Antibody screening method and application thereof

Abstract

The invention provides an antibody screening method and application thereof, and relates to the technical field of biology. The method comprises the following steps: respectively reacting to-be-screened antibodies with a series of antigen reagents with different antigen concentrations to obtain a signal-to-noise ratio of the reaction of each antibody and the antigen reagent with each concentration; the signal-to-noise ratios corresponding to the same antigen concentration are compared, and the antibody with the larger signal-to-noise ratio is better. The method for screening the antibody overcomes the defect that the affinity of the antibody is inaccurately evaluated in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for screening antibodies and an application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Antibody affinity is the non-covalent binding force between the antibody and the antigen epitope. Its strength depends on the degree of fit between the antibody variable region and the antigen epitope. It is an important parameter for measuring antibody quality and reflects the binding efficiency of the antibody with the corresponding antigen. The higher the affinity, the higher the degree of binding between the antibody and the corresponding antigen, which has great guiding value in the application of antibodies. Antibodies are the core raw materials of in vitro diagnostic reagents, and their affinity directly determines the lower limit of the sensitivity of the reagents. Accurately and efficiently screening out high-affinity antibodies is the key means to solve the problem of reagent sensitivity.

[0004] In the traditional antibody screening process, the method of evaluating or comparing affinity is generally to coat an excess of antigen on a solid phase, and when the antigen concentration remains unchanged, the antibody to be evaluated is gradiently diluted, and then reacted with the antigen separately. The inflection point concentration when the antibody reacts with the antigen to the bottom plateau is set as the binding rate of 100%, and the reciprocal value of the antibody concentration corresponding to the binding rate of 50% is the affinity. On the one hand, different antibodies have different plateau concentrations, and it is often necessary to touch different antibody dilutions due to inappropriate concentrations, which increases the cumbersomeness of the operation steps. On the other hand, in the actual application scenarios of antibodies as in vitro diagnostic reagents, it is usually the case that the antibody is excessively coated on the solid phase or coupled with the signal substance, and the concentration of the antigen to be tested changes in a gradient. Therefore, the affinity screened and evaluated by this method is not accurate enough, especially when the affinity levels of the antibodies are close. The traditional method may be due to accuracy reasons, and sometimes there is a deviation from the actual application.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a method for screening antibodies to improve the defect of inaccurate antibody affinity assessment in existing screening methods.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] It should be noted that:

[0009] In the present invention, unless otherwise specified, all implementation methods and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution; all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0010] Herein, unless otherwise stated, various reactions or operation steps may or may not be performed in sequence.

[0011] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual such relationship, order, or importance between such entities or actions, such as numbers (i), (ii), (iii), and "first" and "second", unless otherwise stated.

[0012] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0013] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0014] As used herein, "and / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0015] In a first aspect, a method for screening antibodies is provided, the method comprising reacting the antibodies with a series of antigen reagents having different antigen concentrations, respectively, to obtain a signal-to-noise ratio of the reaction between each antibody and each concentration of the antigen reagent:

[0016] The signal-to-noise ratio of each antibody when it first meets the condition greater than or equal to 2 is the minimum signal-to-noise ratio, and the antigen concentration of the antigen reagent in the reaction corresponding to the minimum signal-to-noise ratio is C min , C min The smaller it is, the better the antibody is. When comparing the signal-to-noise ratio corresponding to the same antigen concentration, the antibody with a larger signal-to-noise ratio is better.

[0017] The signal-to-noise ratio is the ratio of the detection value obtained after the antibody reacts with the antigen reagent to the detection value when the antigen concentration in the antigen reagent is 0.

[0018] In an optional embodiment, when the antibody signal-to-noise ratio satisfies a value greater than or equal to 2, the corresponding minimum antigen concentration is the C min , C min The smaller the antibody, the better.

[0019] In an optional embodiment, the C min When the signal-to-noise ratio is the same, the antibody with a higher signal-to-noise ratio is better.

[0020] In an optional embodiment, the C min When the two antibodies are the same, compare the min When the ratio of larger signal-to-noise ratio to smaller signal-to-noise ratio is greater than or equal to 2, the antibody corresponding to the larger signal-to-noise ratio is better; otherwise, the two antibodies are at the same level.

[0021] In an optional embodiment, the series of antigen reagents with different antigen concentrations are different antigen reagents obtained by gradient dilution of the antigen.

[0022] In an optional embodiment, the concentration of each antigen in the series of antigen reagents with different antigen concentrations is distributed in the range of 1 to 10000 pg / mL.

[0023] In an optional embodiment, the concentration of each antigen in the series of antigen reagents with different antigen concentrations is distributed as 1 pg / mL, 10 pg / mL, 100 pg / mL, 1000 pg / mL and 10000 pg / mL.

[0024] In an optional embodiment, the reacting of the antibody with a series of antigen reagents having different antigen concentrations includes reacting the antibody coated on a solid phase carrier with the antigen reagent, and obtaining the detection value by detecting the signal substance connected to the antigen.

[0025] The signal substance refers to a substance that can provide a detectable signal. Examples of signal substances include, but are not limited to, enzymes, luminescent labels, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, quantum dots, biotin, streptavidin, radionuclides, radiocontrast agents, paramagnetic ions, metals, or photosensitizers.

[0026] In an optional embodiment, the detection value includes fluorescence intensity, luminescence intensity, color depth or radioactivity intensity.

[0027] In an optional embodiment, the solid phase carrier includes: microparticles, microporous plates or carrier membranes.

[0028] In an optional embodiment, the antigen and the signal are connected by a specific binding pair. A specific binding pair refers to two substances having a specific binding relationship, which can be immune or non-immune types. In an optional embodiment, the specific binding pair is selected from an antibody-antigen binding pair, a biotin-avidin binding pair, a biotin-streptavidin binding pair, an enzyme-cofactor binding pair or a receptor-ligand binding pair, preferably a biotin-streptavidin binding pair.

[0029] In an alternative embodiment, the specific binding pair is a biotin-streptavidin binding pair, the antigen is conjugated with biotin, and the signal substance is conjugated with streptavidin. Further optionally, the solid-phase carrier is selected from tosyl magnetic particles, carboxyl magnetic particles or enzyme-linked immunosorbent assay (ELISA) plates. Further optionally, the signal substance includes alkaline phosphatase.

[0030] In an alternative embodiment, the reacting the antibody with a series of antigen reagents having different antigen concentrations respectively includes:

[0031] (a) Coating the antibody to be evaluated on a solid-phase carrier.

[0032] (b) Conjugating the antigen with biotin, and then serially diluting the biotin-conjugated antigen to obtain a series of antigen reagents having different antigen concentrations; optionally, the molar ratio of the antigen to biotin is 1:10 to 1:100.

[0033] (c) Reacting the antibody coated on the solid-phase carrier in step (a) with each of the antigen reagents in step (b).

[0034] (d) Adding a signal substance conjugated with streptavidin to the reaction system in step (c) to react with the antigen-antibody complex in the reaction system, and obtaining a signal value.

[0035] In an alternative embodiment, the method for screening an antibody according to the first aspect is for non-diagnostic and non-therapeutic purposes.

[0036] In a second aspect, there is provided an application of the method for screening an antibody according to the first aspect in the preparation of an in vitro diagnostic kit based on immunoassay technology.

[0037] In an alternative embodiment, the method for screening an antibody is applied to screening an antibody for immunoassay.

[0038] In an alternative embodiment, the immunoassay is a sandwich immunoassay.

[0039] In an alternative embodiment, the screened antibody is used as a capture antibody.

[0040] In an alternative embodiment, the screened antibody includes an anti-Tau antibody (anti-microtubule-associated protein Tau antibody).

[0041] In an alternative embodiment, the screened antibody includes a pTau-181 antibody (a specific antibody against pTau-181 formed after phosphorylation of Tau protein at threonine 181 site) or a pTau-217 antibody (a specific antibody against pTau-217 formed after phosphorylation of Tau protein at threonine 217 site).

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention provides a simple, efficient and accurate method for screening antibodies. The logic and principle of screening are closer to the application scenarios of diagnostic reagents, improve the accuracy of antibody affinity assessment, reduce the workload and cost of later reagent screening of antibodies, and reduce the probability of wrong screening and missed screening. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] pTau-181 antibody affinity assessment and validation

[0046] Example 1-1 Using Tosyl Magnetic Beads as Solid Phase

[0047] (1) The pTau-181 antibody to be evaluated is coated onto Tosyl magnetic beads:

[0048] The five antibodies to be evaluated were added to the reaction buffer (0.1M HEPES, pH 8.0) and the reaction accelerator (1M ammonium sulfate) and incubated in a 37°C incubator for more than 6 hours. After the supernatant was removed by magnetic suction, the blocking agent CE510 (JSR) was added and incubated in a 37°C incubator for more than 6 hours. After the reaction was completed, the mixture was washed twice with TBS-T Buffer (25mM Tris, 140mM NaCl, 0.1% Tween 20, pH 7.4) to obtain Tosyl magnetic beads coated with pTau-181 antibody. The Tosyl magnetic beads coated with pTau-181 antibody were diluted to 0.2mg / mL with TBS-T Buffer to obtain pTau-181 antibody Tosyl magnetic beads working solution.

[0049] (2) Biotinylated pTau-181 antigen and diluted in series:

[0050] The pTau-181 antigen and biotin were mixed thoroughly in a 0.1M PBS buffer (pH 7.4) at a molar ratio of 1:50, placed in a dark place, and reacted at room temperature for 30 minutes. After the reaction was completed, ultrafiltration was repeated 5 times with a preservation solution (0.1M PBS, 0.05% PC300) and a 50kDa ultrafiltration tube to obtain the supernatant, which was the biotin-labeled pTau-181 antigen. The concentration of the biotin-labeled pTau-181 antigen was determined using a micro-protein spectrophotometer. The biotin-labeled pTau-181 antigen was diluted to 0pg / mL (sample diluent), 1pg / mL, 10pg / mL, 100pg / mL, 1000pg / mL, and 10000pg / mL with a sample diluent (produced by Zhuhai Lizhu Reagent Co., Ltd.).

[0051] (3) Reaction test between the antibody to be evaluated and the biotin-labeled antigen:

[0052] Alkaline phosphatase-labeled streptavidin was diluted to 0.2 μg / mL with 50 mM MOPS buffer (containing 1% BSA, pH 7.4).

[0053] The luminescence was measured by an automatic immunoassay analyzer (produced by Zhuhai Lizhu Reagent Co., Ltd., model: LiCreate ML5000Pro A): 50 μL of the prepared pTau-181 antibody magnetic bead working solution and 50 μL of the gradient diluted biotin-labeled pTau-181 antigen were added to the reaction cup, vortexed and reacted at 37°C for 10 minutes. Then, magnetic separation was performed and washed with a cleaning solution (produced by Zhuhai Lizhu Reagent Co., Ltd.). After washing, 50 μL of diluted 0.2 μg / mL alkaline phosphatase-labeled streptavidin was added, vortexed and reacted at 37°C for 10 minutes. Then, magnetic separation was performed and washed with a cleaning solution. After washing, 200 μL of the substrate solution for the automatic immunoassay system (produced by Zhuhai Lizhu Reagent Co., Ltd.) was added to the reaction cup, vortexed and reacted at 37°C for 5 minutes, and the luminescence was measured.

[0054] (4) Calculation and result interpretation:

[0055] The luminescence value of the biotin-labeled antigen at each dilution is the average of the luminescence values ​​measured twice. The ratio of the luminescence value of each strain of the antibody to be evaluated and the antigen at each concentration (i.e., S / N ratio, signal-to-noise ratio) is obtained. The lowest antigen concentration corresponding to the signal-to-noise ratio ≥ 2 is C min , and compared the signal-to-noise ratio of the same antigen concentration and different antibodies. The results are shown in Tables 1 and 2 (C minThe lower the concentration, the better the antibody, and the greater the signal-to-noise ratio at the same antigen concentration, the better the antibody. It is generally believed that at the same concentration, a signal-to-noise ratio greater than or equal to 2 times is considered to be different, and a signal-to-noise ratio less than 2 times is considered to be at the same level. ):

[0056] Table 1 Luminescence values ​​of the reaction between the pTau-181 antibody and the biotin-labeled antigen at various dilutions in Example 1-1

[0057]

[0058] Table 2 Signal-to-noise ratio of the reaction between the pTau-181 antibody and the biotin-labeled antigen at various dilutions in Example 1-1

[0059]

[0060] From the above results, we can see that the antigen concentration corresponding to the signal-to-noise ratio ≥ 2 is the lowest for pTau-181 antibody-2, C min 1pg / mL; followed by pTau-181 antibody-1, pTau-181 antibody-4, pTau-181 antibody-5, C min The signal-to-noise ratio of pTau-181 antibody-4 is about 2 times higher than that of pTau-181 antibody-5 and pTau-181 antibody-1. Finally, pTau-181 antibody-3, C min Therefore, it is inferred that the antibodies are better as follows: pTau-181 antibody-2>pTau-181 antibody-4>pTau-181 antibody-1≈pTau-181 antibody-5>pTau-181 antibody-3.

[0061] Example 1-2 Using carboxyl magnetic beads as solid phase

[0062] (1) The pTau-181 antibody to be evaluated is coated onto carboxyl magnetic beads

[0063] Add ethyl (dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the carboxyl magnetic beads, and incubate at room temperature for 30 minutes. After washing and resuspending with MES buffer (0.1M, pH5.0), add the antibodies to be evaluated, vortex mix, and rotate and incubate at room temperature for 2 hours. After the reaction is completed, add the blocking agent CE510 (JSR) and incubate at room temperature for 3 hours. After the reaction is completed, wash twice with TBS-T Buffer (25mM Tris, 140mMNaCl, 0.1% Tween 20, pH7.4) to obtain carboxyl magnetic beads coated with pTau-181 antibodies. Dilute the carboxyl magnetic beads coated with pTau-181 antibodies with TBS-TBuffer to 0.2mg / mL to obtain pTau-181 antibody carboxyl magnetic beads working solution.

[0064] Steps (2) to (4) are the same as steps (2) to (4) of Example 1-1. The results are shown in Tables 3 and 4:

[0065] Table 3 Luminescence values ​​of the reaction between pTau-181 antibody and biotin-labeled antigen at various dilutions in Example 1-2

[0066]

[0067] Table 4 Signal-to-noise ratio of the reaction between pTau-181 antibody and biotin-labeled antigen at various dilutions in Example 1-2

[0068]

[0069] From the above results, we can see that the lowest antigen corresponding to the signal-to-noise ratio ≥ 2 is pTau-181 antibody-2, with a concentration of 1 pg / mL; followed by pTau-181 antibody-1, pTau-181 antibody-4 and pTau-181 antibody-5, and the minimum antigen concentration C min It is 10pg / mL; it can be further seen that when the antigen concentration is 10pg / mL, the signal-to-noise ratio of pTau-181 antibody-4 is 2 times higher than that of pTau-181 antibody-5 and pTau-181 antibody-1; finally, pTau-181 antibody-3, C min It is 1000 pg / mL. In summary, the antibodies are preferably pTau-181 antibody-2>pTau-181 antibody-4>pTau-181 antibody-1≈pTau-181 antibody-5>pTau-181 antibody-3. The conclusion is consistent with the method of Example 1-1.

[0070] Example 1-3 Using ELISA plate as solid phase

[0071] (1) The pTau-181 antibody to be evaluated is coated onto the luminescent plate:

[0072] Use coating buffer (pH 9.6, Na 2 CO 3 1.59 mg / mL, NaHCO 3 2.93mg / mL) dilute p-Tau181 antibody to 2μg / mL. Add the diluted antibody to a 96-well ELISA plate, 100μL per well, and incubate at 37℃ for 2h. Wash the plate 3 times with PBST buffer, 60s each time, and pat the plate dry. Add 200μL / well of blocking solution, incubate at 37℃ for 2h, and pat dry after washing the plate.

[0073] (2) pTau-181 antigen was labeled with biotin and diluted in a gradient manner, as in step (2) of Example 1-1.

[0074] (3) Reaction test between the antibody to be evaluated and the biotin-labeled antigen

[0075] Add the diluted antigen to the coated ELISA plate, 100 μL per well, incubate at 37±2℃ for 60±3min, wash the plate and pat dry. Add the prepared alkaline phosphatase-labeled streptavidin, 100 μL per well, incubate at 37℃ for 1-2h, wash the plate and pat dry. Add 200 μL of substrate solution for the fully automatic immunoassay system, cover with a sealing film, and incubate at 37℃ for 15±3min; the luminescence is measured with a multifunctional ELISA instrument to obtain the count value.

[0076] (4) Calculation and affinity determination, same as step (4) of Example 1-1, the results are shown in Tables 5 and 6:

[0077] Table 5 Luminescence values ​​of the reaction between pTau-181 antibody and biotin-labeled antigen at various dilutions in Example 1-3

[0078]

[0079] Table 6 Signal-to-noise ratio of the reaction between pTau-181 antibody and biotin-labeled antigen at various dilutions in Example 1-3

[0080]

[0081] From the above results, we can see that the antigen concentration corresponding to the signal-to-noise ratio ≥ 2 is the lowest for pTau-181 antibody-2, C min 1pg / mL; followed by pTau-181 antibody-1, pTau-181 antibody-4, pTau-181 antibody-5, C min The signal-to-noise ratio of pTau-181 antibody-4 is about 2 times higher than that of pTau-181 antibody-5 and pTau-181 antibody-1. Finally, pTau-181 antibody-3, C min is 1000 pg / mL. Therefore, it is inferred that the better antibodies are pTau-181 antibody-2>pTau-181 antibody-4>pTau-181 antibody-1≈pTau-181 antibody-5>pTau-181 antibody-3. The conclusion is consistent with the methods of Examples 1-2 and 1-3.

[0082] Comparative Example 1-1 Comparison of Antibody Affinity by Traditional Methods

[0083] (1) pTau-181 antigen coated ELISA plate

[0084] Use coating buffer (pH 9.6, Na 2 CO 3 1.59 mg / mL, NaHCO 32.93mg / mL) p-Tau181 antigen was diluted to 2μg / mL. The diluted antigen was added to a 96-well ELISA plate, 100μL per well, and incubated at 37℃ for 2h. The plate was washed 3 times with PBST buffer, 60s each time, and the plate was patted dry. Blocking solution was added at 200μL / well, incubated at 37℃ for 2h, and the plate was patted dry after washing.

[0085] (2) Antibody and antigen reaction tests to be evaluated

[0086] Use PBS buffer to dilute the 5 p-Tau181 antibodies to be evaluated in a gradient manner so that the test results show a platform inflection point. If the antibody concentration is not within the dilution range of the platform inflection point, change to a suitable dilution and dilute again. Add the diluted antibody to the coated ELISA plate, 100 μL per well, incubate at 37±2℃ for 60±3min, wash the plate and pat dry.

[0087] Add HRP-labeled goat anti-mouse IgG, 100 μL per well, incubate at 37°C for 1 to 2 h, wash the plate and pat dry.

[0088] Dilute the substrate buffer and substrate solution TMB in a 1:1 ratio, mix thoroughly, add 100 μL to each well, and react at room temperature for 5 to 10 minutes. Add 50 μL of stop solution to each well, mix well, and read the OD450nm value of each well using an ELISA reader.

[0089] (3) Calculation and affinity interpretation

[0090] The OD450nm value of the antibody at each dilution is the average of the luminescence values ​​measured twice. The concentration obtained by dividing the concentration value of the antibody at the platform inflection point by 2 is used as the antibody concentration corresponding to a binding rate of 50%. The reciprocal of the antibody concentration corresponding to a binding rate of 50% is taken as the affinity index. The larger the value, the greater the affinity. The results are shown in Table 7:

[0091] Table 7 Comparative Example 1-1pTau-181 Antibody Affinity Evaluation

[0092]

[0093]

[0094] From the above results, it can be seen that the reciprocal of the antibody concentration corresponding to a binding rate of 50% is pTau-181 antibody-2=pTau-181 antibody-4>pTau-181 antibody-1=pTau-181 antibody-5=pTau-181 antibody-3, and this result is the corresponding antibody affinity.

[0095] Verification Example 1-1

[0096] The pTau-181 antibody to be evaluated was coated on magnetic beads and matched with the enzyme label of the same strain of Tau antibody to prepare a chemiluminescent detection reagent. Five clinical samples with gradient dilutions were tested and the signal-to-noise ratio was calculated to determine the sensitivity.

[0097] (1) Sample preparation: The pTau-181 high-value sample was diluted with the low-value sample to form 5 gradients, and the sample dilution solution was used as the 0 pg / mL sample.

[0098] (2) Immune reaction: Use a fully automatic immunoassay analyzer (produced by Zhuhai Lizhu Reagent Co., Ltd., model: LiCreate ML 5000ProA), add 50 μL of the pTau-181 antibody magnetic bead working solution prepared in Example 1-1 and 50 μL of the sample prepared in step (1) of this verification example to the reaction cup, respectively, and react at 37°C for 10 minutes after vortexing. Then, perform magnetic separation and wash with a cleaning solution (produced by Zhuhai Lizhu Reagent Co., Ltd.). After washing, add a diluted 1 μg / mL alkaline phosphatase-labeled Tau antibody (Zhuhai Lihe Medical Diagnostic Products Co., Ltd.), vortex and react at 37°C for 10 minutes. Then, perform magnetic separation and wash with a cleaning solution. After washing, add 200 μL of the substrate solution for the fully automatic immunoassay system (produced by Zhuhai Lizhu Reagent Co., Ltd.) to the reaction cup, vortex and react at 37°C for 5 minutes. Determine the luminescence amount.

[0099] 3) Calculation and sensitivity interpretation: The luminescence value of each sample is the average of the luminescence values ​​measured twice. Calculate the ratio of the luminescence value of each concentration to 0pg / mL (S / N ratio, signal-to-noise ratio). Compare the lowest concentrations of different antibodies with a signal-to-noise ratio ≥ 2, and measure the signal-to-noise ratio of the same concentration. The lower the concentration, the greater the signal-to-noise ratio, indicating that the sensitivity of the reagent is higher (a signal-to-noise ratio greater than or equal to 2 times is considered to be different, and a signal-to-noise ratio less than 2 times is considered to be at the same level). The results are shown in Tables 8 and 9:

[0100] Table 8 Luminescence values ​​of pTau-181 reagent reacting with various clinical samples

[0101]

[0102]

[0103] Table 9 Signal-to-noise ratio of pTau-181 reagent and clinical samples

[0104]

[0105] From the above results, it can be seen that the sensitivity levels of reagents prepared with different antibodies are as follows: pTau-181 antibody-2>pTau-181 antibody-4>pTau-181 antibody-1≈pTau-181 antibody-5>pTau-181 antibody-3. This sensitivity level is consistent with the antibody affinity results obtained by the methods of Examples 1-1 to 3. If the traditional method is used for evaluation, it is impossible to distinguish the affinity of the two antibodies pTau-181 antibody-2 and pTau-181 antibody-4, and it is impossible to distinguish the affinity of the three antibodies pTau-181 antibody-5, pTau-181 antibody-1 and pTau-181 antibody-3. It can be seen that the methods of Examples 1-1 to 3 can more accurately distinguish the size of antibody affinity than the traditional method, provide more accurate information for subsequent reagent development, and subsequently reduce the workload of screening antibodies during reagent development.

[0106] pTau-217 antibody affinity assessment and validation

[0107] Example 2-1 Using Tosyl Magnetic Beads as Solid Phase

[0108] (1) The pTau-217 antibody to be evaluated is coated onto Tosyl magnetic beads:

[0109] The five antibodies to be evaluated were added to the reaction buffer (0.1M HEPES, pH 8.0) and the reaction accelerator (1M ammonium sulfate) and incubated in a 37°C incubator for more than 6 hours. After the supernatant was removed by magnetic suction, the blocking agent CE510 (JSR) was added and incubated in a 37°C incubator for more than 6 hours. After the reaction was completed, the mixture was washed twice with TBS-T Buffer (25mM Tris, 140mM NaCl, 0.1% Tween 20, pH 7.4) to obtain Tosyl magnetic beads coated with pTau-181 antibody. The Tosyl magnetic beads coated with pTau-217 antibody were diluted to 0.2mg / mL with TBS-T Buffer to obtain the pTau-217 antibody Tosyl magnetic bead working solution.

[0110] (2) Biotinylated pTau-217 antigen and diluted in series:

[0111] The pTau-217 antigen and biotin were mixed thoroughly in a 0.1M PBS buffer (pH 7.4) at a molar ratio of 1:50, placed in a dark place, and reacted at room temperature for 30 minutes. After the reaction was completed, ultrafiltration was repeated 5 times with a preservation solution (0.1M PBS, 0.05% PC300) and a 50kDa ultrafiltration tube to obtain the supernatant, which was the biotin-labeled pTau-217 antigen. The concentration of the biotin-labeled pTau-217 antigen was determined using a microprotein spectrophotometer. The biotin-labeled pTau-217 antigen was diluted to 0pg / mL (sample diluent), 1pg / mL, 10pg / mL, 100pg / mL, 1000pg / mL, and 10000pg / mL with a sample diluent (produced by Zhuhai Lizhu Reagent Co., Ltd.).

[0112] (3) Reaction test between the antibody to be evaluated and the biotin-labeled antigen:

[0113] Alkaline phosphatase-labeled streptavidin was diluted to 0.2 μg / mL with 50 mM MOPS buffer (containing 1% BSA, pH 7.4).

[0114] The luminescence was measured by an automatic immunoassay analyzer (produced by Zhuhai Lizhu Reagent Co., Ltd., model: LiCreate ML5000Pro A): 50 μL of the prepared pTau-217 antibody magnetic bead working solution and 50 μL of the gradient diluted biotin-labeled pTau-217 antigen were added to the reaction cup, vortexed and reacted at 37°C for 10 minutes. Then, magnetic separation was performed and the sample was washed with a cleaning solution (produced by Zhuhai Lizhu Reagent Co., Ltd.). After washing, 50 μL of diluted 0.2 μg / mL alkaline phosphatase-labeled streptavidin was added, vortexed and reacted at 37°C for 10 minutes. Then, magnetic separation was performed and the sample was washed with a cleaning solution. After washing, 200 μL of the substrate solution for the automatic immunoassay system (produced by Zhuhai Lizhu Reagent Co., Ltd.) was added to the reaction cup, vortexed and reacted at 37°C for 5 minutes, and the luminescence was measured.

[0115] (4) Calculation and affinity interpretation:

[0116] The luminescence value of the biotin-labeled antigen at each dilution is the average of the luminescence values ​​measured twice. The ratio of the luminescence value of each antibody to be evaluated and the antigen at each concentration (i.e., S / N ratio, signal-to-noise ratio) is obtained respectively. The lowest antigen concentration corresponding to the signal-to-noise ratio ≥ 2 is C min , and compared the signal-to-noise ratios of the same antigen concentration and different antibody reactions (the signal-to-noise ratios greater than or equal to 2 times were considered different, and those less than 2 times were considered to be at the same level). The results are shown in Tables 10 and 11:

[0117] Table 10 Luminescence values ​​of the reaction between the pTau-217 antibody and the biotin-labeled antigen at various dilutions in Example 2-1

[0118]

[0119] Table 11 Example 2-1 Signal-to-noise ratio of the reaction between pTau-217 antibody and biotin-labeled antigen at various dilutions

[0120]

[0121]

[0122] From the above results, we can see that the antigen concentration corresponding to the signal-to-noise ratio ≥ 2 is the lowest for pTau-217 antibody-3 and pTau-217 antibody-5, C min The signal-to-noise levels of the two were close, followed by pTau-217 antibody-1 and pTau-217 antibody-2. min The signal-to-noise levels of the two are basically the same; finally, pTau-217 antibody-4, C min Therefore, considering the lowest concentration and the signal-to-noise ratio of different concentrations, the best antibodies are: pTau-217 antibody-5≈pTau-217 antibody-3>pTau-217 antibody-2≈pTau-217 antibody-1>pTau-217 antibody-4.

[0123] Example 2-2 Using carboxyl magnetic beads as solid phase

[0124] (1) The pTau-217 antibody to be evaluated is coated onto carboxyl magnetic beads:

[0125] Add ethyl (dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the carboxyl magnetic beads, and incubate at room temperature for 30 minutes. After washing and resuspending with MES buffer (0.1M, pH5.0), add the antibodies to be evaluated, vortex mix, and rotate and incubate at room temperature for 2 hours. After the reaction is completed, add the blocking agent CE510 (JSR) and incubate at room temperature for 3 hours. After the reaction is completed, wash twice with TBS-T Buffer (25mM Tris, 140mMNaCl, 0.1% Tween 20, pH7.4) to obtain carboxyl magnetic beads coated with pTau-217 antibodies. Dilute the carboxyl magnetic beads coated with pTau-217 antibodies with TBS-TBuffer to 0.2mg / mL to obtain pTau-217 antibody carboxyl magnetic beads working solution.

[0126] Steps (2) to (4) are the same as steps (2) to (4) of Example 2-1. The results are shown in Tables 12 and 13:

[0127] Table 12 Luminescence values ​​of the reaction between the pTau-217 antibody and the biotin-labeled antigen at various dilutions in Example 2-2

[0128]

[0129] Table 13 Example 2-2 Signal-to-noise ratio of the reaction between pTau-217 antibody and biotin-labeled antigen at various dilutions

[0130]

[0131]

[0132] From the above results, we can see that the antigen concentrations corresponding to the signal-to-noise ratio ≥ 2 are the lowest for pTau-217 antibody-3 and pTau-217 antibody-5, C min The signal-to-noise levels of the two were close, followed by pTau-217 antibody-1 and pTau-217 antibody-2. min The signal-to-noise level of the two is close to 10pg / mL; finally, pTau-217 antibody-4, C min The concentration of pTau-217 antibody-5 is ≈ pTau-217 antibody-3> pTau-217 antibody-2 is ≈ pTau-217 antibody-1> pTau-217 antibody-4. The conclusion is consistent with the method of Example 2-1.

[0133] Example 2-3 Using ELISA plate as solid phase

[0134] (1) The pTau-217 antibody to be evaluated is coated onto the luminescent plate:

[0135] Use coating buffer (pH 9.6, Na 2 CO 3 1.59 mg / mL, NaHCO 3 2.93mg / mL) dilute p-Tau217 antibody to 2μg / mL. Add the diluted antibody to a 96-well ELISA plate, 100μL per well, and incubate at 37℃ for 2h. Wash the plate 3 times with PBST buffer, 60s each time, and pat the plate dry. Add 200μL / well of blocking solution, incubate at 37℃ for 2h, and pat dry after washing the plate.

[0136] (2) Biotin-labeling the pTau-217 antigen and performing gradient dilution, as in step (2) of Example 2-1.

[0137] (3) Reaction test between the antibody to be evaluated and the biotin-labeled antigen:

[0138] Add the diluted antigen to the coated ELISA plate, 100 μL per well, incubate at 37±2℃ for 60±3min, wash the plate and pat dry. Add the prepared alkaline phosphatase-labeled streptavidin, 100 μL per well, incubate at 37℃ for 1-2h, wash the plate and pat dry. Add 200 μL of substrate solution for the fully automatic immunoassay system, cover with a sealing film, and incubate at 37℃ for 15±3min; the luminescence is measured with a multifunctional ELISA instrument to obtain the count value.

[0139] (4) Calculation and affinity determination, same as step (4) of Example 2-1, the results are shown in Tables 14 and 15:

[0140] Table 14 Luminescence values ​​of the reaction between pTau-217 antibody and biotin-labeled antigen at various dilutions in Example 2-3

[0141]

[0142] Table 15 Signal-to-noise ratio of the reaction between pTau-217 antibody and biotin-labeled antigen at various dilutions in Example 2-3

[0143]

[0144] From the above results, we can see that the corresponding antigen concentrations with a signal-to-noise ratio ≥ 2 are the lowest for pTau-217 antibody-3 and pTau-217 antibody-5, C min The signal-to-noise levels of the two were close, followed by pTau-217 antibody-1 and pTau-217 antibody-2. min The signal-to-noise levels of the two are close. Finally, pTau-217 antibody-4, C min The concentration of the antibody was 100 pg / mL. Therefore, considering the minimum concentration and the signal-to-noise ratio of different concentrations, the optimal antibody is: pTau-217 antibody-5≈pTau-217 antibody-3>pTau-217 antibody-2≈pTau-217 antibody-1>pTau-217 antibody-4. The conclusion is consistent with the method of Example 2-1 and Example 2-2.

[0145] Comparative Example 2-1 Comparison of Antibody Affinity by Traditional Methods

[0146] (1) Elisa plate coated with pTau-217 antigen:

[0147] Use coating buffer (pH 9.6, Na 2 CO 3 1.59 mg / mL, NaHCO 32.93mg / mL) p-Tau217 antigen was diluted to 2μg / mL. The diluted antigen was added to a 96-well ELISA plate, 100μL per well, and incubated at 37℃ for 2h. The plate was washed 3 times with PBST buffer, 60s each time, and the plate was patted dry. Blocking solution was added at 200μL / well, incubated at 37℃ for 2h, and the plate was patted dry after washing.

[0148] (2) Antibody and antigen response tests to be evaluated:

[0149] Use PBS buffer to dilute the 5 p-Tau217 antibodies to be evaluated in a gradient manner so that the test results show a platform inflection point. If the antibody concentration is not within the dilution range of the platform inflection point, change to a suitable dilution and dilute again. Add the diluted antibody to the coated ELISA plate, 100 μL per well, incubate at 37±2℃ for 60±3min, wash the plate and pat dry.

[0150] Add HRP-labeled goat anti-mouse IgG, 100 μL per well, incubate at 37°C for 1 to 2 h, wash the plate and pat dry.

[0151] Dilute the substrate buffer and substrate solution TMB in a 1:1 ratio, mix thoroughly, add 100 μL to each well, and react at room temperature for 5 to 10 minutes. Add 50 μL of stop solution to each well, mix well, and read the OD450nm value of each well using an ELISA reader.

[0152] (3) Calculation and affinity interpretation:

[0153] The OD450nm value of the antibody at each dilution is the average of the luminescence values ​​measured twice. The concentration obtained by dividing the concentration value of the antibody at the platform inflection point by 2 is used as the antibody concentration corresponding to a binding rate of 50%. The reciprocal of the antibody concentration corresponding to a binding rate of 50% is taken as the affinity index. The larger the value, the greater the affinity. The results are shown in Table 16:

[0154] Table 16 Comparative Example 2-1 pTau-217 Antibody Affinity Evaluation

[0155]

[0156] From the above results, it can be seen that the reciprocal of the antibody concentration corresponding to a binding rate of 50% is pTau-217 antibody-3>pTau-217 antibody-5>pTau-217 antibody-1=pTau-217 antibody-2=pTau-217 antibody-4, and this result is the corresponding antibody affinity.

[0157] Verification Example 2-1

[0158] The pTau-217 antibody to be evaluated was coated on magnetic beads and matched with the enzyme label of the same strain of Tau antibody to prepare a chemiluminescent detection reagent, which was used to detect 5 clinical samples with gradient dilutions and calculate the signal-to-noise ratio to determine the sensitivity.

[0159] (1) Sample preparation: The pTau-217 high-value sample was diluted with the low-value sample to form 5 gradients, and the sample dilution solution was used as the 0 pg / mL sample.

[0160] (2) Immune reaction: Use a fully automatic immunoassay analyzer (produced by Zhuhai Lizhu Reagent Co., Ltd., model: LiCreate ML 5000ProA) to add 50 μL of the pTau-217 antibody magnetic bead working solution prepared in Example 2-1 and 50 μL of the sample prepared in step (1) of this verification example to the reaction cup, respectively, and react at 37°C for 10 minutes after vortexing. Then, perform magnetic separation and wash with a cleaning solution (produced by Zhuhai Lizhu Reagent Co., Ltd.). After washing, add a diluted 1 μg / mL alkaline phosphatase-labeled Tau antibody (Zhuhai Lihe Medical Diagnostic Products Co., Ltd.), vortex and react at 37°C for 10 minutes. Then, perform magnetic separation and wash with a cleaning solution. After washing, add 200 μL of the substrate solution for the fully automatic immunoassay system (produced by Zhuhai Lizhu Reagent Co., Ltd.) to the reaction cup, vortex and react at 37°C for 5 minutes. Determine the luminescence amount.

[0161] (3) Calculation and sensitivity interpretation: The luminescence value of each sample is the average of the luminescence values ​​measured twice. Calculate the ratio of the luminescence value of each concentration to 0pg / mL (S / N ratio, signal-to-noise ratio). Compare the lowest concentrations of different antibodies with a signal-to-noise ratio ≥ 2, and measure the signal-to-noise ratio of the same concentration. The lower the concentration, the greater the signal-to-noise ratio (a signal-to-noise ratio greater than or equal to 2 times is considered to be different, and a signal-to-noise ratio less than 2 times is considered to be at the same level), indicating that the sensitivity of the reagent is higher. The results are shown in Tables 17 and 18:

[0162] Table 17 Luminescence values ​​of pTau-217 reagents reacting with various clinical samples

[0163]

[0164] Table 18 Signal-to-noise ratio of pTau-217 reagent and clinical samples

[0165]

[0166] From the above results, it can be seen that the sensitivity levels of reagents prepared by different antibodies are as follows: pTau-217 antibody-5≈pTau-217 antibody-3>pTau-181 antibody-2≈pTau-217 antibody-1>pTau-217 antibody-4. This sensitivity level is closer to the antibody affinity results obtained by the methods of Examples 2-1 to 3. In the reagent application situation, the sensitivity of pTau-217 antibody-5 is the highest. If the traditional method is used for evaluation, it will be misjudged as pTau-217 antibody-3>pTau-217 antibody-5, and it is likely that pTau-217 antibody-5 will be eliminated as an antibody with insufficient affinity, resulting in missed screening. And the traditional method cannot distinguish the affinity of pTau-217 antibody-1, pTau-217 antibody-2 and pTau-217 antibody-4. It can be seen that the methods of Examples 2-1 to 3 can more accurately distinguish the size of antibody affinity than traditional methods, provide more accurate information for subsequent reagent development, reduce the workload of antibody screening during reagent development, and reduce the probability of erroneous antibody elimination.

[0167] Tau antibody affinity assessment and validation

[0168] Example 3-1 Using Tosyl Magnetic Beads as Solid Phase

[0169] (1) The Tau antibody to be evaluated is coated onto Tosyl magnetic beads:

[0170] Add the five antibodies to be evaluated and the Tosyl magnetic beads to the reaction buffer (0.1M HEPES, pH8.0), add the reaction accelerator (1M ammonium sulfate), and incubate in a 37°C incubator for more than 6 hours. After removing the supernatant by magnetic suction, add the blocking agent CE510 (JSR) and incubate in a 37°C incubator for more than 6 hours. After the reaction is completed, wash twice with TBS-T Buffer (25mM Tris, 140mM NaCl, 0.1% Tween 20, pH7.4) to obtain Tosyl magnetic beads coated with Tau antibodies. Dilute the Tosyl magnetic beads coated with Tau antibodies to 0.2mg / mL with TBS-T Buffer to obtain Tau antibody Tosyl magnetic beads working solution.

[0171] (2) Biotinylated Tau antigen and diluted in series:

[0172] Tau antigen and biotin were mixed thoroughly in a 0.1M PBS buffer (pH 7.4) at a molar ratio of 1:50, placed in a dark place, and reacted at room temperature for 30 minutes. After the reaction was completed, ultrafiltration was repeated 5 times with a preservation solution (0.1M PBS, 0.05% PC300) and a 50kDa ultrafiltration tube to obtain the supernatant, which was the biotin-labeled Tau antigen. The concentration of biotin-labeled Tau antigen was determined using a micro-protein spectrophotometer. The biotin-labeled Tau antigen was diluted to 0pg / mL (sample diluent), 1pg / mL, 10pg / mL, 100pg / mL, 1000pg / mL, and 10000pg / mL with a sample diluent (produced by Zhuhai Lizhu Reagent Co., Ltd.).

[0173] (3) Reaction test between the antibody to be evaluated and the biotin-labeled antigen:

[0174] Alkaline phosphatase-labeled streptavidin (Jackson Immunoreacher) was diluted to 0.2 μg / mL with 50 mM MOPS buffer (containing 1% BSA, pH 7.4).

[0175] The luminescence was measured by an automatic immunoassay analyzer (produced by Zhuhai Lizhu Reagent Co., Ltd., model: LiCreate ML5000Pro A): 50 μL of the prepared Tau antibody magnetic bead working solution and 50 μL of the gradient diluted biotin-labeled Tau antigen were added to the reaction cup, vortexed and reacted at 37°C for 10 minutes. Then, magnetic separation was performed and washed with a cleaning solution (produced by Zhuhai Lizhu Reagent Co., Ltd.). After washing, 50 μL of diluted 0.2 μg / mL alkaline phosphatase-labeled streptavidin was added, vortexed and reacted at 37°C for 10 minutes. Then, magnetic separation was performed and washed with a cleaning solution. After washing, 200 μL of the substrate solution for the automatic immunoassay system (produced by Zhuhai Lizhu Reagent Co., Ltd.) was added to the reaction cup, vortexed and reacted at 37°C for 5 minutes, and the luminescence was measured.

[0176] (4) Calculation and result interpretation:

[0177] The luminescence value of the biotin-labeled antigen at each dilution is the average of the luminescence values ​​measured twice. The ratio of the luminescence value of each strain of the antibody to be evaluated and the antigen at each concentration (i.e., S / N ratio, signal-to-noise ratio) is obtained. The lowest concentration corresponding to the signal-to-noise ratio ≥ 2 is C min , and compared the signal-to-noise ratios of the same antigen concentration and different antibody reactions (the signal-to-noise ratios greater than or equal to 2 times were considered different, and those less than 2 times were considered to be at the same level). The results are shown in Tables 19 and 20:

[0178] Table 19 Example 3-1 Luminescence values ​​of the reaction between pTau antibody and biotin-labeled antigen at various dilutions

[0179]

[0180] Table 20 Example 3-1 Signal-to-noise ratio of the reaction between pTau antibody and biotin-labeled antigen at various dilutions

[0181]

[0182] From the above results, we can see that the corresponding antigen concentrations with a signal-to-noise ratio ≥ 2 are the lowest for Tau antibody-1, Tau antibody-2, and Tau antibody-4. min The signal-to-noise level of Tau antibody-1 was higher than that of Tau antibody-2 and Tau antibody-4, and the signal-to-noise level of Tau antibody-2 and Tau antibody-4 was similar; followed by Tau antibody-5, C min is 100 pg / mL; finally, Tau antibody-3, C min Therefore, considering the lowest concentration and the signal-to-noise ratio of different concentrations, the best antibodies are: Tau antibody-1>Tau antibody-2≈Tau antibody-4>Tau antibody-5>Tau antibody-3.

[0183] Example 3-2 Using carboxyl magnetic beads as solid phase

[0184] (1) The Tau antibody to be evaluated is coated onto carboxyl magnetic beads:

[0185] Add ethyl (dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the carboxyl magnetic beads, and incubate at room temperature for 30 minutes. After washing and resuspending with MES buffer (0.1M, pH5.0), add the antibodies to be evaluated, vortex mix, and rotate and incubate at room temperature for 2 hours. After the reaction is completed, add the blocking agent CE510 (JSR) and incubate at room temperature for 3 hours. After the reaction is completed, wash twice with TBS-T Buffer (25mM Tris, 140mMNaCl, 0.1% Tween 20, pH7.4) to obtain carboxyl magnetic beads coated with Tau antibodies. Dilute the carboxyl magnetic beads coated with Tau antibodies to 0.2mg / mL with TBS-T Buffer to obtain Tau antibody carboxyl magnetic beads working solution.

[0186] Steps (2) to (4) are the same as steps (2) to (4) of Example 3-1. The results are shown in Tables 21 and 22:

[0187] Table 21 Example 3-2 Luminescence values ​​of the reaction between pTau antibody and biotin-labeled antigen at various dilutions

[0188]

[0189] Table 22 Example 3-2 Signal-to-noise ratio of the reaction between pTau antibody and biotin-labeled antigen at various dilutions

[0190]

[0191] From the above results, we can see that the corresponding antigen concentrations with a signal-to-noise ratio ≥ 2 are the lowest for Tau antibody-1, Tau antibody-2, and Tau antibody-4. min The signal-to-noise levels of Tau antibody-2 and Tau antibody-4 were basically the same, followed by Tau antibody-5 and C min is 10pg / mL; finally, Tau antibody-3, C min is 100 pg / mL. Therefore, considering the minimum concentration and the signal-to-noise ratio of different concentrations, the antibodies are better as follows: Tau antibody-1>Tau antibody-2≈Tau antibody-4>Tau antibody-5>Tau antibody-3. The conclusion is consistent with the method of Example 3-1.

[0192] Example 3-3 Using ELISA plate as solid phase

[0193] (1) The Tau antibody to be evaluated is coated onto the luminescent plate:

[0194] Use coating buffer (pH 9.6, Na 2 CO 3 1.59 mg / mL, NaHCO 3 2.93mg / mL) dilute Tau antibody to 2μg / mL. Add the diluted antibody to a 96-well ELISA plate, 100μL per well, and incubate at 37℃ for 2h. Wash the plate 3 times with PBST buffer, 60s each time, and pat the plate dry. Add 200μL / well of blocking solution, incubate at 37℃ for 2h, and pat dry after washing the plate.

[0195] (2) Biotin-labeled Tau antigen and graded diluted, same as step (2) of Example 3-1.

[0196] (3) Reaction test between the antibody to be evaluated and the biotin-labeled antigen:

[0197] Add the diluted antigen to the coated ELISA plate, 100 μL per well, incubate at 37±2℃ for 60±3min, wash the plate and pat dry. Add the prepared alkaline phosphatase-labeled streptavidin, 100 μL per well, incubate at 37℃ for 1-2h, wash the plate and pat dry. Add 200 μL of substrate solution for the fully automatic immunoassay system, cover with a sealing film, and incubate at 37℃ for 15±3min; the luminescence is measured with a multifunctional ELISA instrument to obtain the count value.

[0198] (4) Calculation and affinity determination, same as step (4) of Example 3-1, the results are shown in Table 23 and Table 24:

[0199] Table 23 Example 3-3 Luminescence values ​​of the reaction between pTau antibody and biotin-labeled antigen at various dilutions

[0200]

[0201] Table 24 Example - Signal-to-noise ratio of the reaction between 3pTau antibody and biotin-labeled antigen at various dilutions

[0202]

[0203] From the above results, we can see that the corresponding antigen concentrations with a signal-to-noise ratio ≥ 2 are the lowest for Tau antibody-1, Tau antibody-2, and Tau antibody-4. min The signal-to-noise level of Tau antibody-2 and Tau antibody-4 was basically the same; followed by Tau antibody-5, C min is 100 pg / mL; finally, Tau antibody-3, C min is 1000 pg / mL. Therefore, considering the minimum concentration and the signal-to-noise ratio of different concentrations, the antibodies are better as follows: Tau antibody-1>Tau antibody-2≈Tau antibody-4>Tau antibody-5>Tau antibody-3. The conclusion is consistent with the methods of Example 3-1 and Example 3-2.

[0204] Comparative Example 3-1 Comparison of Antibody Affinity by Traditional Methods

[0205] (1) Tau antigen coated ELISA plate: Use coating buffer (pH 9.6, Na 2 CO 3 1.59 mg / mL, NaHCO 3 2.93mg / mL) dilute Tau antigen to 2μg / mL. Add the diluted antigen to a 96-well ELISA plate, 100μL per well, and incubate at 37℃ for 2h. Wash the plate 3 times with PBST buffer, 60s each time, and pat the plate dry. Add 200μL / well of blocking solution, incubate at 37℃ for 2h, and pat dry after washing the plate.

[0206] (2) Reaction test of the antibody and antigen to be evaluated: Use PBS buffer to dilute the 5 Tau antibodies to be evaluated in a gradient manner so that the test results show a platform inflection point. If the antibody concentration is not within the dilution range of the platform inflection point, change to a suitable dilution and dilute again. Add the diluted antibody to the coated ELISA plate, 100 μL per well, incubate at 37±2℃ for 60±3 min, wash the plate and pat dry.

[0207] Add 100 μL of HRP-labeled goat anti-mouse IgG to each well, incubate at 37 °C for 1 - 2 h, wash the plate and pat dry.

[0208] Dilute the substrate buffer and the substrate solution TMB at a ratio of 1:1, mix well, add 100 μL to each well, and react at room temperature for 5 - 10 min. Add 50 μL of the stop solution to each well, mix well, and use an enzyme-linked immunosorbent assay (ELISA) reader to read the OD450nm value of each well.

[0209] (3) Calculation and affinity interpretation:

[0210] The OD450nm value of the antibody at each dilution is the average of the luminescence values measured twice. The concentration obtained by dividing the concentration value of the antibody at the inflection point of the plateau by 2 is used as the antibody concentration corresponding to a binding rate of 50%. Take the reciprocal of the antibody concentration corresponding to a binding rate of 50% as the affinity index, and the larger the value, the greater the affinity. The results are shown in Table 25:

[0211] Table 25 Affinity evaluation of the pTau antibody in Comparative Example 3 - 1

[0212]

[0213] From the above results, it can be seen that the reciprocal of the antibody concentration corresponding to a binding rate of 50% is Tau antibody - 2 > Tau antibody - 4 = Tau antibody - 1 > Tau antibody - 3 = Tau antibody - 5, and this result corresponds to the antibody affinity.

[0214] Verification Example 3 - 1

[0215] Coat magnetic beads with the Tau antibody to be evaluated, and pair it with an enzyme-labeled Tau antibody of the same strain to prepare a chemiluminescence detection reagent. Detect 5 clinically sampled specimens with gradient dilution, and calculate the signal-to-noise ratio. Judge the sensitivity.

[0216] (1) Sample preparation: Gradiently dilute the Tau high-value sample with the low-value sample to obtain 5 gradients, and use the sample diluent as the sample with a concentration of 0 pg / mL.

[0217] (2) Immune reaction: 50 μL of the Tau antibody magnetic bead working solution prepared in Example 3-1 and 50 μL of the sample prepared in step (1) of this verification example were added to the reaction cup, mixed and reacted at 37°C for 10 minutes. Then, magnetic separation was performed and washed with a cleaning solution (produced by Zhuhai Lizhu Reagent Co., Ltd.). After washing, a diluted 1 μg / mL alkaline phosphatase-labeled Tau antibody (Zhuhai Lihe Medical Diagnostic Products Co., Ltd.) was added, mixed and reacted at 37°C for 10 minutes. Then, magnetic separation was performed and washed with a cleaning solution. After washing, 200 μL of the substrate solution for the fully automatic immunoassay system (produced by Zhuhai Lizhu Reagent Co., Ltd.) was added to the reaction cup, mixed and reacted at 37°C for 5 minutes. The luminescence was measured with a photometer to obtain the count value. The actual measurement was performed using a fully automatic immunoassay analyzer (produced by Zhuhai Lizhu Reagent Co., Ltd., model: LiCreate ML 5000ProA).

[0218] (3) Calculation and sensitivity interpretation: The luminescence value of each sample is the average of the luminescence values ​​measured twice. Calculate the ratio of the luminescence value of each concentration to 0pg / mL (S / N ratio, signal-to-noise ratio). Compare the lowest concentrations of different antibodies with a signal-to-noise ratio ≥ 2, and measure the signal-to-noise ratio of the same concentration (signal-to-noise ratios greater than or equal to 2 times are considered to be different, and less than 2 times are considered to be at the same level). The lower the concentration, the greater the signal-to-noise ratio, indicating that the reagent has a higher sensitivity. The results are shown in Tables 26 and 27:

[0219] Table 26 Luminescence values ​​of pTau reagents reacting with various clinical samples

[0220]

[0221] Table 27 Signal-to-noise ratio of Tau reagent and clinical samples

[0222]

[0223] From the above results, it can be seen that the sensitivity levels of reagents prepared with different antibodies are as follows: Tau antibody-1>Tau antibody-2≈Tau antibody-4>pTau antibody-5>Tau antibody-3. This sensitivity level is consistent with the antibody affinity results obtained by the methods of Examples 3-1 to 3. In the reagent application situation, the sensitivity Tau antibody-1>Tau antibody-2, and the traditional method will be misjudged as Tau antibody-2>Tau antibody-1, so Tau antibody-1 may be eliminated as an antibody with insufficient affinity, resulting in missed screening. In addition, the traditional method cannot distinguish between Tau antibody-1 and Tau antibody-4, and cannot distinguish between the affinity of Tau antibody-3 and Tau antibody-5. It can be seen that the methods of Examples 3-1 to 3 can more accurately distinguish the size of antibody affinity than the traditional method, provide more accurate information for subsequent reagent development, and subsequently reduce the workload of screening antibodies during reagent development, and reduce the probability of incorrectly eliminating antibodies.

[0224] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening antibodies, characterized in that: The method comprises reacting the antibodies to be screened with a series of antigen reagents having different antigen concentrations, respectively, to obtain the signal-to-noise ratio of the reaction between each antibody and each concentration of the antigen reagent; Compare the signal-to-noise ratios corresponding to the same antigen concentration. The antibody with a larger signal-to-noise ratio is better. The signal-to-noise ratio is the ratio of the detection value obtained after the antibody reacts with the antigen reagent to the detection value when the antigen concentration in the antigen reagent is 0.

2. The method according to claim 1, characterized in that The minimum antigen concentration corresponding to the antibody signal-to-noise ratio being greater than or equal to 2 is the C min , C min The smaller the antibody, the better.

3. The method according to claim 2, characterized in that Any two antibodies C min When the signal-to-noise ratio is the same, the antibody with a larger signal-to-noise ratio is better; Optionally, the C min When the two antibodies are the same, compare the min When the ratio of larger signal-to-noise ratio to smaller signal-to-noise ratio is greater than or equal to 2, the antibody corresponding to the larger signal-to-noise ratio is better; otherwise, the two antibodies are at the same level.

4. The method according to claim 1, characterized in that The series of antigen reagents with different antigen concentrations are different antigen reagents obtained by gradient dilution of the antigen.

5. The method according to any one of claims 1 to 4, characterized in that: The reaction includes reacting the antibody coated on the solid phase carrier with the antigen reagent, and obtaining the detection value by detecting the signal substance marked on the antigen.

6. The method according to claim 5, characterized in that The solid phase carrier includes: microparticles, microporous plates or carrier membranes; And / or, the signal substance includes one or more of an enzyme, a luminescent label, a fluorescent microsphere, a colored microsphere, a latex microsphere, a colloidal gold, a quantum dot, biotin, streptavidin, a radionuclide, a radiocontrast agent, a paramagnetic ion, a metal and a photosensitizer.

7. The method according to claim 6, characterized in that The antigen and signal are linked via a specific binding pair; Optionally, the specific binding pair is selected from an antibody-antigen binding pair, a biotin-avidin binding pair, a biotin-streptavidin binding pair, an enzyme-cofactor binding pair or a receptor-ligand binding pair.

8. The method according to claim 7, characterized in that The specific binding pair is a biotin-streptavidin binding pair, the antigen is connected to biotin, and the signal substance is connected to streptavidin; Optionally, the solid phase carrier is selected from tosyl magnetic microparticles, carboxyl magnetic microparticles or ELISA plates; Optionally, the signal agent comprises alkaline phosphatase.

9. Use of the method according to any one of claims 1 to 8 in the preparation of an in vitro diagnostic kit based on immunoassay technology.

10. The use according to claim 9, characterized in that: The method is applied to screening antibodies for immunoassays; Optionally, the immunoassay is a double antibody sandwich method; Optionally, the antibodies obtained by the screening are used as capture antibodies; Optionally, the antibody comprises an anti-Tau antibody; Optionally, the antibody comprises pTau-181 antibody or pTau-217 antibody.

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