CN115112897B - Method for identifying biological false positive of antibody detection - Google Patents

Abstract

The invention belongs to the technical field of antigen-antibody detection, in particular to a method for identifying biological false positive of antibody detection, and discloses a method for identifying biological false positive of antibody detection, which is a set of denatured genetic engineering antigen immune technology for solving the problem of clinical sample biological false positive, namely nonspecific reaction. The method can be used for identifying the nonspecific reaction of various antigens at the same time, detecting the immune reaction of specific antibodies, avoiding the influence of biological false positive on clinical serological detection, and has important clinical application value.

Description

A method for identifying biological false positives in antibody detection

Technical Field

The invention belongs to the technical field of antigen-antibody detection, and in particular relates to a method for identifying biological false positives in antibody detection.

Background Art

Syphilis is caused by Treponema pallidum (TP) and is an important public health problem in the world today, especially in high-income countries. At present, although TP can be directly detected to provide a basis for the diagnosis of syphilis, it is often difficult to obtain suitable pathogen detection samples in clinical practice. Therefore, syphilis serological tests (including treponemal and non-treponemal serological tests) are still the preferred method for diagnosing and managing syphilis. Among them, non-treponemal tests are usually used for syphilis screening and monitoring the therapeutic efficacy of syphilis. This method is non-specific for syphilis screening, resulting in biological false positive reactions in samples of patients with certain diseases in clinical practice (such as the elderly, tumors, pregnant women, autoimmune diseases, infectious diseases, etc.), which can easily mislead clinicians. Treponema pallidum serological tests are mainly used for screening and confirmation of syphilis. The detection object is Treponema pallidum-specific antibodies, and the antigen used for detection is Treponema pallidum bacteria or purified natural antigens. Due to the high cost of Treponema pallidum strain reproduction, the difficulty in purifying specific antigens, and the low yield, the large-scale application of its specific detection reagents is limited.

Tuberculosis (TB) is a very serious public health problem in my country. Mycobacterium tuberculosis (Mtb) is the main pathogen of TB. Rapid and accurate diagnosis of Mycobacterium tuberculosis (MTB) infection is of great significance for the control of TB. However, the current serological detection of Mtb is also prone to biological false positives.

At present, there are generally four types of pathogen target antigens used for experimental diagnosis: natural antigens, purified antigens, genetically engineered antigens and artificially synthesized antigens. Among the four antigens, only the natural antigens retain the natural structure of the pathogen target antigens, and the antibodies used for detection are all specific antibodies. The other three antibodies are all natural structures of non-pathogen target antigens. When used to detect antibodies, the specificity will be affected to a certain extent. Therefore, the establishment of an efficient specific antigen screening method is of great significance for clinical medical etiology detection. With the development of biotechnology, genetically engineered antigens have replaced natural antigens for specific serological detection experiments such as clinical syphilis, and automated detection technologies such as enzyme-linked immunosorbent assay (ELISA), immunochromatography (Immunochromatography), chemiluminescence immunoassay (Chemiluminescence Immunoassay, CLIA), and chemiluminescent microparticle immunoassay (chemiluminescent microparticle immunoassay, CMIA) have been established, and the sensitivity of the experiment is also constantly improving, which is convenient for clinical large-scale screening. However, with the widespread use of high-sensitivity detection technology in clinical medicine, the problem of biological false positives in sample testing has also emerged, which has seriously affected the judgment of clinicians on disease diagnosis. The main reason for the above problems is that genetically engineered antigens will form non-specific reaction sites during the renaturation process, or the antigens will cause changes in the higher-order structure of the protein during the purification process, ultimately forming false positive reactions.

Therefore, it is necessary to design a set of denatured genetically engineered antigen immunization technologies to solve the biological false positives of clinical samples, that is, non-specific reactions.

Summary of the invention

In order to overcome the deficiencies of the above-mentioned prior art, the present invention discloses a method for identifying biological false positives in antibody detection, in which a plurality of denatured antigens and non-denatured antigens are simultaneously coated on a carrier in pairs linearly or dottedly, and then the corresponding specific antibodies are detected by immunoassay, which can be used to identify biological false positives of antibodies.

To achieve the above purpose, the technical solution adopted by the present invention is:

A method for identifying biological false positives in antibody detection, the method comprising the following steps:

S1. Dissolve multiple antigens in a denaturing buffer containing a protein disulfide bond reducing agent, centrifuge and collect the supernatant after high-temperature water bath to prepare treated antigens; then dissolve the same antigens in a buffer not containing a protein disulfide bond reducing agent, collect the supernatant after centrifugation to prepare non-treated antigens;

S2, coating the antigen supernatant of step S1 on a solid phase carrier in pairs of treated antigens and untreated antigens of the same type in a linear or dot pattern to prepare a solid phase carrier coated with the antigen;

S3, the solid phase carrier coated with the antigen is blocked with 1% skimmed milk powder and then cut into antigen detection strips;

S4. After soaking the antigen detection strip with the immunoblotting working solution, add the antibody sample to be tested, incubate, wash, add enzyme-labeled secondary antibody, wash, develop, and observe the results;

S5. Result interpretation: If both the untreated antigen and the treated antigen are positive, it means that there are specific antibodies against the coated antigen in the sample; if the untreated antigen is positive and the treated antigen is negative, it means that there are non-specific antibodies against the coated antigen in the sample, that is, there is a biological false positive; if both the untreated antigen and the treated antigen are negative, it means that there are no specific antibodies against the coated antigen in the sample.

Genetically engineered antigens express the primary structure (amino acid sequence) of proteins. The renatured antigens are non-natural structures with non-specific immune reaction sites. The present invention uses a protein disulfide bond reducing agent to open the secondary or tertiary structure of genetically engineered antigens, so that they are restored to the primary structure of proteins. The amino acid sequence-specific antigenic determinants are used as targets to detect specific antibodies, and are paired with non-melted antigens for detection, thereby identifying false positive reactions. Because the specific antigenic determinants generated by the amino acid sequence of the primary structure of proteins are natural structures and are specific, the antibodies detected must also be specific antibodies! However, the secondary or tertiary structure of the renatured antigens must not be natural structures, and they will be misfolded, resulting in biological false positive reactions.

Preferably, the denaturing buffer containing a protein disulfide bond reducing agent and the buffer not containing a protein disulfide bond reducing agent both use a Tris-HCl buffer with a pH of 8.0 and a concentration of 0.025 M.

Preferably, the protein disulfide bond reducing agent includes β-mercaptoethanol, dithiothreitol, and tris(2-chloroethyl)phosphate.

Preferably, the concentration of the protein disulfide bond reducing agent in the denaturation buffer is 0.1%.

Preferably, the high temperature water bath is a 100° C. water bath for 3-5 minutes.

Preferably, the antibodies to be tested include syphilis antibodies and tuberculosis antibodies. Of course, antibodies to be tested for other diseases are also applicable to the present invention.

Preferably, the solid phase carrier comprises NC membrane, PVDF membrane, glass slide, organic plastic sheet, silicon wafer. Specifically, the solid phase carrier is NC membrane.

Preferably, the antibody sample to be tested includes antibody serum or cerebrospinal fluid, and the added amount of the antibody serum or cerebrospinal fluid is 10-20uL.

Preferably, in step S1, the final concentration of each antigen in the denaturing buffer containing a protein disulfide bond reducing agent and the buffer not containing a protein disulfide bond reducing agent is 0.5-1.0 mg/mL.

Preferably, the 1% skim milk powder is 0.05M PBS buffer containing 1% skim milk powder, and the pH of the buffer is 7.4.

Preferably, the blocking time of 1% skimmed milk powder is 1-2 hours.

Preferably, the width of the antigen detection strip is 2-3 mm. Specifically, the width of the antigen detection strip is 2.5 mm.

Preferably, the steps of incubation, washing, adding enzyme-labeled secondary antibody, washing, and color development are conventional immunoblotting methods, specifically:

(1) Incubation: Place the multi-antigen membrane strip in the reaction tank, add 1 mL of working solution to each tank to soak the membrane strip, add 10-20 uL of the antibody sample to be tested, place on a rocking platform, and incubate at room temperature for 60 minutes.

(2) Washing: Discard the liquid in the tank and gently tap it dry on absorbent paper. Then add the working solution and place it on a rocking platform to wash three times, 1 mL per tank each time, and each time for 1 minute.

(3) Add enzyme-labeled secondary antibody: Use a pipette to accurately add 1 mL of working solution to each well, then add specific enzyme-labeled secondary antibody, place on a rocking platform, and incubate at room temperature for 30 minutes.

(4) Washing: Same as (2). That is, discard the liquid in the tank and gently tap it on absorbent paper, then add the working solution and place it on a rocking platform to wash 3 times, 1 mL per tank each time, each time for 1 minute.

(5) Color development: Add 1 mL of developer to each tank, shake well, incubate at room temperature for 5 minutes, discard the liquid in the tank, rinse twice with 0.05 M PBS buffer (PH = 7.4), discard the liquid, and observe the results in the tank.

The present invention also provides a kit for identifying biological false positives in antibody detection, the kit comprising an antigen detection strip, the antigen detection strip being cut from a solid phase carrier coated with an antigen, the antigen detection strip comprising a treated antigen and an untreated antigen, the treated antigen and the untreated antigen being distributed in pairs linearly or in dots, the treated antigen being an antigen that has been subjected to a high temperature water bath in a denaturing buffer containing a protein disulfide bond reducing agent, and the untreated antigen being an antigen that has been treated with a buffer not containing a protein disulfide bond reducing agent.

Preferably, the buffer used in the denaturing buffer containing a protein disulfide bond reducing agent and the buffer not containing a protein disulfide bond reducing agent are both Tris-HCl buffer with a pH of 8.0 and a concentration of 0.025 M; the protein disulfide bond reducing agent includes β-mercaptoethanol, dithiothreitol, and tris(2-chloroethyl) phosphate; and the concentration of the protein disulfide bond reducing agent in the denaturing buffer is 0.1%.

Preferably, the high temperature water bath is a 100° C. water bath for 3-5 minutes.

Preferably, the solid phase carrier includes NC membrane, PVDF membrane, glass slide, organic plastic sheet, silicon wafer.

Preferably, the antigen detection strip is formed by cutting a solid phase carrier coated with an antigen after being blocked with 1% skimmed milk powder for 1 hour.

Preferably, the kit further comprises reagents used for conventional immunoblotting.

The method of using the above kit is as follows:

1) Place the antigen test strip in the reaction tank, add 1mL of working solution to each tank to soak the strip, add 10-20uL of the antibody sample to be tested, place on a rocking platform, and incubate at room temperature for 60 minutes.

2) Washing: Discard the liquid in the tank and gently tap it dry on absorbent paper, then add working solution and place it on a rocking platform to shake and wash 3 times, 1 mL per tank each time, each time for 1 minute.

3) Incubation of enzyme-labeled antibodies: Use a pipette to accurately add 1 mL of working solution to each well, then add specific enzyme-labeled secondary antibodies, place on a rocking platform, and incubate at room temperature for 30 minutes.

4) Washing: Same as step 5.

5) Color development and termination: Add 1 mL of developer to each tank, shake well, incubate at room temperature for 5 minutes, discard the liquid in the tank, rinse twice with 0.05 M PBS buffer (PH = 7.4), discard the liquid, and observe the results in the tank.

6) Interpretation of results:

A: Both the untreated antigen and the treated antigen are positive, indicating that there are specific antibodies against the coated antigen in the sample.

B: The non-treated antigen is positive and the treated antigen is negative, indicating that there are non-specific antibodies against the coated antigen in the sample, that is, there is a biological false positive.

C: Both the untreated antigen and the treated antigen are negative, indicating that there is no specific antibody against the coated antigen in the sample.

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

The present invention discloses a method for identifying biological false positives in antibody detection, wherein multiple denatured antigens and non-denatured antigens are simultaneously coated on a carrier in pairs linearly or dot-shaped, and then the corresponding specific antibodies are detected by immunoassay, which can be used to identify biological false positives of antibodies. The method of the present invention can be used to identify non-specific reactions of multiple antigens at the same time, and can detect the immune reactions of specific antibodies, thereby avoiding the influence of biological false positives on clinical serological detection, and has important clinical application value.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of the antigen linear coating pattern;

FIG2 is a diagram showing the interpretation mode of syphilis antibody test results (A. both non-treated antigen and treated antigen are positive; B. non-treated antigen is positive and treated antigen is negative; C. part of treated antigen is negative, or both non-treated antigen and treated antigen are negative);

Fig. 3 is a diagram of the linear coating pattern of syphilis antigen;

FIG4 is a diagram showing the interpretation of the results of syphilis antibody testing (A. both non-treated antigen and treated antigen are positive; B. non-treated antigen is positive and treated antigen is negative; C. part of the treated antigen is negative, or both non-treated antigen and treated antigen are negative);

FIG5 is an example diagram of syphilis antibody test results (samples 1 and 2 are syphilis positive samples; samples 3, 4, 5, 6, 7, 8, 10, 11, 13, 15, and 16 are syphilis negative samples; sample 9 is a TP45 antigen false positive sample; sample 12 is a TP17 antigen false positive sample; sample 14 is a TP45 and TP17 antigen false positive sample);

Fig. 6 is a diagram of the linear coating pattern of tuberculosis antigen;

FIG7 is a diagram showing the interpretation mode of tuberculosis antibody test results (A. both untreated antigen and treated antigen are positive; B. untreated antigen is positive and treated antigen is negative; C. part of treated antigen is negative, or both untreated antigen and treated antigen are negative);

Fig. 8 is the serum test result of tuberculosis patients;

FIG9 shows the serum test results of autoimmune patients;

FIG10 is the test results of serum in the elderly group;

FIG. 11 shows the test results of the serum of the children group.

DETAILED DESCRIPTION

The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

Example 1 Establishment of a biological false positive identification method for antibody detection

(1) Antigen treatment: Dissolve various antigens in denaturing buffer (i.e., Tris-HCl buffer with a concentration of 0.025 M and pH = 8.0, containing 0.1% protein disulfide bond reducing agent, wherein the reducing agent is selected from β-mercaptoethanol, dithiothreitol or tris(2-chloroethyl)phosphate, etc.) to a final antigen concentration of 0.5-1.0 mg/mL, in a 100°C water bath for 3-5 minutes, and centrifuge at 10,000 rpm to obtain the supernatant for use (for treated antigens); then dissolve the same antigens in Tris-HCl buffer with a concentration of 0.025 M and pH = 8.0 to a final antigen concentration of 0.5-1.0 mg/mL, and centrifuge at 10,000 rpm to obtain the supernatant for use (for untreated antigens).

(2) The above antigens are coated linearly (or dotted) in pairs of non-treated antigens and treated antigens of the same type on a carrier (taking NC membrane as an example), as shown in FIG1 .

(3) The NC membrane coated with the antigen was blocked with 0.05 M PBS buffer (PH=7.4) containing 1% skimmed milk powder for 1 hour, and then cut into 2.5 mm wide antigen membrane strips for detection and set aside.

(4) Incubation of the sample to be tested: Place the antigen membrane strip in the reaction tank, add 1 mL of working solution [0.5% skim milk powder in 0.05 M PBS buffer (PH = 7.4)] to each tank to wet the membrane strip, then add 10-20 uL of the antibody sample to be tested, place on a rocking platform, and incubate at room temperature for 60 minutes.

(5) Washing: Discard the liquid in the tank and gently tap dry on absorbent paper. Then add working solution and place on a rocking platform to wash three times, 1 mL per tank each time, each time for 1 minute.

(6) Incubation of enzyme-labeled antibody: Use a pipette to accurately add 1 mL of working solution to each well, then add the enzyme-labeled secondary antibody, place on a rocking platform, and incubate at room temperature for 30 minutes.

(7) Washing: Same as step 5.

(8) Color development and termination: Add 1 mL of the color developer [0.01% 4-chloro-1-naphthol in 0.05 M PBS buffer (PH = 7.4)] to each tank, shake well, incubate at room temperature for 5 minutes, discard the liquid in the tank, rinse twice with 0.05 M PBS buffer (PH = 7.4), discard the liquid, and observe the results in the tank.

(9) Result interpretation: as shown in Figure 2:

A: Both the untreated antigen and the treated antigen are positive, indicating that there are specific antibodies against the coated antigen in the sample.

B: The non-treated antigen is positive and the treated antigen is negative, indicating that there are non-specific antibodies against the coated antigen in the sample, that is, there is a biological false positive.

C: Both the untreated antigen and the treated antigen are negative, indicating that there is no specific antibody against the coated antigen in the sample.

Example 2 A method for identifying biological false positives in clinical syphilis antibody detection

The method for identifying biological false positives in antibody detection established in Example 1 is used to identify biological false positives in clinical syphilis antibody detection. The specific method comprises the following steps:

(1) Purchase four genetically engineered antigens currently recognized in the world for clinical syphilis-specific antibody diagnosis: TP47, TP45, TP17, and TP15 (purchased from Xiamen Inboma Biotechnology Co., Ltd., but products from different suppliers may have different names) and store them at -20°C for future use.

(2) Treatment of genetically engineered antigens: The above four genetically engineered antigens were dissolved in a denaturing buffer (i.e., Tris-HCl buffer with a concentration of 0.025 M and pH = 8.0, containing 0.1% β-mercaptoethanol) to a final concentration of 0.5-1.0 mg/mL. After being incubated in a 100°C water bath for 3-5 minutes and centrifuged at 10,000 rpm, the supernatant was collected for later use (as the treated antigen); the above four identical genetically engineered antigens were dissolved in 0.025 M Tris-HCl buffer (pH = 8.0) to a final concentration of 0.5-1.0 mg/mL. After being centrifuged at 10,000 rpm, the supernatant was collected for later use (as the untreated antigen).

(3) The above four antigens are linearly coated on the carrier NC membrane in pairs of non-treated antigens and treated antigens of the same type, as shown in FIG3 .

(4) The NC membrane coated with the antigen was blocked with 0.05 M PBS buffer (pH = 7.4) containing 1% skimmed milk powder for 1 hour, and then cut into 2.5 mm wide antigen membrane strips for detection and set aside.

(5) Incubation of the specimen to be tested: Place the antigen membrane strip in the reaction tank, add 1 mL of working solution to each tank to soak the membrane strip, then add 10-20 uL of the clinical specimen to be tested (patient serum or cerebrospinal fluid, from the sample library of our hospital laboratory), place on a rocking platform, and incubate at room temperature for 60 minutes.

(6) Washing: Discard the liquid in the tank and gently tap it dry on absorbent paper. Then add the working solution and place it on a rocking platform to wash three times, 1 mL per tank each time, for 1 minute each time.

(7) Incubation of enzyme-labeled antibodies: Use a pipette to accurately add 1 mL of working solution to each well, add the enzyme-labeled secondary antibody (anti-human IgG or anti-human IgM enzyme-labeled secondary antibody), place on a rocking platform, and incubate at room temperature for 30 minutes.

(8) Washing: Same as step 6.

(8) Color development and termination: Add 1 mL of the color developer to each tank, shake well, incubate at room temperature for 5 minutes, discard the liquid in the tank, rinse twice with 0.05 M PBS buffer (PH = 7.4), discard the liquid, and observe the results in the tank.

10. Result interpretation: As shown in Figures 4 and 5, the result interpretation is as follows:

A: Both the untreated antigen and the treated antigen are positive, indicating that there are specific antibodies against the coated antigen in the sample, that is, there are specific antibodies against syphilis TP47, TP45, TP17, and TP15 antigens in the sample.

B: The non-treated antigen is positive and the treated antigen is negative, indicating that there are non-specific antibodies against the coated antigen in the sample, that is, the antibodies against syphilis TP47, TP45, TP17, and TP15 antigens are biological false positives.

C: Both the untreated antigen and the treated antigen are negative, indicating that there is no specific antibody against the coated antigen in the sample, and the antibody to the antigen is negative. If only the positive control band appears and the other bands do not appear, this sample is a syphilis-negative sample.

Example 3 A method for identifying biological false positives in clinical tuberculosis antibody detection

(1) Dissolve the four antigens, TB4, Ce85c (a secretory protein of Mycobacterium tuberculosis), L88a (a fusion expression fragment of three antigens CFP10+38KD+48KD of Mycobacterium tuberculosis), and Mtb8 (i.e., Rv0379, transferrin of Mycobacterium tuberculosis) in a denaturing buffer (i.e., a Tris-HCl buffer with a concentration of 0.025 M and a pH of 8.0, containing 0.1% β-mercaptoethanol) to a final concentration of 0.5-1.0 mg/mL. After centrifugation at 10000 rpm in a 100°C water bath for 3-5 minutes, remove the supernatant for later use (for treated antigens); dissolve the four identical antigens in a 0.025 M Tris-HCl buffer (pH=8.0) to a final concentration of 0.5-1.0 mg/mL. After centrifugation at 10000 rpm, remove the supernatant for later use (for untreated antigens).

(2) The above four antigens are linearly coated on the carrier NC membrane in pairs of non-treated antigens and treated antigens of the same type (as shown in FIG. 6 ).

(3) The NC membrane coated with the antigen was blocked with 0.05 M PBS buffer (pH = 7.4) containing 1% skimmed milk powder for 1 hour, and then cut into 2.5 mm wide antigen membrane strips for detection and set aside.

(4) Place the antigen membrane strip in the reaction tank, add 1 mL of working solution to each tank to soak the membrane strip, add 10-20 uL of clinical samples to be tested [tuberculosis group, non-tuberculosis disease group (autoimmune patient serum), healthy group serum (elderly serum and children serum) samples, all samples are from the sample library of our hospital laboratory], place on a rocking platform, and incubate at room temperature for 60 minutes.

(5) Washing: Discard the liquid in the tank and gently tap dry on absorbent paper. Then add working solution and place on a rocking platform to wash three times, 1 mL per tank each time, each time for 1 minute.

(6) Incubation of enzyme-labeled antibody: Use a pipette to accurately add 1 mL of working solution to each well, add the ready-to-use enzyme-labeled secondary antibody (anti-human IgG enzyme-labeled secondary antibody), place on a rocking platform, and incubate at room temperature for 30 minutes.

(7) Washing: Same as step 5.

(8) Color development and termination: Add 1 mL of color developer to each tank, shake well, incubate at room temperature for 5 minutes, discard the liquid in the tank, rinse twice with 0.05 M PBS buffer (PH = 7.4), discard the liquid, and observe the results in the tank.

(9) Result interpretation: As shown in Figure 7-11, the result interpretation instructions are as follows:

A: Both the untreated antigen and the treated antigen are positive, indicating that there are specific antibodies against the coated antigen in the sample, that is, there are specific antibodies against tuberculosis TB4, CeB5c, L88a, and Mtb8 antigens in the sample.

B: The non-treated antigen is positive and the treated antigen is negative, indicating that there are non-specific antibodies against the coated antigen in the sample, that is, the antibodies against tuberculosis TB4, CeB5c, L88a, and Mtb8 antigens are biological false positives.

C: Both the untreated antigen and the treated antigen are negative, indicating that there is no specific antibody against the coated antigen in the sample, and the antibody to the antigen is negative. If only the positive control band appears and the other bands do not appear, this sample is a tuberculosis antibody negative sample.

The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims (4)

1. A method for identifying biological false positives for antibody detection for non-diagnostic purposes, comprising the steps of:

S1, respectively dissolving a plurality of antigens in a denaturation buffer solution containing a protein disulfide bond reducing agent, carrying out high-temperature water bath at 100 ℃ for 3-5 minutes, and centrifuging to collect supernatant to prepare a treated antigen; respectively dissolving the same antigen in a buffer solution without protein disulfide bond reducing agent, centrifuging, and collecting supernatant to obtain non-treated antigen; in the denaturation buffer solution containing the protein disulfide bond reducing agent and the buffer solution without the protein disulfide bond reducing agent, the adopted buffer solutions are Tris-HCl buffer solution with the pH of 8.0 and the concentration of 0.025M; the concentration of the protein disulfide bond reducing agent in the denaturation buffer is 0.1%; the final concentration of each antigen in the denaturation buffer solution containing the protein disulfide bond reducing agent and the buffer solution without the protein disulfide bond reducing agent is 0.5-1.0mg/mL;

s2, coating antigen supernatant obtained in the step S1 on a solid phase carrier in pairs linearly or punctiform according to the same kind by using a treated antigen and a non-treated antigen, and preparing a solid phase carrier coated with the antigen;

s3, cutting the solid phase carrier coated with the antigen into antigen detection strips after the solid phase carrier coated with the antigen is sealed by 1% skimmed milk powder;

s4, soaking an antigen detection strip by using an immunoblotting working solution, adding an antibody sample to be detected, and observing a result after incubation, washing, adding an enzyme-labeled secondary antibody, washing and developing;

S5, judging a result: the non-treated antigen and the treated antigen are positive, which indicates that the specific antibody aiming at the coated antigen exists in the sample; non-treatment antigen positive, treatment antigen negative, the existence of non-specific antibody aiming at the coating antigen in the sample, namely the existence of biological false positive; both the non-treated antigen and the treated antigen were negative, indicating that no specific antibodies to the coated antigen were present in the sample.

2. A method for identifying biological false positives for antibody detection for non-diagnostic purposes according to claim 1, wherein the protein disulfide bond reducing agent comprises β -mercaptoethanol, dithiothreitol, tris (2-chloroethyl) phosphate.

3. The method for identifying biological false positives for antibody detection for non-diagnostic purposes according to claim 1, wherein the solid support comprises NC membrane, PVDF membrane, glass slide, organic plastic sheet, silicon sheet.

4. The method for identifying biological false positives for antibody detection for non-diagnostic purposes according to claim 1, wherein the antibody sample to be tested comprises serum or cerebrospinal fluid, and the amount of the added serum or cerebrospinal fluid is 10-20uL.