A composite buffer and its application in h. pylori typing and product

Abstract

The application provides a composite buffer and application and products thereof in Helicobacter pylori typing, and relates to the technical field of immunodiagnosis. The composite buffer provided by the application comprises Tris-HCl, NaCl, bovine serum albumin (BSA), polyethylene glycol nonylphenyl ether (NP40), methyl isothiazolinone (MIT), glycine, perfluorooctanoic acid and Proclin 300. The composite buffer provided by the application is combined with a chemiluminescence determination technology to detect Helicobacter pylori, and has the advantages of high sensitivity, good repeatability and strong anti-interference capability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunodiagnosis, and particularly relates to a compound buffer and application and products thereof in Helicobacter pylori typing. BACKGROUND

[0002] As an important pathogenic factor of chronic gastritis, peptic ulcer and gastric cancer, the precise typing detection of Helicobacter pylori has decisive significance for clinical diagnosis and treatment. With the rapid development of serological detection technology, the immunological analysis method based on plasma samples has become the mainstream diagnostic method. However, the complex matrix components (such as heterophilic antibodies, complement proteins, and lipid substances) in plasma often cause non-specific binding, which seriously affects the sensitivity and specificity of detection. Therefore, the development of an optimized buffer with anti-interference ability has become a key technical breakthrough point for improving detection performance.

[0003] Current Helicobacter pylori typing detection mainly relies on enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay. However, clinical data shows that in the unoptimized buffer, the false positive rate of plasma samples is high, mainly due to the following interference factors:

[0004] 1. Ion intensity fluctuation: Changes in the concentrations of sodium and potassium ions in plasma affect the antibody-antigen binding constant, resulting in poor repeatability of the detection signal;

[0005] 2. pH sensitivity: The activity of most enzyme markers (such as HRP) significantly decreases when the pH deviates from 6.0-7.5, resulting in a decrease in detection sensitivity;

[0006] 3. Matrix effect: Non-specific adsorption of plasma albumin to detection antibodies occupies the binding sites;

[0007] 4. Endogenous enzyme interference: Plasma enzymes such as peroxidase catalyze the decomposition of substrates, generating background noise.

[0008] The prior art CN117867067A provides a buffer for improving the detection sensitivity of bovine viral diarrhea virus, which includes 6-hydroxyhexanoic acid, phosphodiesterase, naphthalene acetic acid, morpholine ethanesulfonic acid, PEG, phenol red, EDTA, keratinase, propyl p-hydroxybenzoate, 5-acetylsalicylic acid, stearic acid, sodium chloride, disodium hydrogen phosphate, sodium caseinate, Tetronic 1370, Proclin300, and kanamycin sulfate, which significantly improves the accuracy of detection.

[0009] Therefore, there is an urgent need to optimize the buffer components used in Helicobacter pylori typing in order to achieve higher sensitivity, accuracy and anti-interference ability. SUMMARY

[0010] To solve the above problems, the present application provides a composite buffer and its application in Helicobacter pylori typing and products.

[0011] In one aspect, the present application provides a composite buffer, which comprises: Tris-HCl, NaCl, BSA, NP40, MIT, glycine, perfluorooctanoic acid and Proclin 300.

[0012] Specifically, the composite buffer comprises: 0.03-0.08M Tris-HCl, 0.1-0.2M NaCl, 0.5-2% (w / v) BSA, 0.05-0.2% (v / v) NP40, 0.05-0.2% (w / v) MIT, 0.1-0.5M glycine, 0.5-1g / L perfluorooctanoic acid and 0.05-0.2% (v / v) Proclin 300.

[0013] Further specifically, the composite buffer comprises: 0.03-0.05M Tris-HCl, 0.1-0.15M NaCl, 0.5-1% (w / v) BSA, 0.05-0.1% (v / v) NP40, 0.05-0.1% (w / v) MIT, 0.1-0.2M glycine, 0.5-0.8g / L perfluorooctanoic acid and 0.05-0.1% (v / v) Proclin 300.

[0014] Further specifically, the composite buffer comprises: 0.05-0.08M Tris-HCl, 0.15-0.2M NaCl, 1-2% (w / v) BSA, 0.1-0.2% (v / v) NP40, 0.1-0.2% (w / v) MIT, 0.2-0.5M glycine, 0.8-1g / L perfluorooctanoic acid and 0.1-0.2% (v / v) Proclin 300.

[0015] In another aspect, the present application provides the application of the above-mentioned composite buffer in Helicobacter pylori typing detection, which comprises: Helicobacter pylori typing detection for non-disease diagnosis or treatment.

[0016] In still another aspect, the present application provides the application of the above-mentioned composite buffer in preparing Helicobacter pylori typing detection products, which comprises: reagent kit, test strip or chip.

[0017] In still another aspect, the present application provides a product for Helicobacter pylori typing detection, which comprises the above-mentioned composite buffer.

[0018] Specifically, the product further comprises a calibrator.

[0019] Further specifically, the standards include but are not limited to: CagA calibrators, VacA calibrators and Urease calibrators.

[0020] Specifically, the products further include reagents required for chemiluminescence assay.

[0021] Further specifically, the reagents required for chemiluminescence assay include at least one of the following: magnetic beads, acridinium ester and lysine quenching solution.

[0022] In another aspect, the present application provides a detection method for H. pylori typing, which is used for non-disease diagnosis or treatment of H. pylori typing detection, and the detection method comprises the following steps:

[0023] (1) preparing magnetic microsphere reagent coated with antigens: preparing antigen-coated magnetic microspheres and dispersing them in the above-mentioned composite buffer;

[0024] (2) mixing the sample to be tested with the magnetic microsphere reagent;

[0025] (3) adding acridinium ester reagent for reaction, detecting the relative luminescence intensity, and judging the typing result of H. pylori, wherein the acridinium ester reagent comprises the above-mentioned composite buffer.

[0026] Specifically, in step (1), the antigens include: CagA antigens, VacA antigens and Urease antigens.

[0027] Compared with the prior art, the present application has the following advantages:

[0028] Using the composite buffer provided by the present application in combination with chemiluminescence assay technology for H. pylori typing detection has the advantages of high sensitivity, good repeatability and strong anti-interference ability. DETAILED DESCRIPTION

[0029] The present application will be further described in detail below in combination with specific examples, and the following examples are not used to limit the present application, but only to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions, and the materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0030] Basic example H. pylori typing detection steps

[0031] The reagents used and their purchase sources are shown in Table 1.

[0032] Table 1

[0033]

[0034] 1. Preparation of magnetic microsphere reagent coated with antigen:

[0035] Mix the magnetic microspheres with CagA antigen, VacA antigen and Urease antigen respectively, and stand for 30 min at 25℃. Then add 2% sucrose to block the excess sites, and react for 20 min at 25℃.

[0036] After washing, add the magnetic microspheres into the complex buffer respectively to obtain the magnetic microsphere reagent coated with CagA antigen, the magnetic microsphere reagent coated with VacA antigen and the magnetic microsphere reagent coated with Urease antigen.

[0037] The complex buffer is prepared according to the formulation of the examples and comparative examples for subsequent experimental verification.

[0038] 2. Preparation of acridinium ester reagent:

[0039] Dissolve the acridinium ester with DMSO to obtain a stock solution with a concentration of 10 μg / μL. Add the acridinium ester to the anti-human IgG antibody, and react for 15 min at 25℃. Then add the lysine quenching solution, and react for 15 min. Dials with 10 mM PBS to obtain the anti-human IgG antibody labeled with acridinium ester, and then dilute with the complex buffer to 0.25 μg / μL to obtain the acridinium ester reagent.

[0040] 3. Detection of H. pylori typing:

[0041] (1) Mix the sample to be tested with the magnetic microsphere reagent, so that the CagA antibody, VacA antibody and Urease antibody in the sample bind to the corresponding antigens (CagA, VacA, Urease) coated on the magnetic microspheres to form antigen-antibody complexes.

[0042] (2) Add the acridinium ester reagent to bind the anti-human IgG antibody to the acridinium ester to form a labeled antibody, then add the lysine quenching solution to react for 20 min, and finally dialyze with 10 mM PBS at pH 7.5. The labeled antibody binds to the antigen-antibody complex to form a magnetic bead-sample-acridinium ester complex.

[0043] (3) Separate the complex by magnetic field, and wash with PBS for 3 times to remove the unbound impurities.

[0044] (4) Detect the relative luminescence intensity of the complex by chemiluminescence immunoassay instrument. Compare the detection results with the Cutoff value (CO) confirmed by the calibrators to determine the positive and negative of the CagA antibody, VacA antibody and Urease antibody in the sample to be tested.

[0045] Examples 1-3

[0046] The composite buffer was prepared according to the components in Table 2.

[0047] Table 2

[0048]

[0049] Comparative Examples 1-3

[0050] The composite buffer was prepared according to the components in Table 3.

[0051] Table 3

[0052]

[0053] Experimental Example 1 Sensitivity

[0054] The positive reference of the three antibodies was added to the serum without H. pylori infection, the stock solution concentration was 1 μg / mL, and after dilution to different concentrations, the detection reagent prepared by different composite buffers was used for typing detection, each round of experiment was repeated for 10 times, the number of positive test results was counted, and the experimental results were shown in Tables 4-6.

[0055] Table 4 Sensitivity of Urease Antibody Detection

[0056]

[0057] Table 5 Sensitivity of CagA Antibody Detection

[0058]

[0059] Table 6 Sensitivity of VaeA Antibody Detection

[0060]

[0061] The experimental results showed that when the composite buffer provided by the application was used for typing detection of H. pylori, it had good sensitivity.

[0062] Experimental Example 2 Reproducibility

[0063] The positive reference of the three antibodies was added to the serum substitute, each antibody was diluted to 50 ng / mL, and after preparation of the preparation by the buffer of each example or comparative example, the sample to be tested was detected for 10 times, the average value and standard deviation of the 10 measurement results were calculated, and the coefficient of variation CV (%) was calculated, and the CV values of each antibody detection were shown in Table 7.

[0064] Table 7 Reproducibility Experimental Results

[0065]

[0066] The experimental results showed that the composite buffer reagent provided by the application had good reproducibility when used for typing detection of H. pylori.

[0067] Experimental Example 3 Anti-interference ability

[0068] Using the buffer and related reagents prepared in Example 1, Comparative Example 1 and Comparative Example 2, reagents were prepared according to the description in the basic example, different samples were detected, each sample was detected for 10 times, and the detection results are shown in Table 8.

[0069] Table 8

[0070]

[0071] The results show that when the composite buffer provided by the application is used for Helicobacter pylori typing detection, human hemoglobin, globulin, fibronectin and transferrin have no interference on the detection results, which shows that the composite buffer provided by the application has strong anti-interference ability.

[0072] Example 4 Clinical experiment

[0073] In the hospital, 112 blood samples were collected, including 12 positive samples of Helicobacter pylori infection and 100 negative samples, and the samples were detected using the buffer and related reagents provided by Example 1 of the application, and C 13 The results of the urea breath test were used as a reference to compare the test results, and each sample was detected for 10 times. The detection results are shown in Table 9.

[0074] Table 9

[0075]

[0076] Sensitivity =

[0077] Specificity =

[0078] As can be seen from Table 9, using C 13 Using the urea breath test results as a reference, the sensitivity of the Helicobacter pylori typing detection method of the application is 100.00%, the specificity is 99%, the total coincidence rate is 99.11%, and the Kappa value is 0.9551. It shows that when the composite buffer provided by the application is used for Helicobacter pylori typing detection, the consistency between the detection results and the actual diagnosis results is very good.

[0079] Comparative Example 1

[0080] Using commercial serological detection methods, the blood samples collected in Example 4 were detected, and the results of sensitivity and specificity are shown in Table 10.

[0081] Table 10

[0082]

[0083] The three kits used in Table 10 are all commercial kits which have obtained medical instrument registration certificate. However, according to the records in Table 10, it can be seen that the sensitivity and specificity of the above three commercial kits for detecting H. pylori are not as good as the present application.

[0084] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the scope of protection of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A composite buffer solution for chemiluminescent immunoassay of magnetic microparticles, characterized in that, The composite buffer solution comprises: 0.03-0.08M Tris-HCl, 0.1-0.2M NaCl, 0.5%-2% (w / v) BSA, 0.05%-0.2% (v / v) NP40, 0.05%-0.2% (w / v) MIT, 0.1-0.5M glycine, 0.5-1 g / L perfluorooctanoic acid, and 0.05%-0.2% (v / v) Proclin 300.

2. The application of the composite buffer solution of claim 1 in Helicobacter pylori typing detection, characterized in that, The application described is for Helicobacter pylori typing detection for non-disease diagnosis or treatment.

3. The application of the composite buffer solution of claim 1 in the preparation of Helicobacter pylori typing detection products, characterized in that, The products include reagent kits, test strips, or chips.

4. A magnetic particle chemiluminescent immunoassay kit for Helicobacter pylori typing detection, characterized in that, The kit includes the composite buffer solution as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The kit also includes calibrators.

6. The reagent kit according to claim 4, characterized in that, The kit also includes chemiluminescence assay reagents.

7. The reagent kit according to claim 6, characterized in that, The chemiluminescence assay reagent includes at least one of the following: magnetic beads, acridine ester, and lysine quenching solution.

8. A method for detecting Helicobacter pylori typing, characterized in that, The method is used for Helicobacter pylori typing detection for non-disease diagnosis or treatment, and includes the following steps: (1) Preparation of antigen-coated magnetic microsphere reagent: Prepare antigen-coated magnetic microspheres and disperse them in the composite buffer solution described in claim 1; (2) The sample to be tested is mixed with the magnetic microsphere reagent; (3) Add acridinium ester reagent to react, detect the relative luminescence intensity, and determine the typing results of Helicobacter pylori.

9. The detection method according to claim 8, characterized in that, In step (1), the antigen includes any one or more of CagA antigen, VacA antigen and Urease antigen.

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