Antibody affinity detection kit, antibody affinity detection method and application thereof

Abstract

The invention relates to an antibody affinity detection kit, an antibody affinity detection method and application thereof, the kit comprises a reagent 1, a reagent 2, a discretizing agent and a contrast solution, the reagent 1 comprises luminescent particles coated with antigen, and the luminescent particles internally comprise optical reaction substances; the reagent 2 comprises a biotinylated anti-human IgG antibody; the contrast solution is a matrix solution of the dispersing agent. According to the scheme provided by the invention, the affinity of the IgG antibody in the to-be-detected sample can be conveniently and efficiently detected, the detection efficiency is improved, and the detection result is accurate and reliable.

Description

Technical Field

[0001] The present application relates to the technical field of in vitro diagnostic reagents, and in particular to an antibody affinity detection kit, an antibody affinity detection method and applications thereof. Background Art

[0002] The interaction forces between antigens and antibodies generally include non-covalent bonds such as hydrogen bonds, hydrophobic effects, electrostatic attraction, and van der Waals forces. However, the non-covalent binding of antigens and antibodies is reversible. For example, the non-covalent binding of antigens and antibodies can be disturbed by using a dispersant to destroy the hydrogen bond network between water molecules and weaken the hydrophobic effect.

[0003] In the related art, the affinity detection method of specific IgG antibodies is generally the urea denaturation ELISA indirect method, and the specific operation is: the pre-made microplate has a specific antigen coating solution, and the sample to be tested is added to the well of the microplate during detection (all wells are made in duplicate), and the unbound substances are washed away after incubation reaction. A discrete agent such as urea is added to the discrete agent well, and a buffer is added to the other control well, and the unbound substances are washed away after incubation reaction. Anti-human IgG labeled with horseradish peroxidase is added, and the unbound substances are washed away after incubation reaction. Substrate solution is added, and stop solution is added after incubation reaction to read the results. As can be seen from the above, the defects of the existing ELISA indirect method for detecting affinity are that it requires manual operation and multiple washings, making the detection process very cumbersome. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related art, the present application provides an antibody affinity detection kit, an antibody affinity detection method and its application, which can conveniently and efficiently detect the affinity of antibodies in a sample to be tested, improve the detection efficiency, and the detection results are accurate and reliable.

[0005] The first aspect of the present application provides an antibody affinity detection kit, which includes reagent 1, reagent 2, a dispersant and a control solution, wherein:

[0006] The reagent 1 includes luminescent particles coated with antigens, and the interior of the luminescent particles contains optically reactive substances;

[0007] The reagent 2 includes a biotinylated anti-human IgG antibody;

[0008] The control solution is a matrix solution of the discrete agent.

[0009] In some embodiments, the dispersing agent contains urea, potassium thiocyanate, guanidine hydrochloride or citric acid.

[0010] In some embodiments, the matrix solution is selected from physiological saline or buffer solution; preferably, the matrix solution is a single component solution.

[0011] In some embodiments, the kit further comprises a negative control and a positive control; preferably, the positive control comprises a low-affinity positive control and a high-affinity positive control.

[0012] In some embodiments, the concentration of the reagent 1 is 6.25 mg / L to 25 mg / L; and / or,

[0013] The concentration of the reagent 2 is 1.25 mg / L to 5 mg / L.

[0014] The second aspect of the present application provides a method for detecting antibody affinity, which comprises: simultaneously detecting equal amounts of test samples treated with equal amounts of control solutions or discrete agents, and confirming the affinity strength of the antibodies in the test samples based on the ratio of the detection signal values.

[0015] In some embodiments, the discrete reagent well signal value / control well signal value is an affinity index, and when the affinity index is <0.3, the affinity of the antibody in the sample to be tested is low; when 0.3≤affinity index<0.5, the affinity of the antibody in the sample to be tested is medium; when the affinity index is ≥0.5, the affinity of the antibody in the sample to be tested is high.

[0016] In some implementations, the steps include:

[0017] Add the sample to be tested, the reagent 1 and the discrete agent to the discrete agent wells respectively and mix them; and add the sample to be tested, the reagent 1 and the control solution in the same amount as that in the discrete agent wells respectively and mix them;

[0018] Incubating the discrete agent wells and the control wells at a preset temperature for a preset period of time;

[0019] Adding equal amounts of the reagent 2 and the reagent 3 to the discrete reagent wells and the control wells, and continuing to incubate for a sufficient reaction; wherein the reagent 3 contains photosensitive particles that can react with active oxygen to produce chemiluminescence;

[0020] The discrete agent wells and the control wells are irradiated with laser to obtain corresponding light signal values ​​and calculate the ratio.

[0021] The third aspect of the present application provides a homogeneous chemiluminescence analyzer, which is loaded with the kit described in the above embodiment and / or performs the above detection method to detect the sample to be tested, so as to analyze the affinity of the specific IgG antibody in the sample to be tested.

[0022] The fourth aspect of the present application provides an application of the above-mentioned kit or the above-mentioned detection method in evaluating the immune effect of a vaccine.

[0023] The technical solution provided by this application may have the following beneficial effects:

[0024] The present application establishes a specific IgG antibody affinity detection method by utilizing the different interference degrees of discrete agents on high-affinity antibodies and low-affinity antibodies, and successfully prepares a photochemiluminescence antibody affinity detection kit, which can conveniently and efficiently detect the IgG antibody affinity in the sample to be tested, without the need for repeated manual washing and complicated operations, thereby improving the detection efficiency, and the test results are accurate and reliable, the detection cost is low, and the applicability is wide. It is an auxiliary means for identifying acute infections of pathogens, and can also be used to evaluate the immune effects of vaccines.

[0025] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. DETAILED DESCRIPTION

[0026] To make the application easy to understand, the application will be described in detail below. However, before describing the application in detail, it should be understood that the application is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing the specific embodiments and are not intended to be restrictive. The implementation of the application is not limited to the following embodiments, and any pro forma modifications and / or changes made to the application will fall within the scope of protection of the application.

[0027] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present application. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present application, subject to any explicitly excluded limits in the specified ranges. Where a specified range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the present application.

[0028] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present application belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present application, preferred methods and materials are now described.

[0029] I. Terminology

[0030] The term "active oxygen" as used in this application refers to a general term for substances composed of oxygen, containing oxygen and active in the body or in the natural environment, mainly an excited oxygen molecule, including the one-electron reduction product of oxygen, superoxide anion, the two-electron reduction product of oxygen, hydrogen peroxide, the three-electron reduction product of oxygen, hydroxyl radicals, as well as nitric oxide and singlet oxygen.

[0031] The term "photosensitive particles" used in this application refers to particles containing sensitizers activated by energy or active compounds that can produce active intermediates such as reactive oxygen that react with luminescent particles. In some specific embodiments of the present application, the photosensitive particles are polymer particles filled with photosensitizers, and the photosensitizers can be photosensitizers known in the art, preferably compounds that are relatively stable to light and do not react effectively with singlet oxygen, such as compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine and chlorophyll, and derivatives of these compounds with 1-50 atomic substituents, which are used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups connected to specific binding pair members. Examples of other photosensitizers known to those skilled in the art can also be used in this application.

[0032] The term "luminescent particles" used in this application refers to particles containing compounds that can react with active oxygen to produce detectable signals. The photosensitive particles are activated by energy or active compounds and release high-energy active oxygen, which is captured by the luminescent particles at a close distance, thereby transferring energy to activate the luminescent particles. In some specific embodiments of the present application, the luminescent particles include a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and / or coated on the surface of the carrier.

[0033] The "carrier" described in the present application is selected from strips, sheets, rods, tubes, holes, microtiter plates, beads, particles and microparticles. It can be microparticles or microparticles known to those skilled in the art. It can be of any size. It can be organic or inorganic. It can be expandable or non-expandable. It can be porous or non-porous. It can be magnetic or non-magnetic. It has any density, but preferably has a density close to that of water, preferably can float in water, and is made of transparent, partially transparent or opaque materials.

[0034] In the present application, the "chemiluminescent compound" is a compound called a marker that can undergo a chemical reaction to cause luminescence, such as by being converted into another compound formed in an electronically excited state. The excited state can be a singlet state or a triplet excited state. The excited state can relax to the ground state to emit light directly, or by transferring the excitation energy to an emission energy acceptor, thereby restoring itself to the ground state. In this process, the energy luminescent particle will be transitioned to an excited state and emit light.

[0035] The "specific binding pair members" mentioned in the present application refer to a pair of substances that can specifically recognize and bind to each other.

[0036] The term "sample to be tested" as used herein refers to a mixture containing or suspected to contain a target molecule to be tested. The sample to be tested that can be used in the present application includes body fluids, such as blood (can be anticoagulated blood commonly seen in the collected blood sample), plasma, serum, urine, semen, saliva, cell culture, tissue extracts, etc. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be tested can be diluted with diluent as needed before use. For example, in order to avoid the HOOK effect, the sample to be tested can be diluted with a diluent before being tested on the machine and then tested on the detection instrument.

[0037] The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity to the target molecule to be detected will be used to detect the target molecule. The target molecule to be detected may be a protein, a peptide, an antibody, or a hapten that can bind to an antibody. The target molecule to be detected may be a nucleic acid or an oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected may be any other substance that can form a specific binding pair member. Other typical examples of target molecules to be detected include: drugs, such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial drugs, antifungal drugs, antiviral drugs, purines, antitumor agents, amphetamines, nitrogen compounds, nucleic acids and prostaglandins, and metabolites of any of these drugs; pesticides and their metabolites; and receptors. The target molecules to be detected also include cells, viruses, bacteria and fungi.

[0038] The term "antibody" as used herein is used in the broadest sense, including antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any desired case, the antibody can be further conjugated to other moieties, such as a member of a specific binding pair member, for example, biotin or avidin (a member of a biotin-avidin specific binding pair member), etc.

[0039] The term "antigen" as used in this application refers to a substance that can stimulate the body to produce an immune response and can combine with the immune response products antibodies and sensitized lymphocytes in vivo or in vitro to produce an immune effect.

[0040] As used herein, the term "binding" refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonds, including but not limited to interactions such as salt bridges and water bridges.

[0041] The term "specific binding" as used in this application refers to the mutual recognition and selective binding reaction between two substances, which is the correspondence of the conformations between the corresponding reactants from the perspective of the three-dimensional structure. Under the technical concept disclosed in this application, the detection method of the specific binding reaction includes but is not limited to: double antibody sandwich method, competition method, neutralization competition method, indirect method or capture method.

[0042] The term "antibody affinity" as used herein refers to the binding strength of the antigen-antibody in the entire system. Antibody affinity gradually increases with the increase in the time and frequency of contact with the antigen. High-affinity antibodies can bind more antigens than low-affinity antibodies and form more stable immune complexes that are less likely to dissociate.

[0043] II. Specific implementation plan

[0044] The present application will be described in more detail below with reference to embodiments.

[0045] In one embodiment, the antibody affinity detection kit of the present application includes reagent 1, reagent 2, a dispersant and a control solution. Reagent 1 includes luminescent particles coated with antigens, and the interior of the luminescent particles contains optically reactive substances; reagent 2 includes biotinylated anti-human IgG antibodies, and the control solution is a matrix solution of the dispersant.

[0046] Among them, in order to specifically test the affinity of specific IgG antibodies of a certain pathogen, the antigen in reagent 1 can be selected accordingly. For example, in order to test the affinity of cytomegalovirus IgG antibodies, the antigen in reagent 1 can be a recombinant cytomegalovirus antigen. For another example, in order to test the affinity of Toxoplasma IgG antibodies, the antigen in reagent 1 can be a recombinant Toxoplasma antigen. For another example, the antigen can also be an antigen corresponding to human immunodeficiency virus, Epstein-Barr virus, rubella virus, herpes simplex virus, measles virus, syphilis spirochete, etc., and so on, which will not be repeated here.

[0047] In some embodiments, the luminescent particles in reagent 1 are composed of a carrier encapsulating an optically reactive substance, and the antibody is coated outside the carrier. In some embodiments, the material of the carrier is selected from natural, synthetic or modified naturally occurring polymers; preferably, it is a synthetic polymer.

[0048] In some embodiments, the material of the carrier can be selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate; preferably selected from polystyrene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate or polyacrylate.

[0049] In some embodiments, the optically reactive substance comprises a chemiluminescent compound and a metal chelate.

[0050] In some embodiments, the chemiluminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, dibutyl diacetyl compounds, dioxines, enol ethers, enamines, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether olefins, aryl imidazoles and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives.

[0051] In some embodiments, the metal of the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium and ruthenium, more preferably europium.

[0052] In some embodiments, the metal chelate comprises a chelating agent selected from the group consisting of NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2,2-dimethyl-4-perfluorobutyryl-3-butanone, 2,2'-bipyridine, bipyridyl carboxylic acid, azacrown ethers, azacryptands, and trioctylphosphine oxide, and derivatives thereof.

[0053] In some embodiments, reagent 1 also includes a buffer for diluting the luminescent particles, and the pH value of the buffer is preferably 6 to 8. The buffer selected for reagent 1 can be, for example, HEPES buffer, tris (Tris), sodium phosphate (PBS), 3-morpholinepropanesulfonic acid (MOPS), morpholineethanesulfonic acid (MES) and other common buffers. Through buffer dilution, the concentration of the luminescent particles coated with the antigen in reagent 1 is 6.25 mg / L to 25 mg / L, for example, 6.25 mg / L, 8 mg / L, 10 mg / L, 12.5 mg / L, 15.25 mg / L, 23 mg / L, 25 mg / L, etc. Based on the differences between raw material batches, the concentration between different batches is allowed to fluctuate within a certain range.

[0054] Reagent 2 includes biotinylated anti-human IgG antibodies for specific binding with the avidinized photosensitive particles in the universal solution, i.e., reagent 3, during detection. In some embodiments, reagent 2 also includes a buffer for diluting the biotinylated anti-human IgG antibodies, and the pH value of the buffer is preferably 6 to 8. The buffer selected for reagent 2 may be, for example, a common buffer such as HEPES buffer and Tris-HCl buffer. For detection items of different types of specific IgG antibodies, the buffer selected in reagent 2 may be the same as or different from the buffer selected for reagent 1. Preferably, the concentration of the biotinylated anti-human IgG antibody of reagent 2 is 1.25 mg / L to 5 mg / L, for example, 1.25 mg / L, 2.5 mg / L, 3.5 mg / L, 4 mg / L, 5 mg / L, etc. Based on the differences between batches of raw materials, the concentrations between different batches are allowed to fluctuate within a certain range.

[0055] The dispersant is used to disrupt the hydrogen bond network between water molecules, weaken the hydrophobic effect, etc. to interfere with the non-covalent binding of the antigen and the specific IgG antibody in the sample to be tested. The dispersant of the present application contains urea, potassium thiocyanate, guanidine hydrochloride or citric acid, etc. It can be understood that different dispersants are used at different concentrations according to the respective characteristics of different types of dispersants. The same dispersant has different reaction times with the sample when used at different concentrations. Based on this, the components and corresponding concentrations of the dispersant can be freely selected and deployed according to the acceptable detection cost and detection time, without limitation. For example, taking the reaction time of the sample to be tested and the dispersant as 20 minutes as an example, when the dispersant is urea, the concentration of urea used is 6 mol / L~8 mol / L; if the dispersant used is guanidine hydrochloride, the concentration used needs to be halved or even reduced more.

[0056] Furthermore, the control solution is a matrix solution of the dispersant, that is, the dispersant can be diluted to a desired concentration using the control solution. By using the same matrix solution as the control solution for the dispersant, a more accurate control effect is achieved when the sample to be tested is tested, and interference of other different substances on the test results is reduced. In some embodiments, the matrix solution is selected from physiological saline or a buffer solution; preferably, the matrix solution is a single component solution; by selecting a single component, interference of other substances on the test results is reduced.

[0057] In some embodiments, in order to facilitate users to complete the test more efficiently, the kit of the present application may also include a negative control and a positive control; preferably, the positive control includes a low-affinity positive control and a high-affinity positive control. By adding a negative control and two positive controls with different affinities to the kit, it is convenient for users to obtain test results more accurately.

[0058] It can be understood that the negative control is a reagent that does not contain the specific IgG antibody in the sample to be tested; the low-affinity positive control is a low-affinity reagent that contains the specific low-affinity IgG antibody of the sample to be tested; and the high-affinity positive control is a high-affinity reagent that contains the specific high-affinity IgG antibody of the sample to be tested. Among them, the specific IgG antibody in each positive control is selected according to the type of IgG antibody in the sample to be tested actually detected.

[0059] The following will further illustrate the preparation method of the antibody affinity detection kit of the present application, which includes:

[0060] S110, after coating the pathogen antigen with luminescent microparticles, dilute with a first buffer to obtain reagent 1.

[0061] The concentration of the luminescent particles coated with pathogen antigens in reagent 1 may be 6.25 mg / L to 25 mg / L. The pH value of the first buffer is preferably 6 to 8. The first buffer may be a universal buffer system suitable for antigen-antibody reactions.

[0062] S120, after labeling the anti-human IgG antibody with biotin, dilute it with the second buffer to obtain reagent 2.

[0063] The concentration of the biotin-labeled anti-human IgG antibody in reagent 2 may be 1.25 mg / L to 5 mg / L. The pH value of the second buffer is preferably 6 to 8. The second buffer may be the same as or different from the first buffer.

[0064] S130, respectively prepare the control solution, negative quality control product, low affinity positive quality control product and high affinity positive quality control product. The specific preparation method of the control solution and each quality control product is as follows:

[0065] In some embodiments, a control solution is configured to obtain a matrix solution for diluting a discrete agent. The control solution can be any buffer system suitable for antigen-antibody reaction. For example, the control solution can be physiological saline, or a PBS buffer with a pH of 7.2 and a concentration of 0.02 mol / L, or a HEPES buffer with a pH of 8 and a concentration of 0.02 mol / L, which are only exemplified here and are not limited.

[0066] In some embodiments, a negative control is obtained by configuration, and the negative control is a solution that does not contain specific IgG antibodies. For example, the negative control can be serum that does not contain specific IgG antibodies, which is only an example and not a limitation.

[0067] In some embodiments, a low-affinity positive control product is configured to obtain a solution containing specific low-affinity IgG antibodies. For example, the low-affinity positive control product can be a serum containing specific low-affinity IgG antibodies, which is only exemplified and not limited.

[0068] In some embodiments, a high-affinity positive control product is configured to obtain a solution containing specific high-affinity IgG antibodies; for example, the high-affinity positive control product can be a serum containing specific high-affinity IgG antibodies, which is only used as an example and is not intended to be limiting.

[0069] S140, dispersing agent is prepared.

[0070] The dispersant can be a solution containing urea, potassium thiocyanate, guanidine hydrochloride or citric acid. According to the selected main component of the dispersant, the prepared control solution is used to dilute to the required concentration.

[0071] After the above reagents are prepared respectively, the components can be packaged separately so that each kit includes the corresponding components.

[0072] As can be seen from the above, the preparation method of the kit of the present application is simple, the materials are easily available, the cost is low, and it is easy to promote.

[0073] The following will further introduce the antibody affinity detection method of the present application, and the sample to be tested is detected according to the above-mentioned antibody affinity detection kit.

[0074] In some embodiments, the method for detecting antibody affinity includes: simultaneously detecting an equal amount of a sample to be tested that has been treated with an equal amount of a control solution or a discrete agent, and confirming the affinity strength of the antibody in the sample to be tested based on the ratio of the detection signal values.

[0075] For example, one of the samples to be tested can be mixed with a discrete agent in an equal amount in a discrete agent well, and another sample to be tested can be mixed with a control solution in a control well, with the discrete agent and the control solution being used in the same amount. According to the principle of homogeneous photochemiluminescence detection, the corresponding detection signal values ​​are obtained by laser irradiation and the ratio is calculated, and the affinity strength of the antibody in the sample to be tested can be confirmed according to the ratio.

[0076] In some embodiments, the following specifically describes a method for detecting antibody affinity, including:

[0077] S210, adding the sample to be tested, reagent 1 and the discrete agent into the discrete agent wells for mixing; and adding the sample to be tested, reagent 1 and the control solution in the same amount as that in the discrete agent wells into the control wells for mixing.

[0078] The detection method of the present application is a multiple-well detection, and multiple-well detection can be performed on a microplate commonly used on a homogeneous chemiluminescence analyzer. Among them, the microplate includes reaction wells, at least one of which is a discrete agent well, and at least another well is a control well. The difference between adding samples in the discrete agent well and the control well is that a discrete agent needs to be added to the discrete agent well, while a control solution is added to the control well.

[0079] It can be understood that the sample loading can be flexibly adjusted according to the actual capacity of the reaction well. For example, when the capacity of the reaction well is 300uL, the following reagent 3 can be used as a universal liquid to fix a uniform loading volume, such as 175uL, the loading volume of the sample to be tested can be 10uL~25uL, and the loading volume of reagent 1, reagent 2, discrete agent and control solution can be 25uL~50uL. The specific loading volume can be flexibly adjusted according to the target molecule to be tested in the sample to be tested. Among them, in order to ensure the accuracy of the test results, the loading volume of the discrete agent and the control solution in the discrete agent well and the control well is the same, the loading volume of the sample to be tested is the same, and the loading volume of reagent 1 is also the same.

[0080] S220, incubating the discrete agent wells and the control wells at a preset temperature for a preset time.

[0081] After the sample is added to the reaction well and mixed evenly, it can be placed in an incubation device for incubation. The preset temperature can be 30°C to 45°C, and the preset incubation time can be 5 minutes to 30 minutes, so that the sample to be tested can fully react with the mixed solution in the reaction well. It can be understood that the specific temperature and time can be flexibly adjusted according to the sample to be tested.

[0082] S230, adding equal amounts of reagent 2 and reagent 3 to the discrete reagent wells and the control wells, and continuing incubation for sufficient reaction; wherein reagent 3 contains photosensitive particles that can react with active oxygen to produce chemiluminescence.

[0083] After step S220 is completed, reagent 2 and reagent 3 are added to the discrete reagent wells, and the same amount of reagent 2 and reagent 3 as those in the discrete reagent wells are added to the control wells. After each is mixed evenly, it can be placed in the incubation device again for incubation. In this step, the incubation temperature can be 30°C to 45°C, and the preset incubation time can be 5 minutes to 30 minutes to allow for sufficient reaction.

[0084] Reagent 3 includes avidinized photosensitive particles and a third buffer, wherein the concentration of the avidinized photosensitive particles is 40 ug / mL to 50 ug / mL, for example, 50 ug / mL. Optionally, the third buffer may be the same as the first buffer.

[0085] S240, irradiating the discrete agent wells and the control wells with lasers, obtaining corresponding light signal values ​​and calculating the ratio.

[0086] The detection principle of the detection method of the present application is: if specific IgG antibodies are present in the sample to be tested, a complex of luminescent particles-antigen-specific IgG antibodies-anti-human IgG antibodies-biotin-avidin-photosensitive particles will be formed in the reaction well under the laser irradiation of the homogeneous chemiluminescence analyzer.

[0087] Specifically, the photosensitive particles and luminescent particles in the reaction well are drawn closer by the formed complex. Under the irradiation of laser (wavelength of 680nm), the sensitizer in the photosensitive particles converts the oxygen in the surrounding environment into more active singlet oxygen. The singlet oxygen diffuses to the luminescent particles and reacts with the chemiluminescent substances in the luminescent particles, further activating the luminescent groups on the luminescent particles, causing them to emit high-energy red light with a wavelength of 520nm to 620nm. The number of photons of the emitted light is converted into a relative light signal value through a single-photon counter and mathematical fitting, which is the detection signal value corresponding to the sample to be tested in the well. It can be understood that if there is no specific IgG antibody in the sample to be tested, the above-mentioned complex will not be formed, and then the light signal value will not be detected.

[0088] Based on the fact that the dispersant can interfere with the non-covalent binding of antigen and antibody, for different degrees of affinity, the immune complex formed by the specific IgG antibody with high affinity is less susceptible to the interference of the dispersant than the immune complex formed by the specific IgG antibody with low affinity, so the attenuation of the light signal value detected corresponding to the specific IgG antibody with high affinity is relatively less. Therefore, in the control well without the addition of the dispersant, the light signal value obtained will be higher than the light signal value of the dispersant well with the addition of the dispersant.

[0089] S250, when the light signal value of the control well is greater than the preset reference value, the specific IgG antibody affinity in the sample to be tested is determined according to the ratio of the light signal value of the discrete reagent well to the light signal value of the control well.

[0090] In some embodiments, when the light signal value in the control well is greater than the preset reference value, the affinity of the specific IgG antibody in the sample to be tested can be calculated, otherwise the specific IgG content in the sample to be tested is too low and no affinity calculation is required. In other words, if the light signal value measured in the control well is very small, it means that the sample to be tested does not contain specific IgG antibodies, and thus there is no need to perform subsequent steps. Among them, the preset reference value can be 1800 to 2500, for example 2000.

[0091] In order to facilitate more accurate judgment of whether the sample to be tested contains specific IgG antibodies, in some embodiments, the kit of the present application also includes a reference sample, which contains the same specific IgG antibodies as the sample to be tested, and the concentration of the specific IgG antibodies is the lowest concentration that can detect the light signal value in the homogeneous chemiluminescence analyzer. That is to say, at least one reference well can be added and designed in step S210, and after adding the reference sample, reagent 1 and the control solution to the reference well respectively and mixing them, the same subsequent steps as the discrete reagent well and the control well are performed. When the light signal value in the reference well is detected in this step, the light signal value of the reference well is used as the preset reference value of the control well.

[0092] In this step, the affinity of the specific IgG antibody in the sample to be tested can be determined based on the ratio of the light signal value of the discrete reagent well to the light signal value of the control well.

[0093] In some embodiments, the discrete reagent well signal value / control well signal value is an affinity index, and when the affinity index is less than 0.3, the affinity of the antibody in the test sample is low; when 0.3≤affinity index<0.5, the affinity of the antibody in the test sample is medium; when the affinity index is greater than or equal to 0.5, the affinity of the antibody in the test sample is high. As can be seen from the above, the detection using the kit of the present application is simple and convenient to operate, and the affinity detection result can be obtained quickly and accurately without complicated manual operations.

[0094] III. Examples

[0095] The following will take the affinity detection of two different antibodies as an example to further illustrate the kit of the present application and its preparation method and detection method.

[0096] Example 1: Preparation and detection of cytomegalovirus CMV IgG antibody affinity detection kit

[0097] 1) Preparation of Cytomegalovirus IgG Antibody Avidity Detection Kit

[0098] (1) Preparation of Reagent 1

[0099] Aldehyde luminescent microparticles and cytomegalovirus recombinant antigen were mixed in a mass ratio of 10:0.2 in a 0.05 mol / L CB buffer environment. After reacting at 37°C for 4 hours, sodium borohydride was added for reduction. After reacting at 2°C to 8°C for 1 hour, the microparticles were washed twice with the first buffer of reagent 1 (0.05 mol / L HEPES buffer with a pH of 8.0, containing protein stabilizers and preservatives), and the volume was fixed to a microparticle concentration of 10 mg / mL. The antigen-coated luminescent microparticles were diluted to 12.5 mg / L using the first buffer of reagent 1 to prepare reagent 1.

[0100] (2) Preparation of Reagent 2

[0101] The mouse anti-human IgG antibody was dialyzed in 0.1mol / L NaHCO3 solution at room temperature for 2h~4h, and the solution was changed 2 times or more. The dialyzed biotin was transferred to a centrifuge tube, and biotin dissolved in DMSO was added in a molar ratio of 1:30. After reacting at 2℃~8℃ for 2h, it was dialyzed with 0.02mol / L PBS buffer at 2℃~8℃ for at least 6h, and the solution was changed 4 times or more. The biotinylated mouse anti-human IgG was diluted to 2.5mg / L using the second buffer of reagent 2 (0.1mol / L Tris-HCl buffer with a pH of 8.0, containing protein stabilizers and preservatives) to prepare reagent 2.

[0102] (3) Preparation of control solution

[0103] Prepare 0.9% NaCl as the control solution.

[0104] (4) Preparation of dispersant

[0105] The 0.9% NaCl in step (3) was used to adjust the volume of urea to 6 mol / L to prepare a dispersant.

[0106] (5) Preparation of negative control products

[0107] CMV IgG negative serum was added with 0.05% Proclin to prepare a negative control.

[0108] (6) Preparation of positive quality control products

[0109] The CMV low-affinity IgG antibody was diluted a certain multiple with CMV IgG negative serum, and 0.05% Proclin was added to prepare the low-affinity positive quality control product 1.

[0110] CMV high-affinity IgG antibody was diluted a certain multiple with CMV IgG negative serum, and 0.05% Proclin was added to prepare high-affinity positive quality control product 2.

[0111] (7) Packaging the components and assembling them into a finished test kit.

[0112] 2) Detect the affinity of CMV IgG antibodies in the sample to be tested

[0113] (1) Repeat the test of the samples. This experiment designed 10 samples containing specific CMV IgG antibodies with different affinities, namely samples 1 to 10. Mix 25μL of the sample to be tested, 25μL of reagent 1 and 25μL of the discrete agent in the discrete agent well and react at 37℃ for 20 minutes; mix 25μL of the sample to be tested, 25μL of reagent 1 and 25μL of the control solution in the control well and react at 37℃ for 20 minutes. Similarly, perform repeat tests on the negative control, positive control 1 and positive control 2 in the kit.

[0114] (2) Add 25 μL of Reagent 2 and 175 μL of Reagent 3 to each discrete reagent well and control well, mix them separately, and react them at 37°C for 15 minutes to fully react.

[0115] (3) In the automatic photochemiluminescence detector Laser irradiation is performed at 500°, and the light signal values ​​of each discrete reagent well and the control well are read respectively. According to the ratio of the light signal values, i.e., the affinity index, it is confirmed that the CMV IgG antibody in the sample to be tested is a low-affinity antibody or a high-affinity antibody.

[0116] 3) Experimental data

[0117] Table 1

[0118]

[0119] 4) Explanation and analysis of experimental data

[0120] (1) The light signal value of the control well containing the negative control is less than 2000, indicating that the IgG content therein is insufficient to be detected and no subsequent affinity calculation is required.

[0121] (2) According to affinity index = discrete agent well signal value / control well signal value; when the affinity index is <0.3, the specific IgG antibody is a low-affinity IgG antibody; when the affinity index ∈ [0.3, 0.5), the specific IgG antibody is a medium-affinity IgG antibody; when the affinity index is ≥0.5, the specific IgG antibody is a high-affinity IgG antibody.

[0122] According to the above rules, it can be seen from the data in Table 1 that samples 3 and 9 are low-affinity CMV IgG antibodies; samples 1 and 8 are medium-affinity CMV IgG antibodies; and samples 2, 4, 5, 6, 7 and 10 are high-affinity CMV IgG antibodies.

[0123] 6. Experimental conclusion

[0124] The kit of the present application can accurately detect the affinity of CMV IgG antibodies.

[0125] Example 2 Preparation and detection of Toxoplasma gondii IgG antibody affinity detection kit

[0126] 1) Preparation of Toxoplasma gondii IgG antibody affinity detection kit

[0127] (1) Preparation of Reagent 1

[0128] Carboxyl luminescent microparticles and Toxoplasma gondii recombinant antigens were mixed at a mass ratio of 10:0.5 in a 0.02 mol / L PBS buffer at pH 7.2. After reacting at 37°C for 4 hours, 100 mg / mL BSA was added for blocking. After reacting at 37°C for 1 hour, the microparticles were washed twice with the first buffer of reagent 1 (0.02 mol / L PBS buffer at pH 7.2, containing protein stabilizers and preservatives), and the volume was adjusted to a microparticle concentration of 10 mg / mL. The luminescent microparticles coated with antigens were diluted to 12.5 mg / L using the first buffer of reagent 1 to prepare reagent 1.

[0129] (2) Preparation of Reagent 2

[0130] Dialyze the mouse anti-human IgG antibody in 0.1mol / L NaHCO3 solution at room temperature for 2h~4h, and change the solution 2 times or more. Transfer the dialyzed biotin to a centrifuge tube, add biotin dissolved in DMSO at a molar ratio of 1:30, react at 2℃~8℃ for 2h, and dialyze with 0.02mol / L PBS buffer at 2℃~8℃ for at least 6h, and change the solution 4 times or more. Use the second buffer of reagent 2 (0.02mol / L PBS buffer with a pH of 7.2, containing protein stabilizers and preservatives) to dilute the biotinylated mouse anti-human IgG to 2.5mg / L to prepare reagent 2.

[0131] (3) Preparation of control solution

[0132] Prepare 0.9% NaCl as the control solution.

[0133] (4) Preparation of discrete agents

[0134] The 0.9% NaCl in step (3) was used to adjust the volume of urea to 6 mol / L to prepare a dispersant.

[0135] (5) Preparation of negative control products

[0136] Toxoplasma IgG negative serum was added with 0.05% Proclin to prepare a negative control.

[0137] (6) Preparation of positive quality control products

[0138] The Toxoplasma gondii low-affinity IgG antibody was diluted a certain multiple with Toxoplasma gondii IgG negative serum, and 0.05% Proclin was added to prepare the low-affinity positive quality control product 1.

[0139] The Toxoplasma gondii high-affinity IgG antibody was diluted a certain multiple with Toxoplasma gondii IgG negative serum, and 0.05% Proclin was added to prepare the high-affinity positive quality control product 2.

[0140] (7) Packaging the components and assembling them into a finished test kit.

[0141] 2) Detection of the affinity of Toxoplasma IgG antibodies in the sample to be tested

[0142] (1) Repeat the test of the samples. This experiment designed 10 samples containing specific Toxoplasma IgG antibodies with different affinities, namely samples 1 to 10. Mix 25 μL of the sample to be tested, 25 μL of reagent 1 and 25 μL of the discrete agent in the discrete agent well and fully react at 37°C for 20 minutes; mix 25 μL of the sample to be tested, 25 μL of reagent 1 and 25 μL of the control solution in the control well and fully react at 37°C for 20 minutes. Similarly, repeat the negative control, positive control 1 and positive control 2 in the kit.

[0143] (2) Add 25 μL of Reagent 2 and 175 μL of Reagent 3 to each discrete reagent well and control well, mix them separately, and react them at 37°C for 15 minutes to fully react.

[0144] (3) In the automatic photochemiluminescence detector Laser irradiation is performed for 500 seconds, and the light signal values ​​of each discrete agent well and the control well are read respectively. According to the ratio of the light signal values, it is confirmed that the Toxoplasma IgG antibody in the sample to be tested is a low-affinity antibody or a high-affinity antibody.

[0145] 3) Experimental data

[0146] Table 2

[0147]

[0148] 4) Explanation and analysis of experimental data

[0149] (1) The signal value of the control well containing the negative control product is less than 2000, indicating that the IgG content to be tested is insufficient to be detected and no subsequent affinity index calculation is required.

[0150] (2) From the data in Table 2, it can be seen that sample 8 is a low-affinity Toxoplasma IgG antibody; samples 2 and 5 are medium-affinity Toxoplasma IgG antibodies; samples 1, 3, 4, 6, 7, 9 and 10 are high-affinity Toxoplasma IgG antibodies.

[0151] 5) Experimental conclusion

[0152] The kit of the present application can detect the affinity of Toxoplasma gondii IgG antibodies.

[0153] It can be seen from the above embodiments that the antibody affinity detection kit of the present application can be designed for different types of specific IgG antibodies to form corresponding affinity detection kits. The operation is simple and quick, and the IgG antibody affinity detection results in the sample to be tested can be obtained efficiently and accurately.

[0154] In addition to the IgG antibodies described in the above examples, the kit of the present application can also be used to detect the affinity of other specific IgG antibodies. For example, when pathogens such as human immunodeficiency virus, Epstein-Barr virus, rubella virus, herpes simplex virus, measles virus, syphilis spirochete, etc. are present, the present application can detect the affinity of the corresponding specific IgG antibodies.

[0155] The kit of the present application can also be used for autoantibody affinity detection for clinical auxiliary diagnosis of immune system diseases, such as affinity detection of anti-double-stranded DNA antibodies, which is helpful for the diagnosis of systemic lupus erythematosus; affinity detection of anti-thyroglobulin antibodies is helpful for the diagnosis of Hashimoto's thyroiditis.

[0156] The kit of the present application can also be used to detect the effectiveness of vaccines, such as evaluating the population immune effect of new coronavirus vaccines and hepatitis E virus vaccines, etc.

[0157] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present application may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. An antibody affinity detection kit, characterized in that: It includes reagent 1, reagent 2, discrete agent and control solution, wherein: The reagent 1 includes luminescent particles coated with antigens, and the interior of the luminescent particles contains optically reactive substances; The reagent 2 includes a biotinylated anti-human IgG antibody; The control solution is a matrix solution of the discrete agent.

2. The kit according to claim 1, characterized in that The dispersant contains urea, potassium thiocyanate, guanidine hydrochloride or citric acid.

3. The kit according to claim 1, characterized in that The matrix solution is selected from physiological saline or buffer solution; preferably, the matrix solution is a single component solution.

4. The kit according to claim 1, characterized in that It also includes negative quality control products and positive quality control products; preferably, the positive quality control products include low-affinity positive quality control products and high-affinity positive quality control products.

5. The kit according to claim 1, characterized in that: The concentration of the reagent 1 is 6.25 mg / L to 25 mg / L; and / or, The concentration of the reagent 2 is 1.25 mg / L to 5 mg / L.

6. A method for detecting antibody affinity, characterized in that: An equal amount of the sample to be tested treated with an equal amount of control solution or discrete agent is simultaneously tested, and the affinity strength of the antibody in the sample to be tested is confirmed based on the ratio of the detection signal values.

7. The detection method according to claim 6, characterized in that: The signal value of the discrete reagent well / the signal value of the control well is an affinity index. When the affinity index is less than 0.3, the affinity of the antibody in the sample to be tested is low; When the affinity index is 0.3≤0.5, the affinity of the antibody in the sample to be tested is medium; When the affinity index is ≥ 0.5, the affinity of the antibody in the sample to be tested is high.

8. The detection method according to claim 6 or 7, characterized in that: Includes steps: Add the sample to be tested, the reagent 1 and the discrete agent to the discrete agent wells respectively and mix them; and add the sample to be tested, the reagent 1 and the control solution in the same amount as that in the discrete agent wells respectively and mix them; Incubating the discrete agent wells and the control wells at a preset temperature for a preset period of time; Adding equal amounts of the reagent 2 and the reagent 3 to the discrete reagent wells and the control wells, and continuing to incubate for a sufficient reaction; wherein the reagent 3 contains photosensitive particles that can react with active oxygen to produce chemiluminescence; The discrete agent wells and the control wells are irradiated with laser to obtain corresponding light signal values ​​and calculate the ratio.

9. A homogeneous chemiluminescence analyzer, which is loaded with the kit according to any one of claims 1 to 5 and / or performs the detection method according to any one of claims 6 to 8 to detect a sample to be tested, so as to analyze the affinity of specific IgG antibodies in the sample to be tested.

10. Use of the kit according to any one of claims 1 to 5, or the detection method according to any one of claims 6 to 8 in evaluating the immune effect of a vaccine.

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