Anti-interference reagent and application thereof

Abstract

The application discloses an anti-interference reagent and application thereof, and relates to the technical field of biological detection. The application provides the reagent which can reduce the interference in the immunodetection sample in the presence of the interference encountered in the existing antigen-antibody sandwich reaction. The reagent comprises 0.1-1 parts of a blocking agent, 10-25 parts of a non-ionic surfactant and 5-20 parts of fat-free protein. The reagent can be directly added into a detection system, can effectively avoid or reduce the false results in the sample test, and can maintain the accuracy and stability of the detection results.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically, to an anti-interference reagent and its application. Background Technology

[0002] In clinical testing, sometimes the test results do not match the patient's clinical symptoms, especially in sandwich reactions of antigen and antibody, such as in chemiluminescence, lateral chromatography, and immunoturbidimetric platforms. If there are some interfering substances in the sample matrix, it will inevitably affect the efficiency of the immune reaction and the accuracy of the reaction results, especially in liquid reactions and when the sample is only diluted by a low factor.

[0003] In actual testing, due to factors such as the patient's condition, dietary habits, and sampling methods, it is difficult to clearly determine whether the interference in the sample is caused by a single factor. In most cases, it is a combination of multiple interfering factors. How to eliminate or reduce false negative or false positive results is one of the urgent problems to be solved today.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-interference reagent and its application.

[0006] This invention is implemented as follows:

[0007] In a first aspect, embodiments of the present invention provide an anti-interference reagent, which, by weight, comprises: 0.1 to 1 part of an inhibitor, 10 to 25 parts of a nonionic surfactant, and 5 to 20 parts of a fat-free protein; wherein the inhibitor comprises an active inhibitor and a passive inhibitor, the active inhibitor being selected from at least one of anti-HAMA polyclonal antibodies and anti-RF polyclonal antibodies; and the passive inhibitor being selected from at least one of mouse IgG, goat IgG, and bovine IgG.

[0008] Secondly, embodiments of the present invention provide the application of the anti-interference reagent as described in the foregoing embodiments in immune detection, wherein the immune detection is not for the diagnosis of a disease or is directly for the purpose of detection.

[0009] Thirdly, the embodiments of the present invention provide the application of the anti-interference reagent as described in the foregoing embodiments in the preparation of sample pad treatment solution.

[0010] Fourthly, embodiments of the present invention provide a sample pad treatment solution, which includes: an anti-interference reagent as described in the foregoing embodiments.

[0011] Fifthly, embodiments of the present invention provide the application of the anti-interference reagent as described in the foregoing embodiments or the sample pad treatment solution as described in the foregoing embodiments in the preparation of immunochromatographic test strips.

[0012] In a sixth aspect, embodiments of the present invention provide an immunochromatographic test strip, which includes a base plate and a sample pad, a conjugation pad, a nitrocellulose membrane and an absorbent pad that are sequentially overlapped and supported on the base plate. The sample pad is obtained by soaking the cellulose membrane in the sample pad treatment solution as described in the foregoing embodiments and then drying it.

[0013] The present invention has the following beneficial effects:

[0014] This invention addresses the interference encountered in existing antigen-antibody sandwich reactions by providing a reagent that can reduce interference in immunoassay samples. By weight, the reagent comprises 0.1 to 1 part of an inhibitor, 10 to 25 parts of a nonionic surfactant, and 5 to 20 parts of a fat-free protein. It can be directly added to the detection system and can effectively avoid or reduce false results in sample testing, maintaining the accuracy and stability of the test results. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The results show the correlation between actual values ​​and plasma test values, as well as the correlation between plasma and whole blood test values.

[0017] Figure 2 The results are for the stability of formulations 4 and 5. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] This invention provides an anti-interference reagent, which, by weight, comprises: 0.1 to 1 part of an inhibitor, 10 to 25 parts of a nonionic surfactant, and 5 to 20 parts of a fat-free protein; wherein the inhibitor comprises an active inhibitor and a passive inhibitor, the active inhibitor being selected from at least one of anti-HAMA polyclonal antibodies and anti-RF polyclonal antibodies; and the passive inhibitor being selected from at least one of mouse IgG, goat IgG, and bovine IgG.

[0020] This invention reduces the impact of interference on test results in sample detection from two aspects. Firstly, it adds anti-interference reagents to bind with interfering substances, reducing further interaction between the interfering substances and the detection microspheres through steric hindrance. Secondly, it seals non-specific portions of the detection microsphere surface, thereby reducing non-specific interference from interfering substances. Active and passive blocking agents can reduce endogenous interference factors, while nonionic surfactants and lipoprotein-free proteins can seal the microsphere surface, reducing the interference of lipids on the test results.

[0021] The concentrations of each component specified above are beneficial for maintaining the stability of the reagent and further improving the accuracy of the detection.

[0022] The weight fractions of the blocker can be in the range of any one or any two of the following: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 parts.

[0023] The weight percentage of nonionic surfactant in the anti-interference reagent can be any one or any two of the following: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 parts.

[0024] The weight fractions of fat-free protein in the anti-interference reagent can be any one or any two of the following: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0025] Preferably, the mass ratio of the active blocking agent to the passive blocking agent is (0.2–0.3):(0.7–1.0), which further increases the stability of the reagent and the accuracy of detection. Specifically, this mass ratio can be any one of 0.2:0.7, 0.2:0.8, 0.2:0.9, 0.2:1.0, 0.3:0.7, 0.3:0.8, 0.3:0.9, or 0.3:1.0.

[0026] Preferably, the passive blocking agent comprises mouse IgG, goat IgG, and bovine IgG;

[0027] Preferably, in the passive blocking agent, the mass ratio of mouse IgG, goat IgG and bovine IgG is 0.1:(0.2-0.4):(0.2-0.4), and this ratio is preferably 0.1:0.3:0.3.

[0028] Preferably, the nonionic surfactant is selected from at least one of Brij35, Tetronic1307 and Tween20.

[0029] Preferably, the fat-free protein is selected from at least one of gelatin and bovine serum albumin.

[0030] The embodiments of the present invention provide the application of the anti-interference reagent as described in any of the foregoing embodiments in immune detection, wherein the immune detection is not for the direct purpose of diagnosing or treating a disease.

[0031] Immunological assays include antigen-antibody sandwich methods, such as those performed in chemiluminescence, lateral chromatography, and immunoturbidimetric platforms.

[0032] The immune detection method is not aimed at diagnosing or treating diseases, but rather at detecting targets in samples and figuring out how to perform the detection more effectively. For example, when the sample to be tested is an environmental sample, the direct purpose of the detection is to determine whether a target substance is present in the sample.

[0033] This invention also provides the application of the anti-interference reagent as described in any of the foregoing embodiments in the preparation of sample pad treatment solution.

[0034] In this article, "sample pad treatment solution" is understood to refer to the sample pad treatment solution required for preparing the sample pad in immunochromatographic test strips.

[0035] This invention provides a sample pad treatment solution, which includes an anti-interference reagent as described in any of the foregoing embodiments.

[0036] Preferably, the effective concentrations of each component of the anti-interference reagent in the sample pad treatment solution are as follows: 0.1–1 mg / mL of blocking agent, 10–25 mg / mL of nonionic surfactant, and 5–20 mg / mL of lipoprotein-free protein. Within this range, the technical effect of the sample pad treatment solution is superior. Specifically, the effective concentration of the blocking agent can be any one or any two of the following: 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, and 1 mg / mL.

[0037] The effective concentration of nonionic surfactants can be any one or any two of the following: 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, and 25 mg / mL.

[0038] The effective concentration of lipoprotein can be any one or any two of the following: 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, and 20 mg / mL.

[0039] The sample pad treatment solution also includes a treatment solution matrix, which can be selected from existing known sample pad treatment solutions. In a preferred embodiment, the treatment solution matrix includes: 2%–10% sucrose, 2%–10% anhydrous ethanol, 0.001%–0.1% preservative, and 79.9%–95.9% phosphate buffer. It should be noted that "%" here refers to a mass-volume ratio; for example, 10% means 10g / 100mL.

[0040] This invention also provides the application of the anti-interference reagent as described in any of the foregoing embodiments or the sample pad treatment solution as described in the foregoing embodiments in the preparation of immunochromatographic test strips.

[0041] In addition, this invention also provides an immunochromatographic test strip, which includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad that are sequentially overlapped and supported on the base plate. The sample pad is obtained by soaking a glass fiber membrane in the sample pad treatment solution as described in the foregoing embodiments and then drying it.

[0042] It is understood that the preparation of all parts except the sample pad can be carried out in accordance with existing publicly available processes. The specific parameters for soaking and drying the sample pad treatment solution can be referenced from existing processes, preferably drying in a vacuum drying oven for 2 hours. As long as the sample pad is prepared using the sample pad treatment solution provided in the embodiments of this invention, it falls within the protection scope of this invention.

[0043] Preferably, the detection target of the immunochromatographic test strip is any one of B-type natriuretic peptide antibody (BNP), N-terminal brain natriuretic peptide precursor antibody, C-reactive protein, and procalcitonin antibody.

[0044] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0045] Example 1

[0046] An anti-interference reagent, the formula of which is shown in Table 1.

[0047] Table 1. Formulation of anti-interference reagents

[0048]

[0049] Seven experimental groups were set up. Each group used an anti-interference reagent prepared with one of the formulations and added it to the treatment solution matrix (5% sucrose, 5% anhydrous ethanol, 0.1% preservative and 89.9% phosphate buffer) to obtain the sample pad treatment solution. The B-type natriuretic peptide (BNP) immunochromatographic test strips were prepared and tested on clinical samples for verification.

[0050] The preparation method of the B-type natriuretic peptide immunochromatographic test strip is as follows:

[0051] (1) Preparation of sample pad: Soak the glass fiber membrane in the sample pad treatment solution, place it in a vacuum drying oven and dry for 2 hours for later use.

[0052] (2) Preparation of conjugate pad: Immerse glass fiber membrane in conjugate pad treatment solution containing fluorescent microspheres for BNP detection, place in vacuum drying oven and dry for 2 hours for later use.

[0053] (3) 2 mg / mL BNP capture antibody and 2 mg / mL goat anti-mouse polyclonal antibody were fixed onto nitrocellulose membranes with a streaking buffer at a flow rate of 0.75 μL / cm as test lines and control lines, respectively. The membranes were dried at 37°C for 2 hours and then stored for later use. The streaking buffer consisted of 10 mM Bis-Tris pH 7.3, 1% sucrose and 1.5% trehalose, 0.5% BSA, and 0.02% Proclin 300.

[0054] (4) Paste the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad in sequence on the PVC base plate, and cut them into 3.2mm wide test strips with a chopper.

[0055] (5) Put the cut test strips into plastic cards and use a shell press to complete the test strips.

[0056] The test results are shown in Table 2.

[0057] Table 2. Sample test results for different formulations

[0058]

[0059] Note: The components of the non-blocking agent are the same as those of the sample pad treatment solution matrix.

[0060] As shown in Table 2, formulations 1 through 7 can effectively reduce the impact of HAMA on the test.

[0061] At the same time, correlation (Ri) was performed between the actual values ​​and the test values ​​under various conditions. 2 The comparison involves using the actual value as x and the test value under each formula as y to establish a linear function relationship. The results are shown in Table 3.

[0062] Table 3. Correlation of samples under different formulations

[0063]

[0064]

[0065] Formulas 4, 5, 6, and 7 showed R values ​​in sample ranges of 0-5000 pg / mL and 0-2000 pg / mL. 2 All values ​​were above 0.95, with a slope close to 0.9-1.1. Compared with the case without an inhibitor, this indicates that the anti-interference reagent formulation can effectively reduce interference in immunoassay samples and improve clinical relevance.

[0066] Example 2

[0067] Thirty plasma and whole blood samples were tested using test strips containing Formula 5, including six samples that were lipemic (marked with *). The test results are shown in Table 4.

[0068] Table 4. Results of plasma and whole blood tests

[0069]

[0070]

[0071] The test results are shown in Table 4, and a correlation comparison is made between the actual values ​​and plasma test values, as well as between plasma and whole blood test values. Figure 1 As shown, the correlation R 2 All values ​​were above 0.95, and the slope was close to 0.9-1.1. This shows that the anti-interference reagent formula can reduce the interference of lipemia on the test results and can be directly used for whole blood testing without being affected by red blood cells.

[0072] Example 3

[0073] After establishing standard curves for test strips of formulas 4, 5, 6, and 7, 36 test strips were placed at room temperature (16-25℃) for testing at different time points. The implementation method is as follows:

[0074] (1) Establish a standard curve: Use the prepared test strips to test 10 concentrations of 5500, 3200, 1600, 1200, 800, 400, 100, 50, 25, and 0 pg / mL. Each concentration is repeated three times and the average value is taken.

[0075] (2) Take out the test strips placed at room temperature at the time points of 1 month, 2 months, 3 months, 6 months, 9 months and 12 months to test the antigen solutions of 94 and 772 pg / mL.

[0076] The test results are shown in Table 5 and Figure 2 As shown.

[0077] Table 5. Room temperature stability of test strips

[0078]

[0079]

[0080] The table uses the coefficient of variation (CV) to represent the fluctuation of antigen concentration at different time points. A small CV indicates that the results at different time points fluctuate less. The CV results show that Formula 5 has relatively smaller fluctuations in results over 12 months compared to other formulas. Figure 2 The dashed line in the figure represents the result of the target value ±15%, and it can also be seen that formulation 5 is relatively more stable than other formulations.

[0081] The results above show that using a combination of anti-interference reagents, namely active and passive blocking agents combined with nonionic surfactants and lipoprotein-free proteins, can effectively reduce interference in immunoassay samples by competing with interfering substances and blocking non-specific parts of the surface of the detection microspheres. At the same time, the combination of anti-interference reagents mixed in a certain proportion can further improve the stability of the test strip, thereby improving the accuracy of the detection.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An interference-resistant reagent, characterized in that, The components are 0.1-1 part of blocking agent, 10-25 parts of non-ionic surfactant and 5-20 parts of fat-free protein by weight; The blocking agent comprises active blocking agent and passive blocking agent, the active blocking agent is anti-HAMA polyclonal antibody, and the mass ratio of the active blocking agent to the passive blocking agent is (0.2-0.3):(0.7-1.0). The passive blocking agent comprises mouse IgG, goat IgG and bovine IgG, and the mass ratio of the mouse IgG, the goat IgG and the bovine IgG in the passive blocking agent is 0.1:(0.2-0.4):(0.2-0.4).

2. The interference-resistant reagent of claim 1, wherein, The non-ionic surfactant is at least one selected from Brij35, Tetronic 1307 and Tween 20.

3. The interference-resistant reagent of claim 1, wherein, The fat-free protein is at least one selected from gelatin and bovine serum albumin.

4. The anti-interference reagent according to any one of claims 1-3 is applied in an immunoassay which is not directly aimed at diagnosis or treatment of a disease.

5. The anti-interference reagent according to any one of claims 1-3 is applied in preparation of a sample pad treatment solution.

6. A sample pad treatment fluid, characterized by, It comprises: The anti-interference reagent according to any one of claims 1-3.

7. The sample pad treatment fluid of claim 6, wherein, In the sample pad treatment solution, the acting concentrations of the components of the anti-interference reagent are as follows: 0.1-1 mg / mL of blocking agent, 10-25 mg / mL of non-ionic surfactant and 5-20 mg / mL of fat-free protein.

8. The anti-interference reagent according to any one of claims 1-3 or the sample pad treatment solution according to claim 6 or 7 is applied in preparation of an immunochromatographic test strip.

9. An immunochromatographic test strip comprising a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad which are successively overlapped and carried on the base plate, characterized in that, The sample pad is obtained by soaking a fiber membrane in the sample pad treatment solution according to claim 6 or 7 and then drying.

10. The immunochromatographic test strip according to claim 9, characterized in that, The detection target of the immunochromatographic test strip is any one of B-type natriuretic peptide antibody (BNP), N-terminal pro-brain natriuretic peptide antibody, C-reactive protein antibody and procalcitonin antibody.

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