Method for detecting false positive by inhibiting antigen-antibody reaction and application thereof
By treating the samples with β-mercaptoethanol reducing agent, combined with spirochete gel agglutination test and affinity index judgment, the problem of false positive results in syphilis diagnosis was solved, and the accuracy of the detection and diagnostic efficiency were significantly improved.
Patent Information
- Application Number
- CN202411807353.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art is difficult to essentially inhibit nonspecific biological false positive interference, resulting in the occurrence of false positive results in syphilis diagnosis.
The samples were treated with β-mercaptoethanol as the reducing agent, and the positive samples were detected through the spirochete gel agglutination test, and the affinity index was calculated again to determine whether it was false positive.
Effectively eliminate false positive results caused by non-specific biological substances, improve the accuracy of testing, simplify the process of syphilis diagnosis, and reduce the mental burden and unnecessary medical measures of false positive patients.
Smart Images
Figure CN119643862A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical antigen-antibody detection, and relates to a method for inhibiting false positive antigen-antibody reaction detection and application thereof. Background Art
[0002] The Treponema pallidum gel agglutination test uses ultrasonically lysed Treponema pallidum as an antigen. Sensitized red gelatin particles bind to Treponema pallidum antibodies in human serum or plasma to produce a red agglutination reaction visible to the naked eye. The Treponema pallidum gel agglutination test (TPPA test) has high sensitivity and specificity and is a good confirmatory test for syphilis diagnosis. Patients with positive TPPA tests will remain positive for life even after anti-syphilis treatment due to the strong replication ability of memory cell antibodies and high sensitivity of specific tests. Therefore, it cannot be used as an indicator for observing the treatment effect. However, according to clinical observations, the serum of patients with early syphilis has a weak positive reaction such as TPPA test, which may turn negative over time. Specific tests can appear one week earlier for primary syphilis than non-specific tests. The first thing that appears in the syphilis process is IgM body, which can be detected from the serum in the second week after syphilis infection, while IgG antibodies can only be detected in the fourth week after infection. However, there are limitations to the experimental method. There are about 60 biological substances that can cause false positives for syphilis, and it is difficult to distinguish each one carefully.
[0003] CN115112897A discloses a method for identifying biological false positives in antibody detection, and proposes a method for identifying false positives by simultaneously coating multiple denatured antigens and non-denatured antigens in pairs linearly or dot-shaped on a carrier, and then detecting the corresponding specific antibodies through an immunoassay. The antigen treatment includes treating the antigen with a denaturing buffer (containing a protein disulfide bond reducing agent) and treating the antigen with a buffer without a reducing agent, and judging the presence of specific antibodies and non-specific antibodies by comparing the reaction results of the treated antigen and the non-treated antigen. This method provides a new experimental design idea. However, it does not essentially suppress the interference of non-specific biological false positives caused by various factors in the patient's body.
[0004] Therefore, there is an urgent need to provide a method that essentially suppresses nonspecific biological false positives. Summary of the invention
[0005] In view of the deficiencies in the prior art and actual needs, the present invention provides a method for inhibiting false positives in antigen-antibody reaction detection and its application, which can inhibit false positives in antigen-antibody reaction detection, effectively eliminate false positive results caused by non-specific biological substances, and improve the accuracy of detection.
[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:
[0007] In a first aspect, the present invention provides a method for inhibiting false positive detection of antigen-antibody reaction, the method comprising: subjecting a sample to be tested to a Treponema pallidum gel agglutination test, screening out positive samples, mixing the positive samples with a reducing agent and allowing them to stand, subjecting the sample to a Treponema pallidum gel agglutination test again after standing, and calculating an affinity index to determine whether it is a false positive.
[0008] The Treponema pallidum gel agglutination test uses ultrasonically lysed Treponema pallidum as the antigen. The sensitized red gelatin particles combine with the Treponema pallidum antibodies in human serum or plasma to produce a red agglutination reaction visible to the naked eye.
[0009] The present invention provides a method for treating a sample by using beta-mercaptoethanol as a reducing agent, which can inhibit false positive detection of antigen-antibody reaction, effectively eliminate false positive results caused by non-specific biological substances, and improve the accuracy of detection.
[0010] The present invention uses affinity index threshold 0 as the judgment of false positive and affinity index threshold 1 as the judgment of true positive, which has a scientific basis, and the judgment is concise and clear, because to judge whether the activity of Treponema pallidum antibody still exists, the affinity index ratio can be calculated by the result of the optimum β-mercaptoethanol 0.035mol / L concentration and 0 concentration β-mercaptoethanol combined with TPPA to judge whether the activity of the detection substance has changed. When the positive titer of the detection substance is consistent and the activity has not changed, the affinity index is equivalent to 1, which is a true positive patient. Once the interfering substance is removed and the titer becomes 0, the activity changes, and the affinity index is equivalent to 0, indicating that it is a false positive. This is extremely easy to judge for observing the false positive caused by the biology of Treponema pallidum disease, and it can be clearly found whether it is a false positive or a true positive through this simple affinity index judgment. This can bring good news to the diagnosis of syphilis patients, because some low-titration false positives that cause the diagnosis of syphilis in patients are followed up for 1 to 6 weeks in clinical practice, and some even need to go through ≥6 months to exclude false positives. The method of the present invention is both fast and simple, and can provide a scientific basis for the diagnosis of different groups of patients. It reduces the mental burden of pseudo-patients and the intervention of unnecessary medical measures, saves medical expenses, and creates favorable conditions for the national economy.
[0011] Preferably, the reducing agent comprises beta-mercaptoethanol.
[0012] Preferably, the volume ratio of the positive sample to the reducing agent is 1:(0.015-0.1).
[0013] The specific point values in the above 0.015-0.1 can be selected as 0.015, 0.025, 0.03, 0.06, 0.07, 0.1, etc.
[0014] Preferably, the standing time is 5-120 min (e.g., 5 min, 10 min, 20 min, 50 min, 100 min, 120 min). Preferably, the final concentration of the reducing agent is 0.022-0.179 mol / L (e.g., 0.022 mol / L, 0.04 mol / L, 0.05 mol / L, 0.08 mol / L, 0.10 mol / L, 0.15 mol / L, 0.179 mol / L).
[0015] Preferably, the affinity index is the ratio of the Treponema pallidum gelatin agglutination result of the test sample treated with a reducing agent to the Treponema pallidum gelatin agglutination result of the test sample not treated with a reducing agent.
[0016] Preferably, the judgment standard is that if the affinity index threshold is greater than 0.5 and less than 1, it is judged as a true positive; if the affinity index threshold is greater than 0 and less than or equal to 0.5, it is judged to be retained; if the affinity index threshold is equal to 0, it is judged as a false positive.
[0017] In a second aspect, the present invention provides a kit for inhibiting false positive detection of antigen-antibody reaction, wherein the kit contains β-mercaptoethanol.
[0018] In a third aspect, the present invention provides application of the method for suppressing false positive antigen-antibody reaction detection described in the first aspect in immune reaction detection.
[0019] Preferably, the immune response detection includes Treponema pallidum gelatin agglutination serological detection, human hepatitis A virus IgG antibody detection or antigen-antibody nonspecific reaction false positive detection.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] The present invention provides a method for treating a sample by using beta-mercaptoethanol as a reducing agent, which can inhibit false positive detection of antigen-antibody reaction, effectively eliminate false positive results caused by non-specific biological substances, and improve the accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The result graph is for determining the optimal final concentration of the reducing agent β-mercaptoethanol;
[0023] Figure 2 The results of the Treponema pallidum gel agglutination test before and after the serum samples were treated with β-mercaptoethanol;
[0024] Figure 3 The results of the method of the present invention for testing 16 known true positive and false positive serum samples are shown in the figure;
[0025] Figure 4This is a diagram of Western immunoblotting results. DETAILED DESCRIPTION
[0026] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.
[0027] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.
[0028] In the present invention, β-mercaptoethanol was purchased from Sigma Trading Co., Ltd.; the Treponema pallidum gel agglutination test (TPPA test) detection kit was from Fuji Rebio Co., Ltd. of Japan, and the Western blot kit provided by EURIOIMUM (Germany) was selected as the gold standard comparison method, and all operations were performed strictly in accordance with the reagent instructions.
[0029] Example 1
[0030] This embodiment uses β-mercaptoethanol to treat serum samples. The source: serum samples come from patients in Changning District Community Hospital, clinically diagnosed as suspected syphilis patients, and the specimens tested positive and negative by Treponema pallidum agglutination test, and then the syphilis treponema IgG antibody westblot immunoblot test of the gold standard method are screened out for true positive, true negative and false positive specimens of different titers. The results of the syphilis treponema gel agglutination test before and after β-mercaptoethanol treatment are compared with the consistency of the syphilis treponema IgG antibody westblot immunoblot test to determine the final dissociation concentration of β-mercaptoethanol. It is also used as the basis for the true and false positive detection of the syphilis treponema agglutination test.
[0031] Sample preparation: Collect and separate serum samples from the above subjects. β-mercaptoethanol treatment of serum samples: Use β-mercaptoethanol storage solution (1.43 mol / L) to treat true positive, true negative and false positive specimens of different titers into a series of different concentrations of β-mercaptoethanol (0.022-0.1 mol / L), with the volume ratio of serum and β-mercaptoethanol being 1:0.0154, 1:0.0245, 1:0.0315, 1:0.07 respectively. Mix with β-mercaptoethanol reducing agent and let stand at room temperature for 15 minutes. After standing, perform TPPA test on the test results and calculate the affinity index to determine whether it is a false positive.
[0032] The principle for selecting the final concentration of β-mercaptoethanol is to use the affinity index of true positives at different titers, the highest positive specimen titer with an affinity index of 1, and the false positive with an affinity index of 0 as the dissociation concentration for the final treatment of serum. The final concentration of β-mercaptoethanol in this experiment is 0.035 mol / L, and the volume ratio of the sample to the reducing agent is 1:0.0245.
[0033] Method for Treponema pallidum gelatin agglutination test:
[0034] Dilution of non-sensitized particles and sensitized particle antigen: Dilute the diluent to the working concentration.
[0035] Sample addition: Add 100 μL of diluent to each well of the microplate, and add 25 μL of the sample to be tested to each reaction well.
[0036] Serum dilution: Dilute the serum to be tested, using a 2-fold serial dilution for 4 or 6 tubes.
[0037] Add the diluted 1:40 (the 3rd tube) serum to the well containing 25 μL of non-sensitized particles, and add the diluted 1:80 (the 4th tube) serum to the well containing 25 μL of sensitized particle antigen, and mix well.
[0038] Incubation: Incubate the mixed samples at room temperature (25 °C) for 2 h.
[0039] Observation of results: After incubation, observe and record the agglutination phenomenon.
[0040] Judgment criteria for reaction images:
[0041] (1) The particles aggregate in a button shape, presenting a round shape with a uniform and smooth outer edge, which is negative (-), simply referred to as 0.
[0042] (2) The particles form a small ring, presenting a round shape with a uniform and smooth outer edge, which is (±), simply referred to as 80.
[0043] (3) The particle ring becomes significantly larger, and its outer edge is uneven and agglutinates杂乱地 (the original text seems incorrect here, assuming it should be "unevenly") in a disorderly manner, which is (+), simply referred to as 80.
[0044] (4) Uniform agglutination occurs, and the agglutinated particles extend in a film-like manner on the bottom as a whole, which is (卄), simply referred to as 80.
[0045] Result judgment criteria
[0046] (1) Positive
[0047] The reaction image of non-sensitized particles (the final dilution factor 1:40 self-control) is judged as (-), and the sensitized particle red gelatin particles (the final dilution factor for determination 1:80 determination) are positive, and the final judgment is positive (simply referred to as 80).
[0048] (2) Negative
[0049] Regardless of the reaction image of the non-sensitized particles, as long as the measurement image of the sensitized red gelatin particles (final dilution factor 1:80) is shown as (-), it is finally judged as negative (abbreviated as 0).
[0050] (3) Retention
[0051] When the reaction image of the non-sensitized particles (final dilution factor 1:40 self-control) was judged as (-) and the measurement image of the sensitized red gelatin particles (final dilution factor 1:80) was judged as (±), the final judgment was retained (abbreviated as 80).
[0052] The concentration determination results are as follows Figure 1 As shown in Figure 2, serum samples treated with β-mercaptoethanol at a final concentration of 0.035 mol / L can effectively correct false positive results and improve diagnostic accuracy. Figure 2 As shown, A is the negative quality control result of Treponema pallidum gel agglutination test, B is the true positive result of Treponema pallidum gel agglutination test after eliminating interference, C is the true negative result of Treponema pallidum gel agglutination test after eliminating interference, B' is the true positive result of Treponema pallidum gel agglutination test, C' is the false positive result of Treponema pallidum gel agglutination test, and D is the positive quality control result of Treponema pallidum gel agglutination test.
[0053] Example 2
[0054] In this example, 16 patients with a history of syphilis infection were selected and tested positive for Treponema pallidum gel agglutination test, of which 2 Treponema pallidum gel agglutination test results were false positives.
[0055] Group 1: The Treponema pallidum gel agglutination test was performed according to the experimental method and judgment criteria described in Example 1, and the final concentration of the reducing agent β-mercaptoethanol was 0.035 mol / L. The samples were 14 true positive samples of the Treponema pallidum gel agglutination test.
[0056] Group 2: The Treponema pallidum gel agglutination test was performed according to the experimental method and judgment criteria described in Example 1, and the final concentration of the reducing agent β-mercaptoethanol was 0.035 mol / L. The samples were 2 false positive samples of Treponema pallidum gel agglutination test.
[0057] The experimental results are as follows Figure 3 : The affinity index threshold of the first group (true positive) experimental results is 1, and the affinity index threshold of the second group (false positive) experimental results is 0.
[0058] Example 3
[0059] Western blot analysis verified the accuracy of the method of the present invention.
[0060] Western blot kit (lot number S210412BD; Tp15kDa, Tp17kDa, TP45kDa, Tp47kDa). The samples were 2 false positive syphilis serology samples and 14 true positive syphilis serology samples. The specific experimental methods were referred to the kit instructions. The results are shown in the figure. Figure 4 As shown, cases 1-2: serum samples of patients with false positive results in the gel agglutination test for Treponema pallidum and no specific antigen bands. Cases 3-16: serum samples of patients with positive results in the gel agglutination test for Treponema pallidum and at least one or more specific antigen bands. The diagnostic results of the gel agglutination test for Treponema pallidum performed by the Western blot detection method and the β-mercaptoethanol treatment sample method on 2 false-positive samples of TPPA and 14 true-positive samples were exactly the same, indicating that the method of the present invention is accurate and feasible.
[0061] In summary, the present invention provides a method for treating a sample using β-mercaptoethanol as a reducing agent, which can inhibit false positive detection of antigen-antibody reaction, effectively eliminate false positive results caused by non-specific biological substances, and improve the accuracy of detection.
[0062] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A method for inhibiting false positive detection of antigen-antibody reaction, characterized in that: The method comprises: performing a Treponema pallidum gel agglutination test on a sample to be tested, screening out a positive sample, mixing the positive sample with a reducing agent and letting it stand, performing a Treponema pallidum gel agglutination test again after standing, and calculating an affinity index to determine whether it is a false positive.
2. The method for inhibiting false positive detection of antigen-antibody reaction according to claim 1, characterized in that: The reducing agent includes beta mercaptoethanol.
3. The method for inhibiting false positive detection of antigen-antibody reaction according to claim 1 or 2, characterized in that: The volume ratio of the positive sample to the reducing agent is 1:(0.015-0.1).
4. The method for inhibiting false positive detection of antigen-antibody reaction according to any one of claims 1 to 3, characterized in that: The standing time is 5-120 min.
5. The method for inhibiting false positive detection of antigen-antibody reaction according to any one of claims 1 to 4, characterized in that: The final concentration of the reducing agent is 0.022-0.179 mol / L.
6. The method for inhibiting false positive detection of antigen-antibody reaction according to any one of claims 1 to 5, characterized in that: The affinity index is the ratio of the syphilis treponema gelatin agglutination result of the test sample treated with a reducing agent to the syphilis treponema gelatin agglutination result of the test sample not treated with a reducing agent.
7. The method for inhibiting false positive detection of antigen-antibody reaction according to any one of claims 1 to 6, characterized in that: The judgment standard is that if the affinity index threshold is greater than 0.5 and less than 1, it is judged as a true positive; if the affinity index threshold is greater than 0 and less than or equal to 0.5, it is judged to be retained; if the affinity index threshold is equal to 0, it is judged as a false positive.
8. A kit for inhibiting false positive detection of antigen-antibody reaction, characterized in that: The kit contains beta-mercaptoethanol.
9. Use of the method for inhibiting false positive detection of antigen-antibody reaction according to any one of claims 1 to 7 in immune response detection.
10. The use according to claim 9, characterized in that: The immune response detection includes syphilis Treponema gelatin agglutination serological detection, human hepatitis A virus IgG antibody detection or antigen-antibody nonspecific reaction false positive detection.