Method for quantitatively detecting antibody concentration in tear

Abstract

The invention provides a method for quantitatively detecting antibody to-be-detected substances or markers in tears, which comprises the following steps: collecting the tears by using a filter paper strip, adding a diluent to extract the tears on the filter paper strip, and then detecting the concentration of the antibody to-be-detected substances or markers in the tears by adopting an ELISA (Enzyme-Linked Immunosorbent Assay) method. The method is simple to operate, low in price and high in sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay analysis, and in particular to a method for quantitatively detecting the concentration of antibody-like test substances in tears. Background Art

[0002] Immunochromatography technology is a new type of immunoassay that appeared in the early 1980s. It is a simple and rapid immunological detection technology based on immunofiltration. It is also an important tool for on-site instant detection. It was first used for the detection of human chorionic gonadotropin (HCG) as a basis for the diagnosis of early pregnancy. It has been widely used in clinical diagnosis, food safety, drug testing, environmental pollution and other fields.

[0003] Immunochromatography technology is mainly carried out with the help of immunochromatography test strips. Its principle is to first fix the specific antibody on a certain zone of the nitrocellulose membrane. When one end of the dry nitrocellulose is immersed in the sample (urine or serum), the sample will move forward along the membrane due to capillary action. When it moves to the area where the antibody is fixed, the corresponding antigen in the sample will specifically bind to the antibody. If immunocolloidal gold or immunoenzyme staining is used, the area can show a certain color, thereby achieving specific immunodiagnosis.

[0004] Enzyme-Linked Immunosorbent Assay (ELISA) appeared in the 1970s. It has the advantages of high specificity, strong sensitivity, simple operation, and no need for expensive instruments and equipment. It has become the most widely used technology in enzyme immunoassay technology. The basic principle of ELISA method includes: adsorbing antigen or antibody on a solid phase carrier, then reacting with the corresponding antibody or antigen to be tested, adding enzyme-labeled antibody or anti-antibody after washing, using the efficient catalytic properties of the enzyme and substrate color development, and using ELISA detector to measure the amount of antigen or antibody. In addition to retaining the high specificity of antigen and antibody reactions, the ELISA method has an amplifying effect on the immunological reaction due to the enzymatic reaction of the enzyme label, and the measurement sensitivity can reach the nanogram (1ng) or even picogram (1pg) level. Commonly used ELISA methods include direct method, indirect method, double antibody sandwich method, direct competition method and capture coating method.

[0005] The composition of tears includes water, electrolytes, lipids, proteins, various immunoglobulins, growth factors and cytokines, among which proteins include albumin, lysozyme and lactoferrin, etc. Certain eye diseases can be diagnosed by measuring specific markers in tears. For example, the measurement of specific IgE, eosinophil cationic protein, IL-4, IL-5 and chemokines can be used to diagnose eye allergies, etc.

[0006] CN105137069A discloses a test paper for detecting allergen-specific IgE antibodies in tear samples. When in use, the tear sample can be dropped onto the sample pad of the test paper, and then the changes in the quality control line and the detection line set on the cellulose membrane are observed, so as to determine whether there are IgE antibodies in the tears and diagnose whether allergic conjunctivitis occurs in the eyes.

[0007] CN103336132A discloses a method for detecting lactoferrin in tears and a special colloidal gold detection card thereof. The detection reaction principle of the lactoferrin colloidal gold detection card is that when the sample solution to be tested is added to the sample addition hole of the detection card, the solution to be tested drives the test object and the gold-labeled antibody in the gold-labeled binding pad to diffuse to the cellulose membrane together through the siphon effect, and finally infiltrates into the absorbent pad end. During the diffusion process, if there is a test object in the sample, the test object and the gold-labeled antibody are combined, and then occupy the antigen binding site on the gold-labeled antibody, preventing the gold-labeled antibody from combining with the detection line (lactoferrin) on the cellulose membrane, so that the detection line does not develop color or develops color very weakly, indicating that the test sample is positive or weakly positive: if there is no test sample in the sample, the gold-labeled antibody is in the process of moving up, and a clear red line is displayed when the test line is encountered, indicating that the test sample is negative. Similarly, the gold-labeled antibody is also combined with the quality control line (sheep anti-mouse IgG) on the cellulose membrane, so that the quality control line is red. The presence or absence of the quality control line color respectively indicates the validity or invalidity of this test card.

[0008] Although the immunochromatographic technique is simple and fast to operate, it is a qualitative diagnostic technique and cannot be used for quantitative determination. At the same time, due to the difficulty of tear extraction technology, there is currently no literature reporting a method for quantitatively detecting the concentration of antibody analytes in tears. Summary of the invention

[0009] The present invention aims to provide a method for quantitatively detecting antibody analytes or markers in tears, which is simple to operate, cheap, highly sensitive, and does not require the use of allergen-specific IgE antibody test paper or special colloidal gold detection card.

[0010] The present invention uses a tear detection filter paper strip to collect tears, and adds a diluent (such as phosphate buffered saline containing Tween, PBST) to dissolve the tears on the filter paper strip.

[0011] The present invention adopts ELISA method to detect the concentration of antibody analytes or markers in tears. The principle is as follows: different capture reagents are coated on an ELISA plate (such as a 96-well ELISA plate) for different analytes, and after blocking, a standard curve sample and a diluent containing tears are added, and the analytes in the tears are captured by specific binding of the capture reagent and the antibody, or by specific binding of the capture reagent and the antigen, and then an enzyme-labeled secondary antibody is added for incubation, and then a substrate (such as 3,3′,5,5′-tetramethylbenzidine (TMB)) of an enzyme (such as horseradish peroxidase (HRP)) is added to generate a color reaction, and the depth of the color is proportional to the concentration of the analyte in the tears, and finally a stop solution is added, and after the reaction is stopped, the OD value is read (for example, the OD value is read at a dual wavelength of 450 / 630 nm, 450 nm is the detection wavelength, and 630 nm is the reference wavelength), and the standard curve is fitted (for example, using a 4-parameter fitting model).

[0012] One aspect of the present invention provides a method for quantitatively detecting an antibody analyte or a marker analyte in tears, the method comprising:

[0013] (1) Collecting tears using filter paper strips or hydrogel strips (preferably tear detection filter paper strips);

[0014] (2) Add a certain volume of diluent to dissolve the tears on the filter paper strip;

[0015] (3) Detecting the concentration of the antibody analyte or marker in the diluent.

[0016] Another aspect of the present invention provides a sample diluent for ELISA, which is a PBST solution, and the diluent contains: 1.4-2.3mM KH2PO4, 6-14mM Na2HPO4·12H2O, 600-900mM NaCl, 2.0-3.5mM KCl, 0.02%-0.1% Tween 20, and a pH of 6.0-9.0.

[0017] Another aspect of the present invention provides an application of a sample diluent for ELISA, characterized in that the sample to be tested by ELISA is diluted with the diluent as described above, and then the diluted sample is subjected to ELISA detection.

[0018] Unless otherwise defined below, the meanings of all technical terms and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. Reference to the technology used herein is intended to refer to the technology commonly understood in the art, including those changes in technology that are obvious to those skilled in the art or replacement of equivalent technology.

[0019] As used herein, the terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended in that they do not exclude other unrecited elements or method steps, even though the other unrecited elements or method steps are not necessarily present (i.e., these terms also encompass the terms "consisting essentially of" and "consisting of").

[0020] As used herein, the term "antibody analyte" includes antibodies, antibody fragments, antibody modifications and antibody fragment modifications.

[0021] As used herein, the term "antibody" is interpreted in the broadest sense and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, so long as they have the desired biological activity.

[0022] As used herein, the term "monoclonal antibody" refers to an antibody that comes from a group of substantially homogeneous antibodies, i.e., the antibodies that make up the group are identical except for a small amount of natural mutations that may be present. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, whereas polyclonal antibodies, in contrast, contain different antibodies for different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by being from a substantially homogeneous group of antibodies and should not be construed as requiring production by a particular method.

[0023] Herein, monoclonal antibodies also specifically include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class or subclass of antibody, and the remaining portion is identical or homologous to another type, class or subclass of antibody, as long as they have the desired biological activity. Chimeric antibodies include primatized antibodies, which contain variable region antigen binding sequences from non-human primates (e.g., ancient monkeys, orangutans, etc.) and human constant region sequences.

[0024] As used herein, the term "antibody fragment" refers to a portion of an antibody, preferably the antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; and single-chain antibody molecules.

[0025] As used herein, the terms "bispecific antibody" and "bifunctional antibody conjugate" are used interchangeably to refer to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a coupling arm, which retains the activity of each antibody and thus has bifunctionality and bispecificity.

[0026] As used herein, the term "multispecific antibody" includes, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities.

[0027] In this article, the term "complete antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2 and CH3). The constant region can be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. A complete antibody is preferably a complete antibody with one or more effector functions.

[0028] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the heavy chain constant region. The five main classes are IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of different classes of antibodies are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.

[0029] The antibody may be an anti-human ErbB2 antibody, and preferably, the CDR1, CDR2 and / or CDR3 of the heavy chain and light chain in the anti-human ErbB2 antibody are the CDR1, CDR2 and / or CDR3 of the heavy chain and light chain of trastuzumab, respectively. The anti-human ErbB2 antibody may be a humanized antibody or a fully human antibody. The antibody is particularly preferably trastuzumab.

[0030] As used herein, the term "antibody modifications" includes, for example, antibody-drug conjugates formed by connecting an antibody to a cytotoxic drug via a bivalent linker; antibody modifications also include, for example, antibody labeling modifications, i.e., antibodies are cross-linked to enzymes, fluorescent dyes, biotin, etc. via different chemical reagents; in addition, antibody modifications also include antibody modifications performed by biological methods, such as glycosylation modifications, antibody constant region Fc amino acid modifications, antibody subclass reconstruction, etc.

[0031] The term "marker" includes other small or large molecules in tears besides antibody analytes, such as lactoferrin, lysozyme, growth factors and cytokines.

[0032] The term "tear detection filter paper strip" refers to tear detection filter paper, tear secretion detection filter paper or tear detection test paper, which is a common tool for measuring the amount of tear secretion in the eyes. This filter paper usually has good water absorption, adhesion, tensile strength and water resistance, does not cause discomfort or irritation to eye tissues, is easy to use and has low cost. Tear detection filter paper strips are usually in the shape of long strips, usually marked with scale lines with millimeters as the unit of length, and their specifications can be: width 5mm, length 30-40mm, mainly used in medical fields such as ophthalmology and dermatology and biochemical experiments. Chinese patents CN2625897Y, CN2845716Y, CN208973862U, and CN209745982U all disclose tear detection filter paper strips, and the contents disclosed in the above patents are introduced into the present invention as a whole. At present, tear detection filter paper strips have been commercialized and are easily purchased on the market.

[0033] The present invention provides a method for quantitatively detecting an antibody analyte or a marker analyte in tears, the method comprising:

[0034] (1) Collecting tears using filter paper strips or hydrogel strips (preferably tear detection filter paper strips);

[0035] (2) Add a certain volume of diluent to dissolve the tears on the filter paper strip;

[0036] (3) Detecting the concentration of the antibody analyte or marker in the diluent.

[0037] In some embodiments of the present invention, the marker is other small molecules or macromolecules in tears other than antibody analytes.

[0038] In some embodiments of the present invention, the marker is lactoferrin, lysozyme, growth factor or cytokine.

[0039] In some embodiments of the present invention, the antibody analyte is selected from an antibody or an antibody modification.

[0040] In some embodiments of the present invention, the antibody modification substance is selected from Antibody-Drug Conjugate.

[0041] In some embodiments of the present invention, the antibody-drug conjugate is represented by the general formula (I):

[0042]

[0043] Wherein: A is an anti-ErbB2 antibody or an active fragment or variant thereof;

[0044] X is N;

[0045] Y is CR 1, and R 1 is H;

[0046] L is a divalent linking group selected from the following:

[0047]

[0048]

[0049] wherein m and n are each an integer selected from 1 to 10 at each occurrence;

[0050] D is a cytotoxic drug group; and

[0051] a is an integer selected from 2-10.

[0052] In some embodiments of the present invention, the anti-ErbB2 antibody may be an anti-human ErbB2 antibody. And preferably, the CDR1, CDR2 and / or CDR3 of the heavy chain and light chain in the anti-human ErbB2 antibody are the CDR1, CDR2 and / or CDR3 of the heavy chain and light chain of trastuzumab, respectively. The anti-human ErbB2 antibody may be a humanized antibody or a fully human antibody. The anti-ErbB2 antibody is particularly preferably trastuzumab.

[0053] In some embodiments of the present invention, a is 2, 3 or 4.

[0054] In some embodiments of the present invention, m and n are 1, 2, 3, 4, 5 or 6 respectively when they appear each time.

[0055] In some embodiments of the present invention, the cytotoxic drug group is derived from a compound of the general formula (D1) or (D2) or a stereoisomer thereof:

[0056]

[0057] in:

[0058] R 2 Selected from -CH2N3, -CONHSO2(cyclopropyl), thiazol-2-yl, -CH3 and -COOH;

[0059] R 3 is selected from H and -OH; and

[0060] R 4 Selected from H, -NH2, Cl, Br, I, -OS(O)2R 6 , where R 6 is H, C1-C8 alkyl, C3-C8 cycloalkyl or C6-C 14 Aryl, wherein the alkyl, cycloalkyl and aryl groups are each optionally substituted with one or 1, 2, 3, 4 or 5 substituents selected from halogen;

[0061]

[0062] Where R 5 Selected from -CH(CH3)N(CH3)C(O)CH2CH2SH and -CH(CH3)N(CH3)C(O)CH2C(CH3)2SH.

[0063] In some embodiments of the present invention, the antibody-drug conjugate is selected from compounds I-1, I-2, I-3, I-4 and I-5:

[0064] in:

[0065] Compound I-1 is an antibody-drug conjugate obtained by forming an amide bond between the terminal carboxyl group of compound IA-1 and the amino group on trastuzumab;

[0066] Compound I-2 is an antibody-drug conjugate obtained by forming an amide bond between the terminal carboxyl group of compound IA-2 and the amino group on trastuzumab;

[0067] Compound I-3 is an antibody-drug conjugate obtained by forming an amide bond between the terminal carboxyl group of compound IA-3 and the amino group on trastuzumab;

[0068] Compound I-4 is an antibody-drug conjugate obtained by forming an amide bond between the terminal carboxyl group of compound IA-4 and the amino group on trastuzumab;

[0069] Compound I-5 is an antibody-drug conjugate obtained by forming an amide bond between the terminal carboxyl group of compound IA-5 and the amino group on trastuzumab;

[0070] The drug / antibody ratio (DAR) of the above compounds I-1, I-2, I-3, I-4 and I-5 is 2.

[0071] The intermediate reacts with the amino group of trastuzumab through the terminal carboxyl group to form an amide bond, which is only a description of the reaction result and is not a limitation on the actual reaction process. The antibody-drug conjugate is usually prepared by deriving the terminal carboxyl groups of intermediates IA-1, IA-2, IA-3, IA-4, and IA-5 to obtain an active intermediate, and then the active intermediate reacts with trastuzumab to form an antibody-drug conjugate.

[0072] The chemical names of the compounds IA-1, IA-2, IA-3, IA-4 and IA-5 are:

[0073] N2-[[4-(4-carboxy-1-piperidinyl)-1,4-dioxobutyl]-L-valyl]-N5-(aminocarbonyl)-N-[4-[(2S)-3-azido-2-[[(2R,3R)-3-[(2S)-|-[(3R,5S)-4-[(N,N-dimethyl-L-valyl-L-valyl)(methyl)amino]-3-methoxy-5-methyl-|-oxoheptyl]-pyrrolidin-2-yl]-3-methoxy-2-methyl-|-oxopropyl]amino]propyl]-phenyl]-L-ornithinamide (Compound IA-1);

[0074] In compound IA-|, N2-[[4-(4-carboxy-1-piperidinyl)-|,4-dioxobutyl]-L-valyl] means that the amino group at the 2-position of ornithine (having formed ornithinamide) is replaced by [[4-(4-carboxy-1-piperidinyl)-1,4-dioxobutyl]-L-valyl], N5-(aminocarbonyl) means that the amino group at the 5-position of ornithine (having formed ornithinamide) is replaced by (aminocarbonyl), and ornithinyl The amide N atom in the amine is replaced by [4-[(2S)-3-azido-2-[[(2R,3R)-3-[(2S)-1-[(3R,5S)-4-[(N,N-dimethyl-L-valyl-L-valyl)(methyl)amino]-3-methoxy-5-methyl-1-oxoheptyl]-pyrrolidin-2-yl]-3-methoxy-2-methyl-1-oxopropyl]amino]propyl]-phenyl].

[0075] N2-[[4-(4-carboxyl--1-piperidinyl)-1,4-dioxobutyl]-L-valyl]-N5-(aminocarbonyl)-N-[4-[(2S)-3-azido-2-[[(2R,3R)-3-[(2S)-|-[(3R,4S,5S)-4-[(N,N-dimethyl-L-valyl-L-valyl)(methyl)amino]-3-methoxy-5-methyl-1-oxoheptyl]-pyrrolidin-2-yl]-3-methoxy-2-methyl-1-oxopropyl]amino]propyl]-phenyl]-L-ornithinamide (Compound IA-2);

[0076] In compound IA-2, the amino group at the 2-position of ornithine (having formed ornithinamide) is replaced by [[4-(4-carboxy-1-piperidinyl)-1,4-dioxobutyl]-L-valyl], the amino group at the 5-position of ornithine (having formed ornithinamide) is replaced by (aminocarbonyl), and the amide N atom in ornithinamide is replaced by [4-[(2S)-3-azido-2-[[(2R,3R)-3-[(2S)-1-[(3R,4S,5S)-4-[(N,N-dimethyl-L-valyl-L-valyl)(methyl)amino]-3-methoxy-5-methyl-1-oxoheptyl]-pyrrolidin-2-yl]-3-methoxy-2-methyl-1-oxopropyl]amino]propyl]-phenyl].

[0077] N2′-[3-[[2-(4-carboxy-1-piperidinyl)-2-oxoethyl]thio]-1-oxopropyl]-N2′-deacetyl-maytansine (Compound IA-3);

[0078] N-[(αR, βR)-β-[(2S)-1-[(3R, 5S)-3-methoxy-5-methyl-4-[(N,N-dimethyl-L-valyl-L-valyl)(methyl)amino]heptanoyl]-pyrrolidin-2-yl]-β-methoxy-α-methyl-propionyl]-O-[(4-carboxy-1-piperidinyl)carbonyl]-N-(cyclopropylsulfonyl)-L-tyrosinamide (Compound IA-4);

[0079] In compound IA-4, [(αR, βR)-β-[(2S)-1-[(3R, 5S)-3-methoxy-5-methyl-4-[(N,N-dimethyl-L-valyl-L-valyl)(methyl)amino]heptanoyl]-pyrrolidin-2-yl]-β-methoxy-α-methyl-propionyl] is a substituent on the amino group of L-tyrosine itself, and (cyclopropylsulfonyl) is a substituent on the amide N atom of L-tyrosinamide;

[0080] N-[(αR, βR)-β-[(2S)-1-[(3R, 4S, 5S)-3-methoxy-5-methyl-4-[(N, N-dimethyl-L-valyl-L-valyl)(methyl)amino]heptanoyl]-pyrrolidin-2-yl]-β-methoxy-α-methyl-propionyl]-O-[(4-carboxy-1-piperidinyl)carbonyl]N-(cyclopropylsulfonyl)-L-tyrosinamide (Compound IA-5);

[0081] In compound IA-5, [(αR, βR)-β-[(2S)-1-[(3R, 4S, 5S)-3-methoxy-5-methyl-4-[(N, N-dimethyl-L-valyl-L-valyl)(methyl)amino]heptanoyl]-pyrrolidin-2-yl]-β-methoxy-α-methyl-propionyl] is a substituent on the amino group of L-tyrosine itself, and (cyclopropylsulfonyl) is a substituent on the amide N atom of L-tyrosinamide.

[0082] In some embodiments of the present invention, in the antibody-drug conjugates I-1, I-2, I-3, I-4 and I-5, all cytotoxic drug groups are conjugated to the light chain of trastuzumab.

[0083] In some embodiments of the present invention, after tears are collected using tear detection filter paper strips in step (1), the volume of collected tears is calculated using the scale on the filter paper strips, and then the filter paper strips are cut into pieces.

[0084] In some embodiments of the present invention, step (2) comprises adding a certain volume of diluent and incubating for a certain period of time under appropriate temperature and rotation speed conditions to dissolve the tear fluid on the filter paper strip.

[0085] In some embodiments of the present invention, the pH of the diluent used in step (2) is in the range of 6.0-9.0, and the NaCl concentration in the diluent is 100-900 mM.

[0086] In some embodiments of the present invention, the pH of the diluent used in step (2) is in the range of 7.0-8.0, and the NaCl concentration in the diluent is 300-800 mM.

[0087] In some embodiments of the present invention, the pH of the diluent used in step (2) is 7.0, and the NaCl concentration is 750 mM.

[0088] In some embodiments of the present invention, the composition of the diluent used in step (2) includes: 1.4-2.3mM KH2PO4, 6-14mM Na2HPO4·12H2O, 100-900mM NaCl, 2.2-3.3mM KCl, 0.02%-0.1% Tween 20, and pH 6.0-9.0.

[0089] In some embodiments of the present invention, the composition of the diluent used in step (2) includes: 1.6-2.0mM KH2PO4, 8-12mM Na2HPO4·12H2O, 300-800mM NaCl, 2.5-3.0mM KCl, 0.03%-0.06% Tween20, and pH 7.0-8.0.

[0090] In some embodiments of the present invention, the composition of the diluent used in step (2) includes: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 750 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, and pH is 7.0-7.4.

[0091] In some embodiments of the present invention, the composition of the diluent used in step (2) includes: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 750 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, and pH is 7.0, 7.2 or 7.4.

[0092] In some embodiments of the present invention, the composition of the diluent used in step (2) includes: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, and pH 7.0-7.4.

[0093] In some embodiments of the present invention, the composition of the diluent used in step (2) includes: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, and pH is 7.0, 7.2 or 7.4.

[0094] In some embodiments of the present invention, the incubation time in step (2) is 1-3 hr.

[0095] In some embodiments of the present invention, the incubation time in step (2) is 1.5-2 hr.

[0096] In some embodiments of the present invention, in step (2), a low adsorption chromatography injection bottle is used to extract the sample in the filter paper.

[0097] In some embodiments of the present invention, the low adsorption chromatography injection bottle used in step (2) is a glass bottle.

[0098] In some embodiments of the present invention, the step (3) detects the concentration of the antibody analyte or marker in the diluent by ELISA.

[0099] In some embodiments of the present invention, the ELISA method in step (3) is selected from the group consisting of direct method, indirect method, double antibody sandwich method and competitive method.

[0100] In some embodiments of the present invention, the ELISA method in step (3) comprises the following steps: coating different capture reagents on the ELISA plate for different analytes, adding a diluent containing tears after blocking, capturing the analyte in the tears through capture reagent-antigen specific binding or capture reagent-antibody specific binding, then adding an enzyme-labeled secondary antibody for incubation, then adding the enzyme substrate to produce a color reaction, and reading the OD value after terminating the reaction.

[0101] In some embodiments of the present invention, the ELISA test in step (3) is performed in a 96-well ELISA plate or a 24-well ELISA plate (preferably a 96-well ELISA plate).

[0102] In some embodiments of the present invention, in step (3), the analyte in the tear fluid is captured by specific binding of the capture reagent and the antigen.

[0103] In some embodiments of the present invention, the capture reagent in step (3) is an anti-toxin small molecule antibody that specifically binds to the toxin small molecule in the antibody-drug conjugate.

[0104] In some embodiments of the present invention, the capture reagent in step (3) is an anti-toxin small molecule + linker antibody that specifically binds to the toxin small molecule + linker in the antibody-drug conjugate.

[0105] In some embodiments of the present invention, in step (3), the analyte in the tear fluid is captured by specific binding of a capture reagent and an antibody.

[0106] In some embodiments of the present invention, the capture reagent in step (3) is an antigen that specifically binds to the antibody portion of the antibody-drug conjugate.

[0107] In some embodiments of the present invention, the toxin small molecule + linker is compound IA-2.

[0108] In some embodiments of the present invention, the anti-toxin small molecule antibody or the anti-toxin small molecule + linker antibody is obtained by coupling KLA, BSA or OVA as a carrier protein to the toxin small molecule or the toxin small molecule + linker, and then immunizing mice.

[0109] In some embodiments of the present invention, the preparation process of anti-toxin small molecule antibodies and anti-toxin small molecule + linker antibodies comprises the following steps:

[0110] ① Coupling of the test sample: Use KLH, BSA or OVA to couple the test sample (toxin small molecule or toxin + linker) to obtain toxin-KLH, toxin-BSA, toxin-OVA, toxin + linker-KLH, toxin + linker-BSA or toxin + linker-OVA;

[0111] ② Preparation of immunogens: mixing the conjugate obtained in step ① with one or more adjuvants such as Freund's complete adjuvant to obtain immunogens of OVA, BSA and KLH;

[0112] ③ Mouse immunization and immune response ELISA evaluation: Use the immunogen obtained in step ② to immunize mice, and use the indirect ELISA method to measure the mouse tail blood titer;

[0113] ④Cell fusion and monoclonal antibody screening: Fusion culture of mouse spleen cells and myeloma cells, selection of positive cell lines, and screening of cell lines that can secrete monoclonal antibodies that recognize proteins;

[0114] ⑤ Preparation of mouse monoclonal antibodies: Based on the cell line affinity results, select candidate monoclonal antibody cell lines for ascites preparation.

[0115] In some embodiments of the present invention, the capture reagent is coated on a 96-well ELISA plate in step (3).

[0116] In some embodiments of the present invention, in step (3), standard curve samples and a diluent containing tears are added after blocking.

[0117] In some embodiments of the present invention, the enzyme-labeled secondary antibody in step (3) is a horseradish peroxidase-labeled secondary antibody, and then the substrate of horseradish peroxidase 3,3',5,5'-tetramethylbenzidine (TMB) is added.

[0118] In some embodiments of the present invention, after the reaction is terminated in step (3), the OD values ​​are read at dual wavelengths of 450 and 630 nm, with 450 nm being the detection wavelength and 630 nm being the reference wavelength.

[0119] In some embodiments of the present invention, a 4-parameter fitting model is used to fit the standard curve in step (3).

[0120] In some embodiments of the present invention, the method comprises the following steps:

[0121] (1) Prepare coating working solution: Prepare capture reagent working solution with carbonate buffer (0.05 M CBS, pH 9.4-9.6);

[0122] (2) Coating: Add the prepared coating working solution to the ELISA plate, seal the plate with a sealing film, and incubate at 2-8°C overnight.

[0123] (3) Washing the plate: discard the coating solution, wash the plate with plate washing solution, and pat the plate wells dry on clean paper;

[0124] (4) Blocking: Add blocking solution to the ELISA plate and seal the plate with a sealing film and incubate;

[0125] (5) Tear filter paper sample processing: record the scale of the tear filter paper strip for tear collection, divide the filter paper strip longitudinally in half from the middle of the end, cut one half into pieces, add an appropriate volume of diluent (pH 7.0, 750 mM NaCl) according to the tear volume, and incubate for 1 to 1.5 h;

[0126]

[0127] (6) Sample preparation: Prepare the sample according to the standard curve sample preparation table;

[0128] (7) Washing the plate: Take out the ELISA plate, wash the plate with plate washing solution, and pat the plate wells dry on clean paper;

[0129] (8) Place the standard curve samples and the treated tear samples into the wells according to the plate map, seal the plate with a sealing film, and incubate;

[0130] (9) Secondary antibody preparation: prepare enzyme-labeled secondary antibody working solution in a certain ratio;

[0131] (10) Washing the plate: Take out the ELISA plate, wash the plate with plate washing solution, and pat the plate wells dry on a clean paper;

[0132] (11) Add enzyme-labeled secondary antibody working solution and incubate;

[0133] (12) Washing the plate: Take out the ELISA plate, wash the plate with plate washing solution, and pat the plate wells dry on clean paper;

[0134] (13) Add TMB substrate solution and react at room temperature in the dark;

[0135] (14) Termination: Add stop solution (1M H2SO4) to terminate the reaction;

[0136] (15) Detection: Use an ELISA reader to read the OD value within 5 min, with a detection wavelength of 450 nm and a reference wavelength of 630 nm.

[0137] The invention provides a sample diluent for ELISA, which is a PBST solution. The diluent comprises: 1.4-2.3mM KH2PO4, 6-14mM Na2HPO4·12H2O, 100-900mM NaCl, 2.2-3.3mM KCl, 0.03%-0.1% Tween 20, and the pH value is 6.0-9.0.

[0138] In some embodiments of the present invention, the composition of the diluent includes: 1.6-2.0 mM KH2PO4, 8-12 mM Na2HPO4·12H2O, 300-800 mM NaCl, 2.5-3.0 mM KCl, 0.03%-0.06% Tween 20, and pH 7.0-8.0.

[0139] In some embodiments of the present invention, the composition of the diluent includes: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 750 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, and pH is 7.0-7.4.

[0140] In some embodiments of the present invention, the composition of the diluent includes: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 750 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, and pH is 7.0, 7.2 or 7.4.

[0141] In some embodiments of the present invention, the composition of the diluent includes: 1.8mM KH2PO4, 10mMNa2HPO4·12H2O, 137mM NaCl, 2.7mM KCl, 0.05% Tween 20, and pH is 7.0-7.4.

[0142] In some embodiments of the present invention, the composition of the diluent includes: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, and pH is 7.0, 7.2 or 7.4.

[0143] The present invention provides an application of a sample diluent for ELISA, wherein the diluent is used to dilute a sample to be tested by ELISA, and then the diluted sample is subjected to ELISA detection.

[0144] Compared with the prior art, the present invention has the following beneficial effects:

[0145] 1. The present invention uses tear detection filter paper strips to collect tears, and then adds a diluent to extract tears. The diluent is a phosphate buffered saline (PBST) containing Tween. The diluent has a high recovery rate for antibody analytes or markers in tears, and can be used to quantitatively detect the concentration of antibody analytes or markers in tears. By investigating the pH value of the diluent, it is found that under neutral acid-base conditions of pH 6.0-8.0 (preferably 7.0-8.0, more preferably 7.0) far from the isoelectric point, the solubility of antibody analytes or markers can be improved, and the recovery rate is high. By investigating the neutral salt concentration of the diluent, it is found that under 100-900mM (preferably 750mM) NaCl conditions, charge adsorption can be reduced to obtain a recovery rate of 80%-100%. By investigating the incubation time for extracting tears, it is found that an incubation time of 1 to 2h (preferably 1.5h) is acceptable.

[0146] 2. The present invention uses the ELISA method to quantitatively detect antibody analytes or markers in tears. The method is simple to operate, cheap, and highly sensitive, and does not require the use of allergen-specific IgE antibody test paper or special colloidal gold detection card.

[0147] 3. In the ELISA method, the sensitivity, throughput and specificity of the detection of the analyte in the sample are improved by optimizing the selection of different coated solid media. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 It is the standard curve diagram of compound I-2. DETAILED DESCRIPTION

[0149] The present invention will be further illustrated in the following examples. These examples are only used to illustrate the present invention, but are not intended to limit the present invention in any way.

[0150] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0151] In the examples and comparative examples, an antibody-drug conjugate (ADC) shown as compound I-2 was used, and all cytotoxic drug groups were coupled to the light chain of trastuzumab.

[0152] The toxin small molecule + linker used in the example is compound IA-2.

[0153] The composition of the carbonate buffer used in the examples includes: Na2CO3 1.59 g, NaHCO3 2.93 g, add deionized water to 900 ml, adjust the pH value to 9.4-9.6, and adjust the volume to 1000 ml.

[0154] In the examples and comparative examples, the meanings of the abbreviations are as shown in the following table.

[0155]

[0156] Example 1

[0157] This example describes the preparation process of anti-toxin small molecule + linker antibody.

[0158] ① Coupling of test sample:

[0159] Weigh 2 mg of toxin small molecule + linker (IA-2) and dissolve it in 462 uL DMSO; dissolve 5 mg KLH in 3 mL PBS. Take 120 uL of DMSO solution with small molecule dissolved, add it to 0.5 mL PBS solution with KLH dissolved, rotate overnight to react; then add 0.5 mL PBS, place in a dialysis bag, dialyze with 1 L PBS, change the solution twice, overnight; after PBS is diluted 30 times, measure OD 230 The results showed that the OD of IA-2-KLH 230 =0.410.

[0160] ② Preparation of immunogen:

[0161] 42 μL of IA-2-KLH was mixed with 600 μL of Freund's complete adjuvant, and then the volume was made up to 1 mL with sterile water, and the mixture was mixed and ultrasonically emulsified to prepare the immunogen.

[0162] ③ ELISA evaluation of mouse immunity and immune response:

[0163] Four BALB / c mice were immunized with the KLH immunogen obtained in step ② (immunization sites were the back subcutaneous tissue and leg muscles).

[0164] The tail blood after immunization was evaluated by indirect ELISA, and the mouse antibody titer recognizing small molecule-coupled KLH obtained after immunization of mice reached 1:50000, which met the experimental requirements.

[0165] ④Cell fusion and monoclonal antibody screening:

[0166] Conventional hybridoma screening was performed. In this study, mouse spleen cells of IA-2-KLH and mouse myeloma cells SP2 / 0 were fused under the action of PEG, cultured, screened, expanded, passaged for stability, and screened for specificity verification to obtain four mouse monoclonal antibody hybridoma cell lines that specifically recognized the IA-2 small molecule. This indicates that the immunogen prepared using IA-2 has good repeatability in obtaining specific hybridomas.

[0167] ⑤ Preparation of mouse monoclonal antibodies:

[0168] Select one clone from the four cell lines obtained in step ④, prepare ascites by conventional methods, and purify with Protein G (for ascites preparation and Protein purification operations, refer to the literature: Gary C. Howard, Matthew R. Kaser, 2020; Zhang Jianmin et al., A Guide to Antibody Preparation and Use (Second Edition). Beijing: Science Press, 2020). The titer of the purified mouse monoclonal antibody reached 0.005 μg / mL, and a total of 41.85 mg of antibody was obtained, with a concentration of 2.79 mg / mL and a volume of 15 mL.

[0169] Example 2

[0170] This example investigates the effect of using a 24-well ELISA plate as a coating medium on the recovery rate.

[0171] Solutions of compound I-2 with different concentrations were dripped onto tear test filter paper strips (produced by Tianjin Jingming New Technology Development Co., Ltd., Tianjin Medical Device No. 20172200301, whatman41# filter paper), the scale of the tear filter paper strips was recorded, the filter paper strips were cut into pieces, and placed in the formulation buffer of each well of a 24-well plate, and then the concentration of compound I-2 in the test dilution was analyzed by ELISA, and the recovery rate was calculated. The results are shown in Table 1.

[0172] The steps are as follows:

[0173] ① Prepare coating working solution: dilute the anti-toxin + linker small molecule antibody prepared in Example 1 to 2 μg / mL using carbonate buffer (0.05M CBS, pH 9.4-9.6);

[0174] ② Coating: Add the prepared coating working solution into a 24-well plate at 400 μL / well and incubate at 37°C for 2 hours;

[0175] ③ Washing: discard the coating solution, wash the plate once with 1000 μL / well of washing solution (PBST), and pat the wells dry on clean paper;

[0176] ④ Blocking: Add blocking solution (5% SM-PBS) to the ELISA plate at 1000 μL / well and incubate at 37°C for 2 h;

[0177] ⑤ Sample pretreatment: 5, 12, and 25 μL of the solution of compound I-2 (1600 ng / mL) were dropped onto tear filter paper;

[0178] ⑥ Standard curve sample preparation: prepare and dilute according to the standard curve sample preparation table;

[0179] ⑦ Wash the plate: Take out the 24-well plate, wash the plate three times with 1000 μL / well of plate washing solution (PBST), and pat the plate wells dry on clean paper;

[0180] ⑧ Sample addition: Place the pre-treated filter paper with the sample in 400 μL / well preparation buffer (0.05% PBST, pH 7.2) of a 24-well plate, add the prepared standard samples to other corresponding wells, and incubate overnight at 25°C on a shaker at 150 rpm / min;

[0181] ⑨ Secondary antibody preparation: prepare Goat Anti-Human IgG-HRP and monkey ads enzyme-labeled secondary antibody working solution at a ratio of 1:3000;

[0182] ⑩ Washing: Take out the 24-well plate, wash the plate 4 times with 1000 μL / well of plate washing solution (PBST), and pat the wells dry on clean paper;

[0183] Add Goat Anti-Human IgG-HRP, monkey ads enzyme-labeled secondary antibody working solution, 400 μL / well, and incubate at 25°C for 1 hr±5 min;

[0184] Washing: Take out the ELISA plate, wash the plate 5 times with 1000 μL / well of plate washing solution (PBST), and pat the wells dry on clean paper;

[0185] Add TMB substrate solution: 400 μL / well, react at room temperature in the dark for 19 min;

[0186] Stop: Add stop solution (1M H2SO4), 400 μL / well, to stop the reaction;

[0187] Detection: Use a microplate reader to read the OD value within 5 minutes. The detection wavelength is 450nm and the reference wavelength is 630nm.

[0188] The composition of PBST used in this example includes: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH 7.2.

[0189] Table 1 24-well plate test results

[0190]

[0191] As shown in Table 1, when 1600 ng / mL of compound I-2 was dripped onto tear test filter paper in different volumes (5, 12, 25 μL), the recovery rates in the 24-well ELISA plate were 26%, 5.1%, and 4.8%.

[0192] Example 3

[0193] In this example, a 96-well ELISA plate was used to investigate the effect of the pH of the diluent on the recovery rate of compound I-2.

[0194] The solutions of compound 1-2 with different concentrations were dripped onto tear test filter paper strips (produced by Tianjin Jingming New Technology Development Co., Ltd., Tianjin Medical Device No. 20172200301, whatman41# filter paper), the tear filter paper strip scale was recorded, the filter paper strips were cut into pieces, and placed in a chromatographic injection glass vial, and the tear dissolution treatment was performed using PBST (phosphate buffer containing Tween) diluents of different pH values, the incubation time was 2h, and ELISA analysis was performed using a 96-well enzyme-labeled plate, the concentration of compound I-2 in the diluent was tested, and the recovery rate was calculated. The results are shown in Table 2.

[0195] The composition of PBST used included: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, and the solution was adjusted to the desired pH using 2 M HCl or 0.5 M NaOH.

[0196] The steps of ELISA analysis are as follows:

[0197] ① Prepare coating working solution: dilute the antitoxin small molecule + linker antibody prepared in Example 1 to 1 μg / mL using carbonate buffer (0.05M CBS, pH 9.4-9.6);

[0198] ② Coating: Add the prepared coating working solution into a 96-well plate at 100 μL / well and let stand at 2-8°C for 16-18 hours;

[0199] ③ Washing: discard the coating solution, wash the plate once with 300 μL / well of washing solution (PBST), and pat the wells dry on clean paper;

[0200] ④ Blocking: Add blocking solution (5% SM-PBS) to the ELISA plate at 300 μL / well and incubate at 37°C for 2 h;

[0201] ⑤ Preparation and treatment of filter paper samples: After preparing the samples at the established concentrations of 2500, 1600, 800, and 400 ng / mL, add 10 μL to the tear filter paper. After the filter paper is dry, cut it into pieces and put it into a sample bottle. Add 390 μL of PBST with different pH values ​​(3, 4, 5, 6, 8, and 9), and incubate it on a shaker at 37°C and 250 rpm for 2 hours. Finally, centrifuge it at 1200 rpm for 5 minutes for later use.

[0202] ⑥ Standard curve sample preparation: prepare and dilute according to the standard curve sample preparation table;

[0203] ⑦ Washing: Take out the ELISA plate, wash the plate three times with 300 μL / well of plate washing solution (PBST), and pat the plate wells dry on clean paper;

[0204] ⑧ Sample addition: add the standard sample and the pretreated sample to the corresponding wells, seal the plate with a sealing film, and incubate at 25°C for 2h;

[0205] ⑨ Secondary antibody preparation: prepare Goat Anti-Human IgG-HRP and monkey ads enzyme-labeled secondary antibody working solution at a ratio of 1:8000;

[0206] ⑩ Washing: Take out the ELISA plate, wash the plate 5 times with 300 μL / well of plate washing solution (PBST), and pat the plate wells dry on clean paper;

[0207] Add Goat Anti-Human IgG-HRP and monkey ads enzyme-labeled secondary antibody working solution, 100 μL / well, and incubate at 25°C for 1 h;

[0208] Washing: Take out the ELISA plate, wash the plate 6 times with 300 μL / well of plate washing solution (PBST), and pat the wells dry on clean paper;

[0209] Add TMB substrate solution: 100 μL / well, react at room temperature in the dark for 6-7 minutes;

[0210] Stop: Add stop solution (1M H2SO4), 100 μL / well, to stop the reaction;

[0211] Detection: Use a microplate reader to read the OD value within 5 minutes, the detection wavelength is 450nm, and the reference wavelength is 630nm.

[0212] Table 2 Results of exploration tests under different pH conditions

[0213]

[0214] As shown in Table 2, the recovery rate is high under pH 6.0-9.0 conditions, and the recovery rate is the highest under pH 8.0 conditions at 37°C for 2h, exceeding 50%, in the range of 55%-69%. This indicates that compound I-2 has a high solubility in the diluent under pH conditions far from the isoelectric point. The recovery rate is slightly reduced when the incubation time is extended by about 20min.

[0215] This example uses a 96-well ELISA plate, and it is found that the 96-well ELISA plate can reduce nonspecific adsorption and sample usage.

[0216] Example 4

[0217] The experimental method of Example 4 is basically the same as that of Example 3, except that the incubation time in step ⑤ is shortened to 1.5 h, and the effect of the dilution solution at different pH and different NaCl concentration levels on the recovery rate is investigated. The results are shown in Table 3.

[0218] Table 3 Test results of different NaCl concentrations

[0219]

[0220] As shown in Table 3, whether the NaCl concentration is 136 mM or 750 mM, the recovery rate is higher than 70% when incubated for 1.5 h at pH 7.0-8.0. Among them, the recovery rate is the highest when incubated at 37°C for 1.5 h at pH 7.0 and NaCl concentration of 750 mM. The recovery rate is high at high NaCl concentration (750 mM) at pH 7.0-8.0.

[0221] Example 5

[0222] This example detects the concentration of compound I-2 in the tears of cynomolgus monkeys at different time points after administration of compound I-2. The steps are as follows:

[0223] (1) Preparation of coating working solution: Dilute the antitoxin small molecule + linker antibody prepared in Example 1 to 1 μg / mL with carbonate buffer (0.05 M CBS, pH 9.4-9.6) to obtain capture reagent working solution, i.e., coating working solution.

[0224] (2) Coating: Add the prepared coating working solution into a 96-well ELISA plate at 100 μL / well, seal the plate with a sealing film, and incubate at 2-8°C for 16-18 hours.

[0225] (3) Washing: Discard the coating solution, wash the plate once with 300 μL / well of plate washing solution (PBST), and pat the wells dry on clean paper.

[0226] (4) Blocking: Blocking solution (5% skim milk powder-PBS) was added to the ELISA plate at 300 μL / well, and the plate was sealed with a sealing film and incubated at 37°C for 2 h.

[0227] (5) Tear filter paper sample processing: Use tear detection filter paper strips (produced by Tianjin Jingming New Technology Development Co., Ltd., Tianjin Medical Device No. 20172200301, whatman41# filter paper) to collect cynomolgus monkey tears, record the scale of cynomolgus monkey tears collected by the tear filter paper strips, divide the filter paper strips in half from the middle, cut the filter paper strips horizontally into pieces with a width of 1 to 2 mm, add 300 μL of PBST (pH 7.0, 750 mM NaCl) according to the tear volume, and incubate on a shaker at 37°C at 200 rpm for 1.5 h.

[0228] (6) Sample preparation: Compound I-2 standard was used to prepare samples according to the standard curve sample preparation table.

[0229] (7) Washing: Take out the ELISA plate and wash the plate three times with PBST at 300 μL / well, and pat the wells dry on clean paper.

[0230] (8) Sample addition: 100 μL / well of standard curve samples and treated tear samples (tear samples were collected from the left eye and right eye at 0, 2, 4, 24, 48, 96, and 168 h after administration of compound I-2) were added, and the plate was sealed with a sealing film and incubated at 25°C for 2 h.

[0231] (9) Secondary antibody preparation: Prepare Goat Anti-Human IgG-HRP and monkey ads enzyme-labeled secondary antibody working solution at a ratio of 1:8000.

[0232] (10) Washing: Take out the ELISA plate and wash the plate five times with 300 μL / well of plate washing buffer (PBST), and pat the wells dry on clean paper.

[0233] (11) Add Goat Anti-Human IgG-HRP and monkey ads enzyme-labeled secondary antibody working solution at 100 μL / well and incubate at 25°C for 1 h±5 min.

[0234] (12) Washing: Take out the ELISA plate and wash the plate six times with 300 μL / well of plate washing buffer (PBST), and pat the wells dry on clean paper.

[0235] (13) Add TMB substrate solution: 100 μL / well and react at room temperature in the dark for 5-8 min.

[0236] (14) Termination: Add stop solution (1M H2SO4), 100 μL / well, to terminate the reaction.

[0237] (15) Detection: Use an ELISA reader to read the OD value within 5 min, with a detection wavelength of 450 nm and a reference wavelength of 630 nm.

[0238] The composition of PBST used included: 1.8 mM KH2PO4, 10 mM Na2HPO4·12H2O, 750 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH 7.0.

[0239] Experimental results:

[0240] The corresponding relationship between the standard curve sample concentration, plate wells, and OD values ​​is shown in Table 4. The standard curve is shown in the attached Figure 1 The results of the detection of the concentration of compound I-2 in the tears collected at different time points after administration are shown in Table 5.

[0241] Table 4

[0242]

[0243]

[0244] Table 5

[0245]

[0246] According to Table 4 and Appendix Figure 1 It can be seen that the quantitative range of compound I-2 in the detection method of the present invention is 2.5 to 160 ng / mL, and the sensitivity can reach 2.5 ng / mL. The concentration of compound I-2 in tears can be detected according to the standard curve.

[0247] The capture reagent, enzyme-labeled secondary antibody and standard curve sample preparation can also be adjusted according to different antibodies to determine the concentration of the antibody analyte in tears.

[0248] Example 6

[0249] This example investigates the recovery rate data of compound I-2 when other filter papers are used to collect cynomolgus monkey tears.

[0250] The filter paper (Sinopharm Chemical Reagent Co., Ltd., catalog number: 92410112S, medium-speed filter paper) was cut into 1 cm*1 cm filter paper pieces, sterilized at high temperature, dried, folded in half, and placed in the lower eyelid of the cynomolgus monkey to absorb tears. The filter paper was cut into pieces, placed in a 24-well ELISA plate, incubated overnight with PBST at pH 7.4, the concentration of the compound in the test solution, and the recovery rate was calculated. The experimental results are shown in Table 6.

[0251] Table 6

[0252]

[0253] As shown in Table 6, quantitative detection can be achieved within the drop concentration range of 100-1600 ng / mL, but the concentration of compound I-2 detected by dropping PBST on the blank filter paper was 0.912 ng / mL, indicating that the filter paper had non-specific adsorption.

[0254] In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including all patents, patent applications, journal articles, books and any other disclosures) is incorporated herein by reference in its entirety.

Claims

1. A method for quantitatively detecting an antibody analyte or a marker analyte in tears, characterized in that: The method comprises the following steps: (1) Collect tears using filter paper strips or hydrogel strips; (2) adding a certain volume of diluent to dissolve the tears on the filter paper strip or hydrogel strip; (3) detecting the concentration of the antibody analyte or marker in the diluent; Wherein, the marker is other small molecules or macromolecules in tears except the antibody analyte.

2. The detection method according to claim 1, characterized in that The filter paper strip is selected from tear detection filter paper strips; the marker is lactoferrin, lysozyme, growth factor or cytokine.

3. The detection method according to claim 1 or 2, characterized in that After collecting tears using the tear detection filter paper strip in step (1), the volume of the collected tears is calculated using the scale on the filter paper strip, and then the filter paper strip is cut into pieces.

4. The detection method according to any one of claims 1 to 3, characterized in that: The step (2) includes adding a certain volume of diluent and incubating for a certain period of time under appropriate temperature and rotation speed conditions to dissolve the tears on the filter paper strip.

5. The detection method according to any one of claims 1 to 4, characterized in that: The pH of the diluent used in step (2) is in the range of 6.0-9.0, and the NaCl concentration in the diluent is 100-900 mM; Preferably, the composition of the diluent used in step (2) includes: 1.4-2.3 mM KH2PO4, 6-14 mM Na2HPO4·12H2O, 100-900 mM NaCl, 2.2-3.3 mM KCl, 0.02%-0.1% Tween 20, and pH 6-9.

6. The detection method according to any one of claims 1 to 5, characterized in that: In the step (2), a low adsorption chromatography injection bottle is used to extract the sample in the filter paper.

7. The detection method according to any one of claims 1 to 6, characterized in that: The step (3) detects the concentration of the antibody analyte or marker in the diluent by ELISA.

8. The detection method according to any one of claims 1 to 7, characterized in that: The ELISA method in step (3) is selected from the group consisting of direct method, indirect method, double antibody sandwich method and competitive method; Preferably, the ELISA method in step (3) comprises the following steps: coating different capture reagents on the ELISA plate for different analytes, adding a diluent containing tears after blocking, capturing the analyte in the tears through capture reagent-antibody specific binding, or through capture reagent-antigen specific binding, then adding enzyme-labeled secondary antibody for incubation, then adding enzyme substrate to produce a color reaction, and reading the OD value after terminating the reaction.

9. The detection method according to any one of claims 1 to 8, characterized in that: In the step (3), the analyte in the tear fluid is captured by specific binding of the capture reagent and the antigen; Preferably, the capture reagent in step (3) is an anti-toxin small molecule antibody that specifically binds to the toxin small molecule in the antibody-drug conjugate; or The capture reagent in step (3) is an anti-toxin small molecule + linker antibody that specifically binds to the toxin small molecule + linker in the antibody-drug conjugate.

10. A sample diluent for ELISA, which is a PBST solution, the composition of the diluent comprising: 1.4-2.3mMKH2PO4, 6-14mM Na2HPO4·12H2O, 100-900mM NaCl, 2.2-3.3mM KCl, 0.02%-0.1% Tween 20, pH 6.0-9.

0.

11. An application of a sample diluent for ELISA, characterized in that: The ELISA sample is diluted with the diluent as claimed in claim 10, and then the diluted sample is subjected to ELISA detection.

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