Detection kit and method for eliminating nonspecific reactions in blood samples

Abstract

The present invention relates to the field of biological detection technology, and in particular to a detection kit and method for eliminating nonspecific reactions in blood samples. The present invention provides a detection kit comprising a first reagent and a second reagent; the first reagent comprises a buffer solution, 0.01-0.5wt% sodium alginate, 0.05-1wt% nonionic surfactant, and 0.001-0.1wt% animal serum; the pH value of the first reagent buffer system is 7.2-7.4; the second reagent comprises an antibody buffer solution labeled with latex microspheres; the pH value of the second reagent buffer system is 7.8-8.0. The present invention provides a detection kit and its application, which can effectively eliminate the interference of nonspecific reactions on the detection results of biochemical indicators by using latex immunoturbidimetry to detect biochemical indicators in serum and plasma, thereby improving the accuracy of the detection results of biochemical indicators.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a detection kit and method for eliminating nonspecific reactions in blood samples. Background Art

[0002] The basic principle of latex immunoturbidimetry is that antigen-antibody complexes are rapidly formed in a special buffer (including enzyme amplification systems, latex amplification systems, nanogold and silver amplification systems, etc.), causing turbidity in the reaction solution. The change in turbidity through aggregation reflects the amount of the substance to be tested in the sample. After calibration of the detection instrument, quantitative detection of the substance to be tested is achieved. Among them, the biochemical indicators used during the test mainly include inflammatory markers (high-sensitive C-reactive protein), myocardial markers (troponin I, creatine kinase isoenzymes), tumor markers (alpha-fetoprotein), trace proteins (cystatin C, β2-microglobulin), and other substances. Patients with symptoms of inflammatory diseases, cardiovascular diseases, tumor screening, and kidney diseases often develop a series of abnormal indicators. The quantitative detection of the substance to be tested by latex immunoturbidimetry can be used as a diagnostic or auxiliary diagnostic indicator.

[0003] However, for patients with autoimmune diseases and underlying diseases, autoimmune antibodies in the human body (such as rheumatoid factor, anti-mitochondrial antibodies, HAMA, etc.) often interfere with the test results, causing non-specific immune reactions and false positives, resulting in inaccurate test results and affecting the clinical judgment of the disease. For non-specific reaction samples, most biochemical analyzers have the ability to pre-treat the samples. Common methods include setting the sample to be automatically diluted before performing routine tests; you can also choose to manually dilute the sample and then convert the result after testing by multiplying the dilution factor. The above method can reduce the impact of non-specific interference to a certain extent, but this method cannot completely eliminate the impact of non-specific interference on the results, especially for substances with lower content. Due to the matrix effect after dilution, the concentration is lower, and there is a certain error in the test results, which in turn affects the accuracy of clinical testing. Summary of the Invention

[0004] The present invention provides a detection kit and method for eliminating nonspecific reactions in blood samples, so as to eliminate nonspecific reactions in blood samples and achieve high-sensitivity, high-accuracy and high-repeatability detection.

[0005] The present invention provides a detection kit comprising a first reagent and a second reagent;

[0006] The first reagent comprises a buffer solution, 0.01-0.5 wt% sodium alginate, 0.05-1 wt% nonionic surfactant, and 0.001-0.1 wt% animal serum; the pH value of the first reagent buffer system is 7.2-7.4;

[0007] The second reagent comprises an antibody buffer solution labeled with latex microspheres; the pH value of the second reagent buffer system is 7.8-8.0.

[0008] The present invention finds that in a detection kit based on latex immunoturbidimetry, when a solution system containing sodium alginate, a nonionic surfactant and animal serum within the above-mentioned dosage range is used as the first reagent, and the pH value of the first reagent buffer system is controlled to be 7.2-7.4, and the pH value of the buffer solution in the second reagent is controlled to be 7.8-8.0, the interference of nonspecific reactions on the detection results of biochemical indicators can be effectively eliminated, and the accuracy and sensitivity of the detection results of biochemical indicators can be significantly improved.

[0009] Preferably, the mass ratio of the sodium alginate to the nonionic surfactant is 1:5 to 2:1.

[0010] In the prior art, when sodium alginate is used in a detection kit based on latex immunoturbidimetry, it is usually used as a suspending agent, and its usage is often higher than 0.01-0.5wt%. However, the present invention unexpectedly discovered that when the usage of the sodium alginate is 0.01-0.5wt%, especially when the sodium alginate and the nonionic surfactant are in the above-mentioned usage relationship, it can also serve as a stabilizer in the first reagent, reducing the adsorption of nonspecific substances while significantly improving the stability of the first reagent, further improving the accuracy of the detection.

[0011] Preferably, the nonionic surfactant is one or more of Triton X-100, PROPETAL 120, PROPETAL 140, Brij-35, OXETAL 800, glyceryl stearate and glyceryl rosin.

[0012] Preferably, the animal serum is one or more of mouse serum, goat serum, calf serum and rabbit serum;

[0013] Preferably, in the first reagent, the nonionic surfactant is PROPETAL 120, and the animal serum is goat serum;

[0014] Alternatively, in the first reagent, the nonionic surfactant is one of PROPETAL140, a mixture of Brij-35 and PROPETAL140, and a mixture of Triton X-100 and OXETAL800, and the animal serum is mouse serum; preferably, the mass ratio of Brij-35 to PROPETAL140 is 1:5 to 1:1; the mass ratio of Triton X-100 to OXETAL800 is 1:2 to 2:1;

[0015] Alternatively, in the first reagent, the nonionic surfactant is a mixture of Brij-35 and PROPETAL120 and one of Triton X-100, and the animal serum is calf serum; preferably, the mass ratio of Brij-35 to PROPETAL120 is 1:2 to 1:1.

[0016] Preferably, the first reagent buffer solution is at least one of a HEPES buffer solution, a Tris buffer solution, a borate buffer solution, and a phosphate buffer solution; the second reagent buffer solution is at least one of a HEPES buffer solution and a Tris buffer solution;

[0017] More preferably, the concentration of the buffer solution in the first reagent and the second reagent is 50-100 mmol / L.

[0018] Preferably, the first reagent further contains 0.1-1 wt% of a high molecular polymer; preferably, the high molecular polymer is one or both of PVA1750 and PEG20000; more preferably, the high molecular polymer is PEG20000.

[0019] And / or, the first reagent further contains 1-3 wt% of an electrolyte; preferably, the electrolyte is a sodium salt; more preferably, the electrolyte is sodium chloride.

[0020] Preferably, the first reagent and the second reagent further contain a preservative respectively; the amount of the preservative in the first reagent and the second reagent is 0.05-2 wt %.

[0021] More preferably, the preservative in the first reagent is sodium azide, and the preservative in the second reagent is Proclin 300.

[0022] The present invention further provides a method for eliminating nonspecific reactions in blood samples, comprising: using latex immunoturbidimetry, sequentially mixing a serum or plasma sample with the first reagent and the second reagent in the above-mentioned detection kit, and then detecting its biochemical indicators.

[0023] Preferably, the rheumatoid factor content in the sample is ≤180 IU / mL, the anti-mitochondrial antibody is ≤100 ng / mL, and the HAMA is ≤100 ng / mL.

[0024] Preferably, the biochemical indicators are one or more of high-sensitivity C-reactive protein, troponin I, creatine kinase isoenzyme, alpha-fetoprotein, cystatin C and β2-microglobulin.

[0025] Preferably, the volume ratio of the first reagent to the second reagent is 1:5 to 5:1.

[0026] More preferably, when detecting high-sensitivity C-reactive protein, the volume ratio of the first reagent to the second reagent is 1:1 to 2:1;

[0027] When detecting troponin I, the volume ratio of the first reagent to the second reagent is 2:1 to 2.5:1;

[0028] When detecting creatine kinase isoenzymes, the volume ratio of the first reagent to the second reagent is 1.5:1 to 3:1;

[0029] When testing alpha-fetoprotein, the volume ratio of the first reagent to the second reagent is 3:1 to 5:1;

[0030] When detecting cystatin C, the volume ratio of the first reagent to the second reagent is 3:1 to 4:1;

[0031] When detecting β2-microglobulin, the volume ratio of the first reagent to the second reagent is 4.5:1 to 5.5:1.

[0032] Preferably, based on the total volume of the first reagent, when detecting high-sensitivity C-reactive protein, the volume fraction of the sample is 1-2%;

[0033] When troponin I, creatine kinase isoenzymes, and cystatin C were measured, the sample volume fractions were all 4–6%;

[0034] When cystatin C was detected, the volume fraction of the sample was 6–8%;

[0035] When β2-microglobulin was detected, the volume fraction of the sample was 1.4-1.6%.

[0036] As a preferred embodiment of the present invention, the detection kit includes a first reagent and a second reagent;

[0037] The first reagent comprises: based on the total amount of the first reagent, 50-75 mmol / L buffer solution, 0.01-0.5 wt% sodium alginate, 0.05-1 wt% nonionic surfactant, 0.001-0.1 wt% animal serum, 0.1-1 wt% PEG20000, 1-3 wt% sodium chloride, and 1.0-1.5 wt% preservative; the pH value of the first reagent buffer system is 7.2-7.4;

[0038] The second reagent includes: based on the total amount of the second reagent, 50-75 mmol / L buffer solution, 0.01-0.04 wt% antibody labeled with latex microspheres, 0.01-0.05 g / L magnesium sulfate, 0.65-0.85 g / L sodium chloride, 0.5-2 g / L bovine serum albumin and 1-2 g / L preservative; the pH value of the second reagent buffer system is 7.8-8.0.

[0039] Based on the above technical solution, the beneficial effects of the present invention are:

[0040] The present invention provides a detection kit and method for eliminating nonspecific reactions in blood samples. By adopting latex immunoturbidimetry to detect biochemical indicators in serum and plasma, the interference of nonspecific reactions on the detection results of biochemical indicators can be effectively eliminated, and the risk of false positive results of the reagent can be effectively reduced, thereby improving the accuracy of the detection results of biochemical indicators. DETAILED DESCRIPTION

[0041] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0042] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0043] Example 1 High-sensitivity C-reactive protein assay reagent for eliminating nonspecific reactions

[0044] This embodiment provides a detection kit including two components: a first reagent R1 and a second reagent R2. The pH value of the first reagent buffer system is 7.4, and the pH value of the second reagent buffer system is 8.0. The reagent composition is shown in Table 1 below.

[0045] Table 1 Example 1 Reagent composition

[0046]

[0047] Detection method: Use calibrators from Biosino Biotechnology Co., Ltd. and the operation steps on the biochemical analyzer are as follows:

[0048] Table 2 Detection methods

[0049]

[0050]

[0051] When the rheumatoid factor content in the test sample is ≤180IU / mL, the anti-mitochondrial antibody is ≤100ng / mL, and the HAMA is ≤100ng / mL, this method can effectively eliminate the interference of nonspecific immune reactions, thereby improving the accuracy of the test results of the high-sensitivity C-reactive protein assay reagent.

[0052] Example 2 Troponin I assay reagent for eliminating nonspecific reactions

[0053] This embodiment provides a detection kit including two components: a first reagent R1 and a second reagent R2. The pH value of the first reagent buffer system is 7.2, and the pH value of the second reagent buffer system is 7.8. The reagent composition is shown in Table 3 below.

[0054] Table 3 Example 2 Reagent Composition

[0055]

[0056]

[0057] Detection method: Use calibrators from Biosino Biotechnology Co., Ltd. and the operation steps on the biochemical analyzer are as follows:

[0058] Table 4 Detection method

[0059]

[0060] When the rheumatoid factor content in the test sample is ≤180IU / mL, the anti-mitochondrial antibody is ≤100ng / mL, and the HAMA is ≤100ng / mL, this method can effectively eliminate the interference of nonspecific immune reactions, thereby improving the accuracy of the test results of the high-sensitivity C-reactive protein assay reagent.

[0061] Example 3 Creatine kinase isoenzyme assay reagent for eliminating nonspecific reactions

[0062] This embodiment provides a detection kit including two components: a first reagent R1 and a second reagent R2. The pH value of the first reagent buffer system is 7.2, and the pH value of the second reagent buffer system is 8.0. The reagent composition is shown in Table 5 below.

[0063] Table 5 Example 3 Reagent Composition

[0064]

[0065]

[0066] Detection method: Use calibrators from Biosino Biotechnology Co., Ltd. and the operation steps on the biochemical analyzer are as follows:

[0067] Table 6 Detection method

[0068]

[0069] When the rheumatoid factor content in the test sample is ≤180IU / mL, the anti-mitochondrial antibody is ≤100ng / mL, and the HAMA is ≤100ng / mL, this method can effectively eliminate the interference of nonspecific immune reactions, thereby improving the accuracy of the test results of the creatine kinase isoenzyme assay reagent.

[0070] Example 4 Alpha-fetoprotein assay reagent for eliminating nonspecific reactions

[0071] This embodiment provides a detection kit including two components: a first reagent R1 and a second reagent R2. The pH value of the first reagent buffer system is 7.4, and the pH value of the second reagent buffer system is 7.8. The reagent composition is shown in Table 7 below.

[0072] Table 7 Example 4 Reagent Composition

[0073]

[0074]

[0075] Detection method: Use calibrators from Biosino Biotechnology Co., Ltd. and the operation steps on the biochemical analyzer are as follows:

[0076] Table 8 Detection method

[0077]

[0078] When the rheumatoid factor content in the test sample is ≤180IU / mL, the anti-mitochondrial antibody is ≤100ng / mL, and the HAMA is ≤100ng / mL, this method can effectively eliminate the interference of nonspecific immune reactions, thereby improving the accuracy of the test results of the alpha-fetoprotein assay reagent.

[0079] Example 5 Cystatin C assay reagent for eliminating nonspecific reactions

[0080] This embodiment provides a detection kit including two components: a first reagent R1 and a second reagent R2. The pH value of the first reagent buffer system is 7.2, and the pH value of the second reagent buffer system is 8.0. The reagent composition is shown in Table 9 below.

[0081] Table 9 Example 5 Reagent Composition

[0082]

[0083]

[0084] Detection method: Use calibrators from Biosino Biotechnology Co., Ltd. and the operation steps on the biochemical analyzer are as follows:

[0085] Table 10 Detection method

[0086]

[0087] When the rheumatoid factor content in the test sample is ≤180IU / mL, the anti-mitochondrial antibody is ≤100ng / mL, and the HAMA is ≤100ng / mL, this method can effectively eliminate the interference of nonspecific immune reactions, thereby improving the accuracy of the test results of the cystatin C assay reagent.

[0088] Example 6 β2-microglobulin assay reagent for eliminating nonspecific reactions

[0089] This embodiment provides a detection kit including two components: a first reagent R1 and a second reagent R2. The pH value of the first reagent buffer system is 7.4, and the pH value of the second reagent buffer system is 7.8. The reagent composition is shown in Table 11 below.

[0090] Table 11 Reagent composition of Example 6

[0091]

[0092] Detection method: Use calibrators from Biosino Biotechnology Co., Ltd. and the operation steps on the biochemical analyzer are as follows:

[0093] Table 12 Detection method

[0094]

[0095]

[0096] When the rheumatoid factor content in the test sample is ≤180IU / mL, the anti-mitochondrial antibody is ≤100ng / mL, and the HAMA is ≤100ng / mL, this method can effectively eliminate the interference of nonspecific immune reactions, thereby improving the accuracy of the test results of the β2-microglobulin assay reagent.

[0097] Comparative Example 1

[0098] This comparative example provides a detection kit having the same components and detection method as Example 1, except that goat serum is not added to the first reagent and is replaced by an equal amount of BSA.

[0099] Comparative Example 2

[0100] This comparative example provides a detection kit having the same components and detection method as those of Example 3, with the only difference being that the pH value of the solution in the first reagent R1 is changed to 7.0.

[0101] Comparative Example 3

[0102] This comparative example provides a detection kit, which is the same as the method in Example 3, except that the nonionic surfactant in the first reagent is replaced by an equal amount of a cationic surfactant, and the cationic surfactant is hexadecyltrimethylammonium bromide.

[0103] Comparative Example 4

[0104] This comparative example provides a detection kit, which is the same as the method in Example 3, except that the amount of sodium alginate used is 0.8 wt %.

[0105] Comparative Example 5

[0106] This comparative example uses clinically approved detection reagents.

[0107] Test Example 1

[0108] This group uses comparative example 1 to verify the technical effect of embodiment 1 of the present application, as shown in Table 13 for details.

[0109] Table 13 Tests of samples with different concentrations

[0110]

[0111] Test Example 2

[0112] This group uses multiple groups of comparative examples to verify the technical effects of Example 3 of the present application, see Table 14 for details.

[0113] Table 14 Tests of samples with different concentrations

[0114]

[0115] Test Example 3

[0116] Comparative Example 5 (clinically approved test reagent) and Example 2 were used to simultaneously measure a series of interfering sample concentrations. The measured values ​​of the samples were taken as the baseline values. The deviation percentage between the measured values ​​of the test reagent and the baseline values ​​in the interfering samples was calculated. A deviation of 10% or greater was considered to indicate interference. The test results are shown in Table 15.

[0117] Table 15 Experimental data of Example 2

[0118]

[0119]

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A detection kit, characterized in that comprising a first reagent and a second reagent; The first reagent includes a buffer solution, 0.01-0.5 wt% sodium alginate, 0.05-1 wt% nonionic surfactant, and 0.001-0.1 wt% animal serum; the pH value of the first reagent buffer system is 7.2-7.4; The second reagent includes an antibody buffer solution labeled with latex microspheres; the pH value of the second reagent buffer system is 7.8-8.0; The mass ratio of sodium alginate to nonionic surfactant is 1:5 to 2:1; In the kit for detecting high-sensitivity C-reactive protein, the nonionic surfactant is PROPETAL120 and the animal serum is goat serum; In the kit for detecting troponin I, the nonionic surfactant is a mixture of Brij-35 and PROPETAL140, the animal serum is mouse serum, and the mass ratio of Brij-35 to PROPETAL140 is 1:5 to 1:1; In the kit for detecting creatine kinase isoenzymes, the nonionic surfactant is a mixture of Brij-35 and PROPETAL120, the animal serum is calf serum, and the mass ratio of Brij-35 to PROPETAL120 is 1:2 to 1:1; In the kit for detecting alpha-fetoprotein, the nonionic surfactant is a mixture of Triton X-100 and OXETAL800, the animal serum is mouse serum, and the mass ratio of Triton X-100 to OXETAL800 is 1:2 to 2:1; In the kit for detecting cystatin C, the nonionic surfactant is Triton X-100, and the animal serum is calf serum; In the kit for detecting β2-microglobulin, the nonionic surfactant is PROPETAL 140, and the animal serum is mouse serum.

2. The detection kit according to claim 1, characterized in that The buffer solution of the first reagent is at least one of a HEPES buffer solution, a Tris buffer solution, a borate buffer solution and a phosphate buffer solution; the buffer solution of the second reagent is at least one of a HEPES buffer solution and a Tris buffer solution.

3. The detection kit according to claim 2, characterized in that The concentration of the buffer solution in the first reagent and the second reagent is 50-100 mmol / L.

4. The detection kit according to claim 1, characterized in that The first reagent also contains 0.1-1 wt% PEG20000; And / or, the first reagent further contains 1-3 wt % of sodium chloride.

5. The detection kit according to claim 1, characterized in that The first reagent and the second reagent also contain a preservative respectively; the amount of the preservative in the first reagent and the second reagent is 0.05-2wt%.

6. The detection kit according to claim 5, characterized in that The preservative in the first reagent is sodium azide, and the preservative in the second reagent is Proclin 300.

7. A method for eliminating nonspecific reactions in a blood sample, characterized in that: include: The serum or plasma sample is mixed with the first reagent and the second reagent in the detection kit according to any one of claims 1 to 6 in sequence by latex immunoturbidimetry, and then the biochemical indicators are detected.

8. The method for eliminating nonspecific reactions in a blood sample according to claim 7, wherein: The rheumatoid factor content in the sample is ≤180 IU / mL, the anti-mitochondrial antibody is ≤100 ng / mL, and the HAMA is ≤100 ng / mL.

9. The method for eliminating nonspecific reactions in a blood sample according to claim 8, characterized in that: The volume ratio of the first reagent to the second reagent is 1:5~5:

1.

10. The method for eliminating nonspecific reactions in a blood sample according to claim 9, wherein: When detecting high-sensitivity C-reactive protein, the volume ratio of the first reagent to the second reagent is 1:1 to 2:1; When testing troponin I, the volume ratio of the first reagent to the second reagent is 2:1 to 2.5:1; When detecting creatine kinase isoenzymes, the volume ratio of the first reagent to the second reagent is 1.5:1 to 3:1; When testing alpha-fetoprotein, the volume ratio of the first reagent to the second reagent is 3:1 to 5:1; When detecting cystatin C, the volume ratio of the first reagent to the second reagent is 3:1~4:1; When detecting β2-microglobulin, the volume ratio of the first reagent to the second reagent is 4.5:1 to 5.5:

1.

11. The method for eliminating nonspecific reactions in a blood sample according to claim 10, wherein: Based on the total volume of the first reagent, when detecting high-sensitivity C-reactive protein, the volume fraction of the sample is 1-2%; When troponin I, creatine kinase isoenzymes, and cystatin C were measured, the volume fraction of the sample was 4–6%; When cystatin C was detected, the volume fraction of the sample was 6–8%; When β2-microglobulin was detected, the volume fraction of the sample was 1.4~1.6%.

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