Application of protein cross-linking agent in antibody immunodetection

Abstract

The invention provides application of a protein cross-linking agent in preparation of an antibody reagent for immunodetection. The protein cross-linking agent is added into the antibody reagent, and after the protein cross-linking agent is covalently connected with the antibody, natural conformation of the antibody reagent generates certain rigidity and is not easy to stretch and inactivate, vibration of groups in molecules is reduced, exposure of hydrophobic sites of the antibody is reduced, and the stability of the antibody can be obviously improved on the premise that the detection value of an antibody sample is not influenced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of in vitro diagnosis, and particularly relates to application of a protein cross-linking agent in antibody immunodetection, a detection kit containing the protein cross-linking agent, and a method for improving stability of an antibody in immunodetection. BACKGROUND

[0002] In immunodetection, an antibody, particularly an antibody quality control product, is stored in a 2-8℃ environment, and progressive chemical degradation, particularly oxidation, occurs, which interferes with a normal immunoreaction, thereby causing deviation of a result. A method for improving stability of an antibody quality control product is usually chemical modification of a specific site of an antibody molecule or use of a freeze-drying process, but these methods are usually relatively complex or cannot achieve an ideal stability effect. Development of a new method for improving stability of an antibody quality control product is of great significance to immunodetection of an antibody. SUMMARY

[0003] In order to solve the problems in the prior art, the present application provides a new method for improving stability of an antibody, which can obviously improve stability of an antibody without affecting an antibody measurement value by adding a protein cross-linking agent to the antibody.

[0004] A first aspect of the present application provides application of a protein cross-linking agent in preparation of an antibody reagent for immunodetection. The detection is a non-disease diagnosis detection.

[0005] In some embodiments, the antibody reagent comprises at least one of an antibody quality control product, an antibody standard product, an antibody calibration product or an antibody reference product.

[0006] In some embodiments, the protein cross-linking agent is selected from a homologous cross-linking agent capable of reacting with an amino group and / or a sulfhydryl group of the antibody.

[0007] In some embodiments, the homologous cross-linking agent comprises two identical activated reaction groups and a spacer arm located between the activated reaction groups.

[0008] In some embodiments, the activated reaction group is selected from succinimide, maleimide, aminocarbodiimide, a methylol group or an imido ester.

[0009] In some embodiments, the spacer arm is selected from polyethylene glycol.

[0010] By adding the cross-linking agent to the antibody, since the spacer arm of the cross-linking agent covalently connects a high molecule such as a protein, a certain rigidity is generated in a natural conformation of the cross-linking agent, the high molecule is not easy to stretch and inactivate, and vibration of internal groups of the molecule is reduced; a hydrophobic site of the protein is reduced, thereby achieving the purpose of improving stability of the antibody.

[0011] In some embodiments, the protein crosslinker is selected from Bis-(PEG)n-NHS, n is an integer selected from 2-30, preferably an integer selected from 8-15. In some specific embodiments, n is selected from 2, 4, 5, 6, 8, 10, 12, 15, 18, 20, 25, 30, or any value therebetween.

[0012] In some embodiments, the protein crosslinker is used at a concentration of 0.02-0.2 mM, preferably 0.05-0.1 mM. In some embodiments, the protein crosslinker is used at a concentration of 0.02 mM, 0.04 mM, 0.05 mM, 0.08 mM, 0.1 mM, 0.12 mM, 0.15 mM, 0.18 mM, 0.2 mM, or any value therebetween.

[0013] In some embodiments, the antibody comprises at least one of IgG, Fab, Fab', F(ab')2, Fv, scFv, or VH. Preferably, the antibody is IgG.

[0014] In some embodiments, the antibody comprises an antibody against hepatitis C virus or an antibody against human immunodeficiency virus.

[0015] In some embodiments, the immunoassay comprises an immunochromatographic immunoassay, an enzyme-linked immunosorbent assay, or a chemiluminescent immunoassay.

[0016] In some embodiments, the immunoassay comprises a photochemiluminescence assay, a magnetic particle chemiluminescence assay, a colloidal gold immunochromatographic assay, a latex immunochromatographic assay, a radioimmunoassay, or a time-resolved fluorescence immunoassay.

[0017] A second aspect of the present disclosure provides a method for improving the stability of an antibody in a solution, comprising the step of mixing a protein crosslinker with the antibody solution.

[0018] In some embodiments, the protein crosslinker is selected from a homologous crosslinker capable of reacting with the amino group and / or the sulfhydryl group of the antibody.

[0019] In some embodiments, the homologous crosslinker comprises two identical activated reactive groups and a spacer arm located between the activated reactive groups.

[0020] In some embodiments, the activated reactive group is selected from succinimide, maleimide, aminocarbodiimide, a hydroxymethyl group, or an imidoester.

[0021] In some embodiments, the spacer arm is selected from polyethylene glycol.

[0022] By adding a cross-linking agent in the antibody, due to the covalent connection of macromolecules such as proteins after the spacer arm of the cross-linking agent, its natural conformation produces a certain rigidity and is not easy to stretch and inactivate, reducing the vibration of the internal groups of the molecule; reducing the exposure of protein hydrophobic sites, thereby achieving the purpose of improving the stability of the antibody.

[0023] In some embodiments, the protein cross-linking agent is selected from Bis-(PEG)n-NHS, n is selected from an integer from 2 to 30, preferably an integer from 8 to 15. In some specific embodiments, n is selected from 2, 4, 5, 6, 8, 10, 12, 15, 18, 20, 25, 30 or any value therebetween.

[0024] In some embodiments, the concentration of the protein cross-linking agent used is 0.02-0.2 mM, preferably 0.05-0.1 mM. In some embodiments, the concentration of the protein cross-linking agent used is 0.02 mM, 0.04 mM, 0.05 mM, 0.08 mM, 0.1 mM, 0.12 mM, 0.15 mM, 0.18 mM, 0.2 mM or any value therebetween.

[0025] In some embodiments, the antibody comprises at least one of IgG, Fab, Fab', F(ab')2, Fv, scFv or VH. Preferably, the antibody is IgG.

[0026] In some embodiments, the antibody comprises an antibody against hepatitis C virus or an antibody against human immunodeficiency virus.

[0027] The third aspect of the present disclosure provides an antibody reagent comprising an antibody and a protein cross-linking agent.

[0028] In some embodiments, the protein cross-linking agent is selected from a homologous cross-linking agent capable of reacting with the amino group and / or thiol group of the antibody.

[0029] In some embodiments, the homologous cross-linking agent comprises two identical activated reactive groups and a spacer arm between the activated reactive groups.

[0030] In some embodiments, the activated reactive group is selected from succinimide, maleimide, aminocarbodiimide, hydroxymethyl or imido ester.

[0031] In some embodiments, the spacer arm is selected from polyethylene glycol.

[0032] In some embodiments, the protein cross-linking agent is selected from Bis-(PEG)n-NHS, n is an integer selected from 2-30, preferably an integer selected from 8-15. In some specific embodiments, n is selected from 2, 4, 5, 6, 8, 10, 12, 15, 18, 20, 25, 30 or any value therebetween.

[0033] In some embodiments, the antibody reagent comprises at least one of an antibody quality control, an antibody standard, an antibody calibrator or an antibody reference.

[0034] In some embodiments, the protein cross-linking agent is used at a concentration of 0.02-0.2 mM, preferably 0.05-0.1 mM. In some embodiments, the protein cross-linking agent is used at a concentration of 0.02 mM, 0.04 mM, 0.05 mM, 0.08 mM, 0.1 mM, 0.12 mM, 0.15 mM, 0.18 mM, 0.2 mM or any value therebetween.

[0035] In some embodiments, the antibody comprises at least one of IgG, Fab, Fab', F(ab')2, Fv, scFv or VH. Preferably, the antibody is IgG.

[0036] In some embodiments, the antibody comprises an antibody against hepatitis C virus or an antibody against human immunodeficiency virus.

[0037] A fourth aspect of the present disclosure provides a detection kit comprising the antibody reagent of the third aspect.

[0038] The present application, without affecting the antibody measurement, by adding a protein cross-linking agent in the antibody sample, makes the antibody conformation produce certain rigidity, not easy to stretch and reduce the internal group vibration; at the same time, after adding the cross-linking agent, the protein forms a complex, the protein molecule is stable to the hydrophobic cluster, reduces the hydrophobic region exposed on the protein surface, reduces the non-specific reaction, and can obviously improve the stability of the antibody quality control. Compared with chemical modification of specific sites of antibody molecules or use of freeze-drying process, the method is simple and easy to operate, and is convenient for screening and verification. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below with examples. The specific examples described herein are only used to explain the present application, and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0040] The term "protein crosslinker" as used herein refers to a class of small molecule compounds with two or more reactive ends for specific groups (-NH2, -COOH, -HS, etc.) that can couple two or more molecules separately, thereby binding these molecules together. Protein crosslinkers include homobifunctional, heterobifunctional, and photoreactive crosslinkers. Homobifunctional crosslinkers have two identical activated reactive groups, including but not limited to bis-(PEG)n-succinimidyl ester. Heterobifunctional crosslinkers have two different activated reactive groups, including but not limited to succinimidyl-(PEG)n-maleimide.

[0041] The term "standard" as used herein, also known as standard substance or standard sample, refers to a uniform and stable substance with one or more specified property values, which is suitable for a particular purpose of measurement or nominal property inspection. As a "measuring instrument" in the analytical measurement industry, standard substance plays an irreplaceable role in calibrating measuring instruments and devices, evaluating measurement methods, measuring material properties, assessing the technical level of analysts, and product quality control, etc.

[0042] The term "calibrator" as used herein refers to a measurement standard for in vitro diagnostic instruments or detection systems, which is a major tool for realizing the clinical detection of in vitro diagnostic reagents and supervising the accuracy and consistency of test results, and is also a physical measurement standard for ensuring value transfer. The calibrator is designated by the company to calibrate a certain detection system (instrument + reagent + method procedure), and is artificially assigned a calibration value considering the matrix effect. Therefore, the calibrator must be specific to a certain detection system.

[0043] The term "quality control" as used herein refers to a quality control substance for in vitro diagnostics, which aims to evaluate or verify measurement precision and measurement accuracy, or analyze deviations in performance characteristics due to changes in reagents or analytical instruments. The quality control cannot be used for calibration, and has high requirements for stability and inter-bottle difference. When selecting quality control, several concentrations, a wide range of concentration distribution, preferably medical decision levels, and quality control with upper and lower limit values of the reportable range should be selected.

[0044] The term "reference" as used herein refers to a reference material with one or more sufficiently uniform and well-defined properties for calibrating measuring devices, rating measurement methods, or assigning values to materials or substances.

[0045] As a carrier of metrological standards, the standard is the top of the value traceability chain, and is used for quality control and evaluation of in vitro diagnostic products, with higher accuracy, uniformity, and stability than calibrators and quality controls. Calibrators are used to calibrate detection systems, and the traceability of their values is crucial. Quality control is mainly used for measurement precision detection, with greater emphasis on uniformity and stability.

[0046] The following examples are provided to aid the understanding of the present application, but the true scope of the application is set forth in the appended claims. It is understood that modifications and changes can be made therein without departing from the spirit of the application.

[0047] The cross-linking agents used in the embodiments of the present application were purchased from Xi'an Kangfunuo Biotechnology Co., Ltd. The hepatitis C virus antibody detection kit (chemiluminescence method) and the human immunodeficiency virus antibody HIV (1+2 type) antibody detection kit (chemiluminescence method) were purchased from Keminbo Yang Diagnostics Technology (Shanghai) Co., Ltd. The HCV sheep polyclonal antibody was purchased from Shanghai Suoxi Biotechnology Co., Ltd. Unless otherwise specified, the reagents used in the embodiments of the present application are conventional reagents in the art, which can be commercially available or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. The methods used are conventional methods in the art, and the experimental conditions used are conventional experimental conditions in the art. Please refer to the relevant experimental manual or manufacturer's instructions.

[0048] Example 1, preparation of quality control

[0049] 1. Preparation of Anti-HCV quality control

[0050] (1) Preparation of three-level Anti-HCV quality control: dilute HCV sheep polyclonal antibody with Anti-HCV qualitative reference diluent, the average value of level 1 is not higher than 0.5, the target value of level 2 is 4, the range is 3.6≤S / CO≤4.4, the target value of level 3 is 15, and the range is 13.5≤S / CO≤16.5. After preparation, stir at 2-8℃ for at least 30 minutes, and stand for at least 8 hours.

[0051] (2) Divide the prepared quality control of each level into aliquots, one of which is Anti-HCV quality control without adding cross-linking agent, and the rest are aliquots, each of which is added with homologous cross-linking agent: Bis-PEG2-NHS, Bis-PEG8-NHS, Bis-PEG10-NHS, Bis-PEG15-NHS, Bis-PEG25-NHS, and each cross-linking agent is set at four concentrations (0.025mM, 0.05mM, 0.1mM, 0.15mM), and heterologous cross-linking agent: SM(PEG)6, the concentration of cross-linking agent is 0.1mM.

[0052] 2. Anti-HIV quality control preparation process

[0053] (1) Pretreatment of Anti-HIV type I positive blood

[0054] Place the Anti-HIV type I positive blood in a 58±2℃ water bath, and continuously inactivate for 90 minutes.

[0055] (2) Three levels of Anti-HIV quality control preparation: Anti-HIV type I positive serum is diluted with Anti-HIV qualitative reference diluent, the mean value of level 1 is not higher than 0.5, the target value of level 2 is 4, the range is 3.6≤S / CO≤4.4, the target value of level 3 is 15, and the range is 13.5≤S / CO≤16.5. After preparation, it needs to be stirred at 2-8°C for at least 30 minutes, and stand for at least 8 hours.

[0056] (3) Divide the prepared quality control of each level into several parts, one of which is Anti-HIV quality control without adding cross-linking agent, and the rest are respectively added with homologous cross-linking agents: Bis-PEG2-NHS, Bis-PEG8-NHS, Bis-PEG10-NHS, Bis-PEG15-NHS, Bis-PEG25-NHS, each cross-linking agent is set at 4 concentrations (0.025mM, 0.05mM, 0.1mM, 0.15mM respectively), and heterologous cross-linking agent: SM(PEG)6, the concentration of cross-linking agent is 0.1mM.

[0057] Example 2, the effect of adding cross-linking agent on the measured value of Anti-HCV and Anti-HIV quality control

[0058] The prepared quality control was detected by using qualified Anti-HCV antibody detection kit (chemiluminescence method) and Anti-HIV antibody HIV (1+2 type) antibody detection kit (chemiluminescence method) respectively, Anti-HCV quality control of three levels without adding cross-linking agent and Anti-HIV quality control of three levels with adding cross-linking agent were tested respectively, and whether the addition of cross-linking agent would affect the quality control value was evaluated. The results are shown in Tables 1 and 2 respectively.

[0059] Table 1

[0060]

[0061] Table 2

[0062]

[0063] As shown in Tables 1 and 2: the addition of cross-linking agent Bis-(PEG)10-NHS does not affect the quality control value, while the addition of cross-linking agent SM(PEG)6 will make the quality control values of Anti-HCV quality control and Anti-HIV quality control in level two and level three lower.

[0064] Bis-(PEG)n-NHS is a homologous crosslinking agent, its reactive group is NHS ester, which can react with amino group on the protein to form amide bond; and PEG spacer has unique advantages, providing higher stability, lower accumulation tendency and lower immunogenicity. SM(PEG)n is a heterologous crosslinking agent, its reactive group is NHS ester and maleimide, which can react with amino and sulfhydryl groups, NHS ester reacts with amino to form amide bond, and maleimide group reacts with sulfhydryl to form stable thioether bond.

[0065] Example 3, Effect of Crosslinking Agent on Accelerated Stability of Antibody Quality Control

[0066] Take three levels of Anti-HCV quality control without adding crosslinking agent and three levels of Anti-HCV quality control with adding crosslinking agent, respectively, and place them in a 2-8°C refrigerator and a 37°C constant temperature incubator for 7 days. Use qualified hepatitis C virus antibody detection kit (chemiluminescence method) for accelerated stability evaluation, and compare with the value measured on day 0. The results are shown in Table 3 below.

[0067] Table 3

[0068]

[0069]

[0070] The results of Table 3 show that the addition of crosslinking agent Bis-PEG(8-15)-NHS with a final concentration of 0.05-0.1 mM has a significant improvement effect on the stability performance of Anti-HCV project quality control; the addition of crosslinking agent Bis-PEG10-NHS with a final concentration of 0.1 mM has the best improvement effect on the stability performance of Anti-HCV project quality control. The addition of different concentrations and different spacer lengths of homologous crosslinking agent has different effects on the stability of antibody quality control; the longer the spacer of the crosslinking agent, the stronger the flexibility and the smaller the steric hindrance, but the problem that more potential non-specific binding sites will be generated follows.

[0071] Example 4, Real-time Stability Evaluation of Anti-HCV Quality Control and Anti-HIV Quality Control by Crosslinking Agent

[0072] Detection method: On day 3, 7, 14, 30, 60, 90, 180, 270, Anti-HCV quality control (level 1 / level 2 / level 3) and Anti-HIV quality control (level 1 / level 2 / level 3) after adding crosslinking agent Bis-PEG10-NHS (crosslinking agent final concentration 0.1 mM) were measured repeatedly 3 times, and the average value was calculated. According to the measurement drift of each test sample, the stability of the quality control (level 1 / level 2 / level 3) was evaluated.

[0073] Analysis method:

[0074] 1) Plot the average value of the detected quality control (level 1 / level 2 / level 3) (y-axis) versus time (x-axis) and perform linear regression.

[0075] 2) The P-Value obtained by F distribution (F>F0.05(df1, df2)) shows the credibility of the null hypothesis "signal has no relationship with the detection date"; P<0.05 indicates that the null hypothesis is rejected at the probability of 5% of making the first type of error, that is, "the measured value is related to the detection date", which indicates the variability of the measured value over time, which is an unfavorable phenomenon for stability.

[0076] The real-time stability results of Anti-HCV quality control and Anti-HIV quality control added with crosslinking agent Bis-PEG10-NHS (the final concentration of crosslinking agent is 0.1 mM) are shown in Tables 4 and 5, respectively.

[0077] Table 4

[0078]

[0079]

[0080] Table 5

[0081]

[0082]

[0083] The results of Tables 4 and 5 show that after adding the crosslinking agent to the Anti-HCV project quality control and the Anti-HIV project quality control, the 270-day real-time stability quality control measured value basically does not change over time, and the fitting P value also shows that the measured value change is not significant, and the effect of improving the stability of the antibody quality control is best.

[0084] As can be seen from the above examples, the addition of crosslinking agent Bis-PEG10-NHS to the antibody quality control, and the final concentration of the crosslinking agent is 0.1 mM, has the best effect on improving the stability of the antibody quality control.

[0085] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. Use of a protein cross-linker in the preparation of an antibody reagent for immunoassay, Preferably, the antibody reagent comprises at least one of an antibody quality control, an antibody standard, an antibody calibrator or an antibody reference.

2. A method for improving the stability of an antibody in solution, comprising the step of mixing a protein cross-linker with the antibody solution.

3. Use according to claim 1 or method according to claim 2, characterized in that, The protein cross-linker is selected from a homologous cross-linker; Preferably, the homologous cross-linker comprises two identical activated reactive groups and a spacer arm between the activated reactive groups; Preferably, the activated reactive groups are selected from succinimide, maleimide, carbodiimide, methylol or imidoester; Preferably, the spacer arm is selected from polyethylene glycol; More preferably, the protein cross-linker is selected from Bis-(PEG)n-NHS, n is an integer selected from 2-30, preferably an integer selected from 8-15.

4. The use or method according to any one of claims 1 to 3, characterized in that, The protein cross-linker is used at a concentration of 0.02-0.2 mM, preferably 0.05-0.1 mM.

5. The use or method according to any one of claims 1 to 4, characterized in that, The antibody comprises at least one of IgG, Fab, Fab', F(ab')2, Fv, scFv or VH; Preferably, the antibody is IgG; Preferably, the antibody comprises an antibody to hepatitis C virus or an antibody to human immunodeficiency virus.

6. The use according to any one of claims 1, 3-5, characterized in that, The immunoassay comprises an immunochromatographic immunoassay, an enzyme-linked immunosorbent assay or a chemiluminescent immunoassay; Preferably, the immunoassay comprises a photochemiluminescent assay, a magnetic particle chemiluminescent assay, a colloidal gold immunochromatographic assay, a latex immunochromatographic assay, a radioimmunoassay or a time-resolved fluorescence immunoassay.

7. An antibody reagent, the antibody reagent comprising an antibody and a protein cross-linker.

8. The antibody reagent of claim 7, wherein, The protein cross-linker is selected from a homologous cross-linker; Preferably, the homologous cross-linker is capable of reacting with amino groups and / or thiol groups of the antibody; Preferably, the homologous cross-linker comprises two identical activated reactive groups and a spacer arm between the activated reactive groups; Preferably, the activated reactive groups are selected from succinimide, maleimide, carbodiimide, methylol or imidoester; Preferably, the spacer arm is selected from polyethylene glycol; More preferably, the protein cross-linker is selected from Bis-(PEG)n-NHS, n is an integer selected from 2-30, preferably an integer selected from 8-15.

9. The antibody reagent according to claim 7 or 8, characterized in that, The antibody reagent comprises at least one of an antibody quality control, an antibody standard, an antibody calibrator or an antibody reference; Preferably, the protein cross-linker is used at a concentration of 0.02-0.2 mM, preferably 0.05-0.1 mM; The antibody comprises at least one of IgG, Fab, Fab', F(ab')2, Fv, scFv or VH; Preferably, the antibody is IgG; Preferably, the antibody comprises an antibody to hepatitis C virus or an antibody to human immunodeficiency virus.

10. A test kit comprising the antibody reagent of any one of claims 7-9.

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