
  • the test location can comprise any suitable test reagent 2.
  • the test location can comprise a test reagent 2 that competes with a conjugated target for binding to a labeled binding reagent, e.g., a fluorescently labeled binding reagent, for the conjugate portion of the conjugated target.
  • a labeled binding reagent e.g., a fluorescently labeled binding reagent
  • the conjugated target is a biotinylated target polypeptide, e.g., a biotinylated target antibody
  • the test reagent 2 comprises a biotin moiety, or an analog or a fragment thereof.
  • the test reagent 2 can comprise any suitable biotin moiety, or an analog or a fragment thereof.
  • the test reagent 2 can comprise a biotinylated protein, e.g., a biotinylated bovine serum albumin (biotinylated BSA).
  • the conjugated target is a target polypeptide-enzyme conjugate, e.g., a target polypeptide-HRP conjugate
  • the test reagent 2 comprises a corresponding enzyme moiety, e.g. , a HRP moiety, or an analog or a fragment thereof.
  • the conjugated target is a target polypeptide-drug conjugate, e.g., a target antibody drug conjugate (ADC), and the test reagent 2 comprises a corresponding drug moiety, or an analog or a fragment thereof.
  • ADC target antibody drug conjugate
  • the test device comprises two test locations, test location 1 comprising a test reagent 1 that binds to the conjugated target to form a sandwich comprising the test reagent l-the conjugated target-a labeled binding reagent, e.g., a fluorescently labeled binding reagent, for the conjugate portion of the conjugated target, and test location 2 comprising a test reagent 2 that competes with the conjugated target for binding to a labeled binding reagent, e.g., a fluorescently labeled binding reagent, for the conjugate portion of the conjugated target.
  • the test device comprises one test location that comprises both the test reagent 1 and the test reagent 2 described above.
  • the conjugated target is a biotinylated target polypeptide, e.g., a biotinylated target antibody
  • the labeled binding reagent e.g., the fluorescently labeled binding reagent
  • the binding portion that binds to biotin can comprise a streptavidin, avidin or Neutravidin moiety.
  • the conjugated target is a target polypeptide-enzyme conjugate, e.g., a target polypeptide-HRP conjugate
  • the labeled binding reagent e.g., the fluorescently labeled binding reagent
  • the conjugated target is a target polypeptide-drug conjugate, e.g., a target antibody drug conjugate (ADC)
  • the labeled binding reagent e.g., the fluorescently labeled binding reagent, comprises a binding portion that binds to the drug, e.g. , an anti-drug antibody or aptamer.
  • the matrix can have any suitable form or shape.
  • the matrix can be in the form of a strip or a circle.
  • the matrix can also have suitable number of elements.
  • the matrix can be made of a single element or can comprise multiple elements.
  • the test device can further comprise a sample application element upstream from and in fluid communication with the matrix.
  • the sample application element can be made of any suitable materials, such as nitrocellulose, glass fiber, polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene flouride, ethylene vinylacetate, acrylonitrile or polytetrafluoro-ethylene.
  • the matrix and the sample application element can comprise the same or different materials.
  • the test device can further comprise a liquid absorption element downstream from and in fluid communication with the matrix.
  • the liquid absorption element can be made of any suitable materials, such as paper or cellulose materials.
  • the test can further comprise a control location.
  • the control location can comprise a binding reagent that binds to a labeled binding reagent, e.g. , a fluorescently labeled binding reagent, for the conjugate portion of the conjugated target.
  • the conjugated target is a biotinylated target polypeptide, e.g., a biotinylated target antibody
  • the control location comprises a binding reagent that binds to a binder that binds to biotin.
  • Any suitable binding reagent that binds to a binder that binds to biotin can be used.
  • the control location can comprise an anti-streptavidin, anti- avidin or anti-Neutravidin antibody or aptamer.
  • the conjugated target is a target polypeptide-enzyme conjugate, e.g., a target polypeptide-HRP conjugate
  • the control location can comprise a binding reagent that binds to a binder that binds to the enzyme or HRP. Any suitable binding reagent that binds to a binder that binds to the enzyme or HRP can be used.
  • the control location can comprise an antibody or aptamer that binds to an anti-enzyme or anti- HRP antibody or aptamer.
  • the conjugated target is a target polypeptide-drug conjugate, e.g., a target antibody drug conjugate (ADC)
  • the control location can comprise a binding reagent that binds to a binder that binds to the drug.
  • ADC target antibody drug conjugate
  • Any suitable binding reagent that binds to a binder that binds to the drug can be used.
  • the control location can comprise an antibody or an aptamer that binds to an anti-drug antibody or aptamer.
  • the matrix is supported by a solid backing.
  • a solid backing can be made of any suitable material, e.g., solid plastics.
  • a labeled reagent e.g. , a fluorescently labeled binding reagent
  • a liquid e.g., a sample liquid and/or additional liquid
  • a portion of the matrix, upstream from the test location can comprise a dried labeled binding reagent, e.g., a dried, fluorescently labeled binding reagent, for the conjugate portion of the conjugated target, the labeled or fluorescently labeled reagent being capable of being moved by a liquid sample and/or a further liquid to the test location and/or the control location to generate a detectable signal.
  • a dried labeled binding reagent e.g., a dried, fluorescently labeled binding reagent
  • the conjugated target is a biotinylated target polypeptide, e.g., a biotinylated target antibody
  • the dried, labeled or fluorescently labeled binding reagent comprises a reagent that binds to biotin.
  • Any suitable binding reagent that binds to biotin can be used.
  • the labeled or fluorescently labeled binding reagent can comprise a fluorescently labeled streptavidin, avidin or Neutravidin moiety.
  • the conjugated target is a target polypeptide-enzyme conjugate, e.g., a target polypeptide-HRP conjugate
  • the dried, labeled or fluorescently labeled binding reagent can comprise a reagent that binds to the enzyme or HRP.
  • Any suitable binding reagent that binds to the enzyme or HRP can be used.
  • the labeled or fluorescently labeled binding reagent can comprise a labeled or fluorescently labeled anti enzyme or anti-HRP antibody or aptamer.
  • the conjugated target is a target polypeptide-drug conjugate, e.g., a target antibody drug conjugate (ADC)
  • the dried, labeled or fluorescently labeled binding reagent can comprise a reagent that binds to the drug.
  • Any suitable binding reagent that binds to the drug can be used.
  • the labeled or the fluorescently labeled binding reagent can comprise a labeled or fluorescently labeled anti-drug antibody or aptamer.
  • the dried, labeled reagent can be located at any suitable places on the test device. In one example, the dried, labeled reagent is located downstream from a sample application place on the test device. In another example, the dried, labeled reagent is located upstream from a sample application place on the test device.
  • the type of the labeled reagent can be determined based on the intended assay formats. For example, if the test device is to be used in a sandwich assay, the labeled reagent should be capable of binding, and preferably capable of specifically binding, to the analyte or another substance that binds to the analyte. The same labeled reagent can also be used for certain competitive binding assays. For other types of the competitive binding assays, the labeled reagent should be an analyte or an analyte analog linked to a detectable label.
  • the test device can further comprise, upstream from the test location, a conjugate element that comprises the dried, labeled or fluorescently labeled binding reagent.
  • the conjugate element can be located at any suitable places on the test device. In one example, the conjugate element is located downstream from a sample application place on the test device. In another example, the conjugate element is located upstream from a sample application place on the test device.
  • the label can be a soluble label, such as a colorimetric, radioactive, enzymatic, luminescent, fluorescent, magnetic, SERS, or FRET label.
  • the label can also be a particle or particulate label, such as a particulate direct label, or a colored particle label.
  • Exemplary particle or particulate labels include colloidal gold label, latex particle label, nanoparticle label and quantum dot label.
  • the labels such as colorimetric, radioactive, enzymatic, luminescent or fluorescent label, can be either a soluble label or a particle or particulate label.
  • the fluorescently labeled binding reagent comprises a soluble fluorescent label, e.g. , an organic dye, rhodamine, or fluorescein.
  • the fluorescently labeled binding reagent comprises a particle fluorescent label, e.g., a europium polystyrene bead, a quantum dot (bead), a latex bead with an organic dye, or an upconversion nanoparticle.
  • the labeled reagent is dried in the presence of a material that stabilizes the labeled reagent, facilitates solubilization or resuspension of the labeled reagent in a liquid, and/or facilitates mobility of the labeled reagent.
  • a material that stabilizes the labeled reagent facilitates solubilization or resuspension of the labeled reagent in a liquid, and/or facilitates mobility of the labeled reagent.
  • the material can be a protein, e.g. , a meta-soluble protein, a peptide, a polysaccharide, a sugar, e.g., sucrose, a polymer, a gelatin or a detergent. See e.g., U.S. patent Nos. 5,120,643 and 6,187,598.
  • the fluorescently labeled binding reagent is dried in the presence of a material that: a) stabilizes the fluorescently labeled binding reagent; b) facilitates solubilization or resuspension of the fluorescently labeled binding reagent in a liquid; and/or c) facilitates mobility of the fluorescently labeled binding reagent.
  • a material that: a) stabilizes the fluorescently labeled binding reagent; b) facilitates solubilization or resuspension of the fluorescently labeled binding reagent in a liquid; and/or c) facilitates mobility of the fluorescently labeled binding reagent.
  • a material can be a protein, e.g. , a casein or BSA, a peptide, a
  • polysaccharide a sugar, a polymer, e.g., polyvinylpyrrolidone (P VP-40), a gelatin, a detergent, e.g., Tween-20, a buffer, an amino acid, and PEG.
  • P VP-40 polyvinylpyrrolidone
  • PEG polyvinylpyrrolidone
  • the present test devices can be used with any suitable sample liquid.
  • a sample liquid alone is used to transport the analyte and/or the labeled or the fluorescently labeled binding reagent to the test location.
  • a developing liquid is used to transport the analyte and/or the labeled or the fluorescently labeled binding reagent to the test location.
  • the test device can further comprise a housing that covers at least a portion of the test device, wherein the housing comprises a sample application port to allow sample application upstream from or to the test location and an optic opening around the test location to allow signal detection at the test location.
  • the optic opening can be achieved in any suitable way.
  • the optic opening can simply be an open space.
  • the optic opening can be a transparent cover.
  • the housing can cover the entire test device. In still other embodiments, at least a portion of the sample receiving portion of the matrix or the sample application element is not covered by the housing and a sample is applied to the portion of the sample receiving portion of the matrix or the sample application element outside the housing and is then transported to the test location.
  • the housing can comprise any suitable material.
  • the housing can comprise a plastic material.
  • the housing whether in part or in its entirety, can comprise an opaque, translucent and/or transparent material.
  • the present invention provides for a test device wherein the liquid sample has moved laterally along the test device to generate detectable signal(s) at the test and/or control location(s).
  • the present disclosure provides a kit for quantitatively assessing a conjugated target, which kit comprises 1) a lateral flow test device as described above; and 2) a control sample, a diluting buffer and/or an instruction for using said kit.
  • the present disclosure provides a system for quantitatively assessing a conjugated target, which system comprises: 1) a lateral flow test device or a kit as described above; and 2) a reader, e.g., a fluorescence reader, configured for detecting a detectable signal at the test location.
  • the reader or the fluorescence reader can use any suitable measurement route or mechanism.
  • the reader or the fluorescence reader can be configured for a scanning measurement.
  • the reader or the fluorescence reader can be configured for a static imaging measurement.
  • the reader or the fluorescence reader comprises: a) a receiving member adapted to receive a test device described above; b) an excitation module configured to illuminate the test device, when the test device is placed in the receiving member; c) a camera module for capturing an image of the illuminated test device placed in the receiving member; d) a processor for receiving the captured image from the camera module and analyzing a detectable signal at the test location of the test device; and e) memory storing firmware, the firmware including a brightness compensation module configured to adjust the intensity of an image of a test device captured by the camera module, in order to emulate a uniform field of illumination over the test device. (See e.g., WO 2017/063045 Al).
  • the brightness compensation module can he configured to adjust the intensity of the captured image based on an illumination
  • the reader or fluorescence reader is a camera-based reader, e.g., a CCD-camera based reader.
  • the reader or fluorescence reader is a bench-top reader.
  • the reader or fluorescence reader is a hand-held reader.
  • the reader or fluorescence reader can have any suitable reading time.
  • the reader or fluorescence reader can have a reading time from about 0.1 second to about 30 seconds per test device, e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 second(s), or any subrange thereof.
  • the present test device, kit or a system can be configured for any suitable applications.
  • the present test device, kit or a system can be configured for assessing conjugation level of a target, e.g., biotinylation level of a target polypeptide or target antibody, enzyme conjugation level of a target polypeptide or target antibody, or drug conjugation level of a target polypeptide or target antibody.
  • the present disclosure provides a method for quantitatively assessing a conjugated target, which method comprises: a) contacting a liquid sample containing a conjugated target comprising a target portion and a conjugate portion with a lateral flow test device as described above, e.g., a lateral flow test device as described in Section B above, wherein the liquid sample is applied to a site of the test device upstream of the test location; b) transporting the conjugated target and/or a labeled or fluorescently labeled binding reagent for said conjugate portion of said conjugated target to the test location; and c) assessing a detectable signal at the test location for quantitatively assessing said conjugated target.
  • the liquid sample and the labeled reagent are premixed to form a mixture and the mixture is applied to the test device.
  • the labeled reagent can be provided or stored in a liquid and then can be premixed with a sample liquid to form a mixture and the mixture is applied to the test device.
  • the labeled reagent can be dried in a location or container not in fluid communication with the test device, e.g. , in a test tube or well such as a microtiter plate well.
  • the sample liquid can be added to the container, e.g., the test tube or well, to form the mixture and the mixture can then be applied to the test device.
  • the liquid sample and the fluorescently labeled binding reagent can be premixed to form a mixture and the mixture is applied to the test device.
  • the present methods can further comprise a washing step after the mixture is applied to the test device.
  • the washing step can be conducted in any suitable matter.
  • the washing step can comprise adding a washing liquid after the mixture is applied to the test device.
  • the test device can comprise a liquid container comprising a washing liquid and the washing step can comprise releasing the washing liquid from the liquid container.
  • the test device can comprise a dried labeled binding reagent, e.g., a dried fluorescently labeled binding reagent, for the conjugate portion of the conjugated target upstream to the test location before use and the dried labeled reagent can be solubilized or resuspended and transported to the test location by the liquid sample.
  • the dried labeled reagent can be located at any suitable location on the test device.
  • the dried labeled or fluorescently labeled binding reagent can be located downstream from the sample application site, and the dried labeled or fluorescently labeled binding reagent can be solubilized or resuspended and transported to the test location by the liquid sample.
  • the labeled or dried fluorescently labeled binding reagent can be located upstream from the sample application site, and the dried labeled or fluorescently labeled binding reagent can be solubilized or resuspended and transported to the test location by another liquid.
  • the analyte and/or the labeled or fluorescently labeled binding reagent is solubilized or resuspended and transported to the test location by the liquid sample alone.
  • the analyte and/or labeled or fluorescently labeled binding reagent are solubilized or resuspended, and transported to the test location by another liquid, e.g., a developing liquid.
  • the analyte and/or labeled or fluorescently labeled binding reagent are solubilized or resuspended and transported to the test location by both the sample liquid and another liquid, e.g., a developing liquid.
  • the detectable signal can be assessed by any suitable methods or means.
  • the detectable signal is assessed by a reader, e.g., a fluorescent reader.
  • the reader can comprise any suitable number of photodetector(s).
  • the reader or fluorescent reader can comprise a single photodetector or multiple photodetectors.
  • the present methods can be conducted in any suitable time or duration. In some embodiments, the present methods can be conducted or completed in about 5-30 minutes, e.g. , about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 minutes, or any subrange thereof.
  • the present methods can be conducted to quantitatively assess a conjugated target in in any suitable sample liquid.
  • the liquid sample can comprise a conjugation reaction liquid, a cell lysate, a cell culture medium, an in vitro transcription product, an in vitro translation product, a polypeptide purification fraction, and/or a sample isolated or derived from a subject.
  • sample liquid can be used directly or can be processed, e.g. , enriched, purified, or diluted, before use.
  • the present methods can be used for any suitable purpose.
  • the present methods can be used to assess conjugation level of a target.
  • the present methods are used to assess biotinylation level of a target polypeptide or target antibody.
  • the present methods are used to assess enzyme conjugation level of a target polypeptide or target antibody.
  • the present methods are used to assess drug conjugation level of a target polypeptide or target antibody.
  • the present methods can be used assess any suitable conjugation level of a target.
  • the present methods can be used to assess conjugation level of a target from about 0.1 to about 200 conjugates per target, e.g., from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,
  • the present methods can be used to assess biotinylation level of a target polypeptide or target antibody from about 0.1 to about 200 biotin per polypeptide or target antibody target, e.g., from about 0.1, 0.2,
  • LFIA lateral flow immunochromatography based assay
  • LFIA offers several advantages over other detection methods for its lower cost, user friendliness, and shorter time to results[9].
  • LFIA has been used in point-of-care diagnosis and detection for over twenty years and its applications include food contaminant detection, pesticides, drug abuse screenings besides detections of clinical relevant analytes.
  • LFIA traditionally is purely visual for result inquiry and is intended for qualitative detection, it has been improved to the point where full quantification is a reality[l0].
  • U.S. patent publication No. US 2016/0282343 Al describes a quantitative LFIA method for human hormone levels in in-vitro fertilization embryo transfer patients.
  • the challenges for quantitative LFIA include appropriate labels/reporters for stable signal and high sensitivity. Quantitation of LFIA also requires a designated device (reader) and companion software for data acquisition and calculation.
  • fluorescent reporters including fluorescence dye (in silica/polystyrene microparticles), quantum dot (beads), and fluorescent europium (III) nanoparticles [11-15].
  • the fluorescent reporters offer better discrimination of reporter- specific signal over visually detectable reporters (mostly colloidal gold). Fluorescent reporter signal is measured directly by the intensity of the emitted light at a defined wavelength range, while the reflectance-based measurement (colorimetric) cannot exclude non-reporter-specific background signals by spectral resolutions because the image artifacts originating from dust-speckles, scratches or uneven lighting conditions are not limited at any specific wavelength range.
  • higher analytical sensitivities have been reported with fluorescent reporters when compared to visually detectable reporters [16, 17].
  • Quantitative LFIAs require readers that contain image analysis computer software and specialized instrumentations that detects fluorescent particles on the test strips.
  • the features for an ideal reader include affordable, sensitive, specific, user-friendly, rapid and robust.
  • two measurement strategies (scanning and static imaging) are used for the signal detection and quantification.
  • a scanning measurement the optical sensor moves in relation to the test strip longitudinally.
  • a static imaging measurement a multi-pixel image is generated.
  • the main benefit of the scanning measurement over the static imaging methods includes higher detectability of the reporter [18, 19].
  • the static imaging measurement has the capability to analyze two-dimensional images. This can be used to recognize errors originating from imaging artifacts, but more importantly, it enables the analysis of multiplex arrays printed on the LFA strips[20].
  • Sandwich format LFIA requires two capture reagents for one analyte molecule to be detected. It usually gives higher sensitivity but yield false negative results at higher analyte concentrations due to over saturation of the capture antibodies.
  • Competitive LFIA uses one capture reagent and the analyte (analog) to detect the analyte that would compete for the capture reagent. It tends to be more quantitative at higher analyte concentrations. To achieve a wider dynamic range and higher activity for analyte quantitation, both formats can be integrated into one assay[22].
  • Fluorescent beads Europium beads (Ex365nm, Em6l0nm) with diameter of 200nm were purchased from Bang’s Laboratory.
  • LFIA test strips were assembled by mounting the four LFIA elements (sample pad, conjugate pad, nitrocellulose membrane, absorbent pad) consecutively on a solid backing. SA was conjugated to fluorescent beads and printed on the conjugate pad. The test line was printed with protein A/G. The competition line was printed with biotinylated BSA. The control line was printed with rabbit anti-SA IgG. The strip was enclosed into a plastic cassette (housing).
  • activated biotin (NHS-LC-LC-biotin) dissolved in DMSO was added to the protein of interest dissolved in PBS to the desired ratio.
  • biotinylated antibodies with bioti Ab ratios of 0.1, 0.2, 0.5, 1, 2, 4, 8, 20, 40, 60, 100, 200 were made.
  • the reactions were kept on a shaker at room temperature for at least 2 hours and continued at 4°C overnight before being dialyzed with PBS.
  • the concentrations of dialyzed biotinylated Abs were determined by Bradford method (Thermofisher Catalog #1856209).
  • Fluorescence Rapid Test Reader ONOWTM The quantitative lateral flow reader was custom designed and manufactured by Lumos Diagnostics Pty Ltd (Australia) branded as QNOWTM. Part of the reader and the software for controlling the reader is described in WO 2017/063045A1. The algorism for calculation of the final results were developed by Qoolabs.
  • QNOWTM is a CCD-camera based reader. Its sensitivity, dynamic range, reading time and consistency of reading were compared to two other readers on the market (Table 1). QNOWTM reader has a better sensitivity and wider detection range, with lower intra-test variations.
  • G001241 Strip running and signal reading. Sixty (60) ul of sample at the Ab concentration of 1 ug/ml was added to the sample window of the cassette to start the running of the strip. After 15 min, the fluorescent signals on the strip were quantified by QNOW reader.
  • Biotinylated antibody with biotin to antibody ratios of 0.1, 0.2, 0.5, 1, 2, 4, 8, 20, 40, 60, 100 were made separately. Each sample was diluted to lpg/ml of antibody concentration and was applied to the QNow test strips. After the reactions were completed (about 15 minutes to 30 minutes), the test strips were read with the QNow reader. Signals of each line (control, Tl and T2 lines) were recorded and used to construct standard curves, which x-axis was the biotin to antibody ratio, and y-axis was the signal. The standard curve may be separated into several segments, and each segment has its own formula extrapolated from the known points, and was stored in the QNow software.
  • the sample was also diluted to 1 pg/ml (the same concentration of antibody as the standard) and applied to a test strip. After reaction was completed, the test strip was read with QNow reader, and the software would calculate the biotinylation ratio using the captured signals of the unknown sample and the pre-stored formula.
  • Sample Results L _ Streptavidin-Protein A lateral flow assay for quantitation of biotin/antibody ratio.
  • the free biotin or non-labeled antibodies would not interfere with the reaction and would not produce any signal.
  • antibody ratio of less than 8:1 the intensity of T line was proportional to the ratio, higher biotin content would result in higher signal on the T line ( Figure 2).
  • the bioti Ab ratio was calculated according to the formula obtained empirically. The sensitivity of the assay can reach 40 fmole of biotin or below without washing steps, and only uses 60ng or less of biotinylated antibody.
  • biotin antibody ratio of higher than 4:1, the intensity of T line was reverse proportional to the ratio, higher biotin content would result in weaker T line.
  • the biotimAb ratio was calculated according to the formula obtained empirically. This assay was able to quantitate biotinylation at higher range.
  • biotin When a sample containing biotinylated antibodies was added to the sample pad, the biotin will bind on the streptavidin europium beads and migrate to the test line T2, where the antibodies will be captured by the protein A, forming a fluorescent line. Excess streptavidin/biotin-antibody will continue to migrate to the Tl line, where the biotin in the sample will compete with the Biotin-BSA for binding to the streptavidin europium beads. More biotin on the antibody will cause reduced signal on the Tl line due to competition of the Biotin in the sample to the Biotin-BSA for binding of streptavidin europium beads.
  • the bioti Ab ratio was calculated according to the formula obtained empirically. This assay is able to quantitate biotinylation at a wide dynamic range from 0.1:1 to 200:1 of biotin: antibody ratio. It is necessary to use a reader such as QNowTM to cover the dynamic range.
  • Each antibody molecule has an average of about 130 lysines and arginines, which make it the theoretical upper limit of biotin: antibody ratio at 130:1 when using NHS/EDC conjugation method using the free amine group on the lysine and arginine.
  • Table 2 Comparison of biotin: Ab ratio determined by
  • the QNowTM rapid test is able to quantitate the antibody biotinylation in a wide dynamic range. To our knowledge, it is the first rapid test for quantitation of antibody biotinylation. Due to various binding affinities of protein A to different antibody
  • subclass/isotypes protein A and protein G could be used in combination to further improve the accuracy.
  • Subclass/isotype specific secondary antibodies may also be used to substitute protein A/G as the capture for biotinylated antibodies.
  • Non-related Biotin-conjugated proteins other than BSA may be used on the competition test line. The same concept is applicable to quantify other bioconjugations, such as ADC, HRP,
  • ADC drug- antibody- ratio
  • DAR drug- antibody- ratio
  • ADC aims to deliver the highly cytotoxic drugs directly at the tumor site to decrease the minimum effective dose and at the same time elevate the maximum tolerated dose[24].
  • ADC potency increases with increasing DAR, however plasma clearance accelerates as well, possibly due to increased ADC hydrophobicity derived from the conjugated drug.
  • common methods for the detection of payload of ADC include ELISA[25], LC[26], LC-MS[27] and solid-phase extraction (SPE)[28].
  • LC and SPE based methods utilize the hydrophobicity of drug to separate or extract payload from ADC for analysis, normally requiring extra sample preparation to get rid of the antibody portion to avoid detection interferences from this part.
  • ELISA offers a high degree of specificity and sensitivity but suffers from lengthy experiment time and requirement for skillful handling.
  • a method that can be rapidly deployed with minimal sample preparation, yet offer the specificity and sensitivity for payload detection of ADC is highly desirable.
  • LFIA shares the same immunological principle with ELISA with a much faster and simpler procedure and is a promising candidate.
  • HRP conjugation is widely used to modify Ab for westernblot/ ELTSA/immuno histochemical detection.
  • a standard method to quantify HRP conjugates is quite cumbersome.
  • HRP amount is quantified by comparing to a HRP standard curve and protein amount is quantified by OD280 nm or dye methods (Bradford or Lowry).
  • OD280 nm or dye methods Bradford or Lowry

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Abstract

La présente invention concerne des dispositifs de dosage à écoulement latéral, et des utilisations associées, pour évaluer quantitativement une conjugaison cible. Dans certains aspects, la présente invention concerne l'utilisation d'un dosage immunologique à écoulement latéral pour évaluer quantitativement le niveau de biotinylation, le niveau de conjugaison d'enzyme ou le niveau de conjugaison de médicament d'un polypeptide cible ou d'un anticorps cible.
PCT/US2019/045497 2018-08-10 2019-08-07 Dosage à écoulement latéral pour évaluer une conjugaison cible Ceased WO2020033539A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112394052A (zh) * 2020-12-14 2021-02-23 中南民族大学 一种基于上转换纳米材料与bhq-1共振能量转移检测人附睾蛋白4的方法
CN113533282A (zh) * 2021-07-15 2021-10-22 中国科学院苏州生物医学工程技术研究所 一种基于均相时间分辨荧光的生物素定量测定方法
CN113841050A (zh) * 2020-03-17 2021-12-24 菲尔梅迪株式会社 侧流分析条带和使用该条带的分子诊断方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150293086A1 (en) * 2014-04-14 2015-10-15 Abreos Biosciences, Inc. Lateral flow immunoassay
US20160282343A1 (en) * 2012-08-15 2016-09-29 Immunolab LLC Quantitative lateral flow assay strips for quantitative analysis of an analyte, kits containing such strips and methods of manufacture and use of same
US20170234867A1 (en) * 2012-10-31 2017-08-17 Astute Medical, Inc. Quantitative lateral flow assay
US20180106799A1 (en) * 2015-04-06 2018-04-19 Bludiagnostics, Inc. A test device for detecting an analyte in a saliva sample and method of use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160282343A1 (en) * 2012-08-15 2016-09-29 Immunolab LLC Quantitative lateral flow assay strips for quantitative analysis of an analyte, kits containing such strips and methods of manufacture and use of same
US20170234867A1 (en) * 2012-10-31 2017-08-17 Astute Medical, Inc. Quantitative lateral flow assay
US20150293086A1 (en) * 2014-04-14 2015-10-15 Abreos Biosciences, Inc. Lateral flow immunoassay
US20180106799A1 (en) * 2015-04-06 2018-04-19 Bludiagnostics, Inc. A test device for detecting an analyte in a saliva sample and method of use

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, C ET AL.: "A Fast and Sensitive Quantitative Lateral Flow Immunoassay for CrylAb Based on a Novel Signal Amplification Conjugate", SENSORS, vol. 12, no. 9, 2012, pages 11684 - 11696, XP055683213, DOI: 10.3390/s120911684 *
WANG, Y ET AL.: "Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids", BIOSENSORS, vol. 2, no. 1, March 2012 (2012-03-01), pages 32 - 42, XP055592461, DOI: 10.3390/bios2010032 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113841050A (zh) * 2020-03-17 2021-12-24 菲尔梅迪株式会社 侧流分析条带和使用该条带的分子诊断方法
CN112394052A (zh) * 2020-12-14 2021-02-23 中南民族大学 一种基于上转换纳米材料与bhq-1共振能量转移检测人附睾蛋白4的方法
CN112394052B (zh) * 2020-12-14 2022-10-14 中南民族大学 基于上转换纳米材料与bhq-1共振能量转移的上转换荧光探针
CN113533282A (zh) * 2021-07-15 2021-10-22 中国科学院苏州生物医学工程技术研究所 一种基于均相时间分辨荧光的生物素定量测定方法
CN113533282B (zh) * 2021-07-15 2024-05-28 中国科学院苏州生物医学工程技术研究所 一种基于均相时间分辨荧光的生物素定量测定方法

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