An accurate, wide linear range serum amyloid A test kit

Abstract

The invention discloses an accurate serum amyloid A detection kit with a wide linear range, and relates to the field of biotechnology. The detection kit comprises reagent R1, reagent R2, a calibrator and a quality control product, and the reagent components are as follows: reagent R1: buffer, inorganic salt ions, accelerator, surfactant, isopropanol, preservative; reagent R2: buffer, sheep anti-human serum amyloid A antibody latex microspheres, bovine serum albumin, glycerol, D-trehalose, preservative. The invention has high accuracy and a wide linear range, and can be applied to blood cell analyzers and special protein analyzers for detection at the same time.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an accurate serum amyloid A detection kit with a wide linear range. Background Art

[0002] Serum amyloid A (SAA) is a precursor of tissue amyloid A and one of the acute phase response proteins. The level of SAA increases rapidly after the body is damaged by infection, trauma, inflammation, etc. In recent years, with the in-depth study of SAA gene regulation, protein structure and biological function, it has been found that SAA is also involved in the pathogenesis of chronic inflammatory diseases.

[0003] Serum amyloid A (SAA) is known for its role in secondary amyloidosis. It was isolated and identified from serum in 1976 and is a member of a highly conserved acute phase protein family. SAA, like C-reactive protein (CRP), is an acute phase protein that plays a role in inflammatory defense, immune response, and lipid metabolism, and can be used for the diagnosis, treatment, and prognosis follow-up of many diseases.

[0004] A large number of studies have confirmed that SAA has the following biological functions: (1) SAA has pro-inflammatory activity. When the mass concentration of SAA is as low as 10 μg / L, it has the ability to induce chemokines and chemotactic leukocyte migration; (2) SAA has been described as an angiogenic protein. When the mass concentration of SAA is greater than 10-60 mg / L, it can stimulate endothelial cell proliferation, adhesion, invasion and the formation of capillary-like structures, stimulating the formation of new blood vessels in the body; (3) At physiological levels, SAA acts as an opsonin for Gram-negative bacteria by binding to outer membrane protein A, thereby enhancing the phagocytosis of bacteria. (4) SAA has antiviral activity, but this antiviral activity may be limited to hepatitis C virus; (5) SAA has the ability to induce matrix-degrading enzymes; (6) There are contradictory descriptions of the role of SAA in regulating cholesterol transport: SAA (10 mg / L) is reported to inhibit the efflux of cholesterol from cells and reduce the delivery of cholesterol to damaged cells, while other reports show that SAA (10 mg / L) promotes cholesterol efflux. Nevertheless, more functions of SAA need to be further studied.

[0005] If the SAA concentration is less than 10 mg / L, the possibility of viral or bacterial infection is small; if it is 10-100 mg / L, it is a viral infection. If the concentration remains at 10-100 mg / L after 12-24 hours, it is likely to be a viral infection. If the concentration is 100-500 mg / L, it is likely to be an acute bacterial infection. A 30% decrease in SAA 24 hours after antibiotic treatment indicates that the treatment is effective and the prognosis is good. If the concentration is greater than 500 mg / L, it is a viral infection (severe) or bacterial infection. A 30% decrease in SAA 24 hours after antibiotic treatment indicates that the treatment is effective and the prognosis is good.

[0006] At present, the clinical method for detecting serum amyloid A is immunoturbidimetry, including transmission turbidimetry and scattering turbidimetry. At present, the instruments using scattering turbidimetry are mainly blood cell analyzers and special protein analyzers. After testing, it was found that there are large differences in the reagents used by the two instruments. Their linear range is narrow, and it is difficult to make consistent judgments in the high value range. For example, the linear range of SAA detection on the blood cell analyzer can only reach 300mg / L, while the special protein analyzer can reach 500mg / L. The clinical judgment of the two test results will be different. Summary of the invention

[0007] The invention provides an accurate serum amyloid A detection kit with a wide linear range, which can be applied to both a blood cell analyzer and a special protein analyzer for detection.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The present invention provides an accurate serum amyloid A detection kit with a wide linear range, comprising a reagent R1, a reagent R2, a calibrator and a quality control product, wherein:

[0010] The composition and content of the reagent R1 are as follows:

[0011]

[0012] The composition and content of the reagent R2 are as follows:

[0013]

[0014] The composition and content of the calibrator are as follows:

[0015]

[0016] The composition and content of the quality control product are as follows:

[0017]

[0018] Furthermore, the composition and content of the reagent R1 are as follows:

[0019] The composition and content of the reagent R2 are as follows:

[0020]

[0021]

[0022] The composition and content of the calibrator are as follows:

[0023]

[0024] The composition and content of the quality control product are as follows:

[0025]

[0026] Furthermore, the buffer is a combination of two or more of PBS buffer, glycine buffer, Tris buffer, MES buffer, and Goods buffer, and the buffer in reagent R1 and reagent R2 has a temperature of 25° C. and a pH value of 7.5.

[0027] Furthermore, the inorganic salt ions are one or more combinations of sodium chloride, potassium chloride, magnesium chloride and lithium chloride.

[0028] Furthermore, the accelerator is one or more combinations of polyethylene glycol-2000, polyethylene glycol-6000, and polyethylene glycol-8000.

[0029] Furthermore, the surfactant is one or more combinations of dodecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide and hexadecyltrimethylammonium chloride.

[0030] Furthermore, the preservative is procaine hydrochloride.

[0031] Furthermore, the ratio of the reagent R1 to the reagent R2 is 4:1.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) High accuracy: the relative deviation of the serum amyloid A test kit of the present invention is less than 5%;

[0034] 2) Wide linear range: The invention adds isopropanol to increase solubility, so that the linear range of the kit can reach 0-600 mg / L, which significantly increases the linear range;

[0035] 3) Strong versatility: The serum amyloid A detection kit of the present invention can be used for both blood cell analyzers and special protein analyzers without significant differences. DETAILED DESCRIPTION

[0036] In order to make the technical problems to be solved, technical solutions and advantages of the present invention more clear, they are described in detail below in conjunction with specific embodiments.

[0037] Embodiment 1:

[0038] The serum amyloid A detection kit of the present invention comprises reagent R1, reagent R2, a calibrator and a quality control product, wherein:

[0039] The composition and content of the reagent R1 are as follows:

[0040]

[0041] The composition and content of the reagent R2 are as follows:

[0042]

[0043] The composition and content of the calibrator are as follows:

[0044]

[0045]

[0046] The composition and content of the quality control product are as follows:

[0047]

[0048] The concentration of this quality control product is 10 mg / L.

[0049] Detection method: Use a blood cell analyzer (such as Zhizhen Bio C531, etc.) and a special protein analyzer (such as Guosai Aristo) to test quality control samples, whole blood samples, standard substances, and linear substances, respectively, and record the test results.

[0050] Embodiment 2:

[0051] The serum amyloid A detection kit of the present invention comprises reagent R1, reagent R2, a calibrator and a quality control product, wherein:

[0052] The composition and content of the reagent R1 are as follows:

[0053]

[0054] The composition and content of the reagent R2 are as follows:

[0055]

[0056]

[0057] The composition and content of the calibrator are as follows:

[0058]

[0059] The composition and content of the quality control product are as follows:

[0060]

[0061] The concentration of this quality control product is 10 mg / L.

[0062] The detection method is the same as that of Example 1.

[0063] Embodiment 3:

[0064] The serum amyloid A detection kit of the present invention comprises reagent R1, reagent R2, a calibrator and a quality control product, wherein:

[0065] The composition and content of the reagent R1 are as follows:

[0066]

[0067]

[0068] The composition and content of the reagent R2 are as follows:

[0069]

[0070] The composition and content of the calibrator are as follows:

[0071]

[0072] The composition and content of the quality control product are as follows:

[0073]

[0074] The detection method is the same as that of Example 1.

[0075] Control group 1:

[0076] A serum amyloid A (immunoscatter turbidimetry) detection kit commonly available on the market from a certain company was used, which is suitable for the C531 blood cell analyzer.

[0077] Control group 2:

[0078] The serum amyloid A (immunoscattering turbidimetry) detection kit commonly available on the market was used and was suitable for the Aristo special protein analyzer.

[0079] Accuracy test:

[0080] The reagents of Example 1, Example 2, Example 3 and two control groups were used to detect the quality control product (target value was 10 mg / L) and the standard substance (NIBSC 92 / 680 concentration was 156 mg / L) using a C531 blood cell analyzer and an Aristo special protein analyzer, respectively, and their relative deviations were calculated. The test results are shown in Tables 1, 2, 3, and 4:

[0081] Table 1 C531 test quality control results

[0082] frequency Example 1 Example 2 Example 3 Control group 1 Control group 2 1 10.2 9.9 10.3 10.5 12.2 2 10.2 10.0 10.4 10.5 12.6 3 10.1 9.8 10.2 10.6 12.5 average value 10.2 9.9 10.3 10.5 12.4 Relative deviation 2％ -1％ 3％ 5％ 24％

[0083] Table 2 Aristo test quality control results

[0084] frequency Example 1 Example 2 Example 3 Control group 1 Control group 2 1 10.1 9.8 10.2 15.3 10.8 2 10.2 9.9 10.3 15.7 10.8 3 10.0 9.9 10.2 15.6 10.7 average value 10.1 9.9 10.2 15.5 10.8 Relative deviation 1％ -1％ 2％ 55％ 8％

[0085] Table 3 C531 test results of standard substances

[0086] frequency Example 1 Example 2 Example 3 Control group 1 Control group 2 1 158 153 160 168 179 2 159 150 159 165 181 3 158 152 159 169 182 average value 158 152 159 167 181 Relative deviation 1.28％ -2.56％ 1.92％ 7.05％ 16.03％

[0087] Table 4 Aristo test standard substance results

[0088] frequency Example 1 Example 2 Example 3 Control group 1 Control group 2 1 157 150 160 200 175 2 159 151 158 202 178 3 154 153 159 198 171 average value 157 151 159 200 175 Relative deviation 0.64％ -3.21％ 1.92％ 28.21％ 12.18％

[0089] Relative deviation (%) = (measured mean value - target value of quality control product or standard substance) / target value of quality control product or standard substance × 100%.

[0090] As can be seen from Tables 1 to 4, when the reagents obtained in Examples 1, 2, and 3 are used to detect quality control products and standard substances by C531 blood cell analyzer and Aristo special protein analyzer, the relative deviations from the quality control products and standard substances are all less than 5%. The reagent in Example 1 is the best overall, and is better than the reagents in Control Group 1 and Control Group 2. At the same time, the reagents in Control Group 1 and Control Group 2 have very different results when detected by C531 blood cell analyzer and Aristo special protein analyzer, respectively, and cannot be applied to both instruments at the same time. This shows that the serum amyloid A detection kit of the present invention can be used for detection by C531 blood cell analyzer and Aristo special protein analyzer at the same time, and has high accuracy.

[0091] Correlation test:

[0092] 40 clinical whole blood samples were tested simultaneously on a C531 blood cell analyzer and an Aristo special protein analyzer using the reagents of Example 1, Example 2, Example 3 and Control Group 1 and Control Group 2, respectively. The test results are shown in Tables 5 and 6.

[0093] Table 5C531 correlation test results

[0094]

[0095]

[0096]

[0097] Table 6 Aristo correlation test results

[0098]

[0099]

[0100]

[0101] Relative deviation (%)=(measurement mean value of the example-measurement mean value of the control group) / measurement mean value of the control group×100%.

[0102] As can be seen from Table 5, the correlation coefficients of the reagents obtained in the above Examples 1, 2 and 3 with the control group 1 on the C531 blood cell analyzer are 0.9927, 0.9946 and 0.9935, respectively; as can be seen from Table 6, the correlation coefficients of the reagents obtained in the above Examples 1, 2 and 3 with the control group 2 on the Aristo special protein analyzer are 0.9996, 0.9996 and 0.9996, respectively, indicating that the present invention has a great correlation with the control groups of the applicable models, further demonstrating the reliability of the serum amyloid A detection kit of the present invention.

[0103] Linear range detection:

[0104] The linear high value whole blood sample (concentration 600 mg / L) was linearly diluted with the linear low value whole blood sample (concentration 2 mg / L) and mixed into 6 dilution concentrations (xi), as shown in Table 7. The above samples were tested respectively with the reagents of Example 1, Example 2, Example 3 and Control Group 1 and Control Group 2, and each dilution concentration was tested 3 times, and the mean value (yi) of the test results of each dilution concentration was calculated respectively.

[0105] Table 7 Linear dilution

[0106] Low value samples 0 0.2 0.4 0.6 0.8 1 High value samples 1 0.8 0.6 0.4 0.2 0 Theoretical concentration X 0.8X+0.2Y 0.6X+0.4Y 0.4X+0.6Y 0.2X+0.8Y Y

[0107] Note: X-represents the concentration of high-value samples and Y-represents the concentration of low-value samples

[0108]

[0109] Use the dilution concentration (xi) as the independent variable and the mean value of the test result (yi) as the dependent variable to find the linear regression equation. Calculate the correlation coefficient (r) of the linear regression.

[0110] The reagents of Example 1, Example 2, and Example 3 were tested using a C531 blood cell analyzer and an Aristo special protein analyzer, respectively. The reagents of Control Group 1 were tested using a C531 blood cell analyzer and the reagents of Control Group 2 were tested using an Aristo special protein analyzer. The test results are shown in Tables 8-15:

[0111] Table 8 C531 Example 1 Linear range test results

[0112]

[0113] Table 9 Aristo Example 1 Linear Range Test Results

[0114]

[0115] Table 10C531 Example 2 Linear range test results

[0116]

[0117]

[0118] Table 11 Aristo Example 2 Linear Range Test Results

[0119]

[0120] Table 12C531 Example 3 Linear range test results

[0121]

[0122] Table 13 Aristo Example 3 Linear Range Test Results

[0123]

[0124] Table 14C531 control group 1 linear range test results

[0125]

[0126] Table 15 Aristo control group 2 linear range test results

[0127]

[0128] It can be seen from Tables 8 to 15 that the linear correlation coefficients r of the kits of Examples 1, 2, and 3 detected on the C531 blood cell analyzer and the Aristo special protein analyzer are all greater than or equal to 0.9999, and the linear high values ​​can be detected; while the linear correlation coefficient r of the kit of Control Group 1 detected on the C531 blood cell analyzer is 0.9234, and the linear correlation coefficient r of the kit of Control Group 2 detected on the Aristo special protein analyzer is 0.9985, and the linear high values ​​detected by the kits of Control Group 1 and Control Group 2 can only reach about 300 mg / L, which shows that the kits of Examples 1, 2, and 3 have a wider linear range, and there is no obvious difference when used on the blood cell analyzer and the special protein analyzer at the same time.

[0129] In summary, the serum amyloid A detection kit of the present invention has high accuracy and a wide linear range, and can be applied to both blood cell analyzers and special protein analyzers for detection.

[0130] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An accurate serum amyloid A detection kit with a wide linear range, comprising reagent R1, reagent R2, calibrators and quality control products, wherein: The composition and content of the reagent R1 are as follows: The composition and content of the reagent R2 are as follows:

2. According to the accurate and wide linear range serum amyloid A detection kit of claim 1, the composition and content of the calibrator are as follows:

3. According to the accurate and wide linear range serum amyloid A detection kit of claim 1, the composition and content of the quality control product are as follows:

4. The accurate serum amyloid A detection kit with a wide linear range according to any one of claims 1 to 3, characterized in that: The buffer is a combination of two or more of PBS buffer, glycine buffer, Tris buffer, MES buffer, and Goods buffer. The temperature of the buffer in the reagent R1 and the reagent R2 is 25° C., and the pH value is 7.

5.

5. The accurate and wide linear range serum amyloid A detection kit according to claim 1, characterized in that: The inorganic salt ions are one or more combinations of sodium chloride, potassium chloride, magnesium chloride and lithium chloride.

6. The accurate serum amyloid A detection kit with a wide linear range according to claim 1, characterized in that: The accelerator is one or more combinations of polyethylene glycol-2000, polyethylene glycol-6000 and polyethylene glycol-8000.

7. The accurate serum amyloid A detection kit with a wide linear range according to claim 1, characterized in that: The surfactant is one or more combinations of dodecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide and hexadecyltrimethylammonium chloride.

8. The accurate serum amyloid A detection kit with a wide linear range according to any one of claims 1 to 3, characterized in that: The preservative is procaine hydrochloride.

9. The accurate serum amyloid A detection kit with a wide linear range according to claim 1, characterized in that: The ratio of the reagent R1 to the reagent R2 is 4:1.

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