Triglyceride kit for eliminating cross contamination between reagents and preparation method of triglyceride kit

Abstract

The invention relates to the field of in vitro diagnostic reagents. The invention discloses a triglyceride kit for eliminating cross contamination between reagents. The triglyceride kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components: a first buffer solution, a first surfactant, a second surfactant, a stabilizer, a first biological enzyme, a second biological enzyme, a third biological enzyme and an enzymatic substrate; and the R2 reagent comprises the following components: a second buffer solution, a catalytic enzyme, a chromogenic substrate and a chromogenic agent. The invention also provides a preparation method of the triglyceride kit for eliminating cross contamination between reagents. A biological enzyme method is used as a detection principle, the stability of a reaction system and biological enzyme is further improved through combined use of various surfactants, and research finds that after CHAPS or cholate is removed in a reagent R1, compounded sodium tripolyphosphate and dioctyl sodium sulfosuccinate are added, so that the stability of the reaction system can be improved, and the stability of the reaction system is improved; and cross contamination with a bile acid detection kit is eliminated.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and in particular to a triglyceride test kit capable of eliminating cross contamination between reagents and a preparation method thereof. Background Art

[0002] Triglycerides are organic compounds, fat molecules formed by long-chain fatty acids and glycerol. They are one of the most important components of blood lipids and a key indicator in blood lipid testing. They are used to assess risk factors for individuals with high cholesterol and are of great significance. Abnormally elevated serum triglyceride levels may be associated with a variety of diseases, including primary and secondary hyperlipoproteinemia, atherosclerosis, diabetes, kidney disease, and fatty liver. Atherosclerosis, in particular, can lead to serious cardiovascular and cerebrovascular diseases. Conversely, decreased serum triglyceride levels may be associated with conditions such as hyperthyroidism and adrenal insufficiency.

[0003] Conventional determination methods include dry chemical colorimetry and oxidase methods. The dry chemical colorimetry method is highly stable, intuitive, and rapid, but it cannot quantify the test results, is easily interfered by external factors, and is expensive, which has significant limitations in disease diagnosis. The oxidase method is highly efficient, sensitive, and specific, and can accurately quantify the level of total triglycerides. It is particularly suitable for various biochemical automatic analyzers and is conducive to the clinical promotion of rapid serum triglyceride testing.

[0004] Because enzymes have strict requirements for the external environment, bile salt surfactants (such as bile salts and CHAPS) are often added to the reaction system to improve the stability of the enzyme. For example, Chinese invention patent application number CN04498586A discloses a highly stable single-reagent serum triglyceride detection reagent, which includes 4-hydroxyethylpiperazineethanesulfonic acid-sodium glutamate buffer, bovine serum albumin, triethanolamine dodecyl sulfate, sodium perbenzoate, coenzyme FAD, polyvinyl alcohol AH-26, disodium ethylenediaminetetraacetic acid, sodium cholate, magnesium sulfate heptahydrate, 4-aminoantipyrine, N,N-dimethylaniline, Triton X-100, lipoprotein lipase, glycerol kinase, peroxidase, glycerol-3-phosphate oxidase, and sodium azide. While the bile salt added plays an important role in maintaining the stability of the reaction system and enzyme, its addition can also affect the clinical detection of other serum indicators. Clinically, related diseases such as fatty liver and alcoholic liver disease require simultaneous testing of triglycerides and bile acids. The presence of bile salt surfactants in traditional triglyceride assay kits can affect bile acid detection. Simply removing these bile salt surfactants can affect the measured values ​​and the stability of the reagents. Currently, most experiments place interfering and interfered-with items in different modules of a biochemical analyzer. The analysis items must be carefully positioned and programmed, and the instrument must be carefully maintained to reduce the incidence of cross-infection. This not only increases the workload of the experimenter, but also only reduces interference, not completely eliminating it. Therefore, a triglyceride assay kit with strong cross-contamination resistance, good stability, and strong anti-interference capabilities is needed. Summary of the Invention

[0005] The present invention aims to address the shortcomings of the existing technology by providing a triglyceride test kit and its preparation method that eliminate cross-contamination between reagents. Based on the bioenzymatic method, the present invention further improves the stability of the reaction system and the bioenzyme by combining multiple surfactants. Research has found that removing 3-[(3-cholamidopropyl)dimethylaminopropyl]-1-propanesulfonic acid inner salt (hereinafter referred to as "CHAPS") or bile salt from reagent R1 and then adding a compound of sodium tripolyphosphate and sodium dioctyl sulfosuccinate (hereinafter referred to as "AOT") improves the stability of the reaction system and eliminates cross-contamination with the bile acid detection kit.

[0006] To solve the above problems, the present invention provides the following technical solutions: In one aspect, the present invention provides a triglyceride kit for eliminating cross contamination between reagents, comprising an R1 reagent and an R2 reagent; wherein: The R1 reagent includes the following components: a first buffer, a first surfactant, a second surfactant, a stabilizer, a first biological enzyme, a second biological enzyme, a third biological enzyme and an enzymatic substrate; The R2 reagent includes the following components: a second buffer, a catalytic enzyme, a color-developing substrate, and a color-developing agent.

[0007] As described above, the triglyceride kit for eliminating cross contamination between reagents, the R1 reagent includes the following components at the following concentrations: First buffer 7-8 g / L; pH 7.0-8.0; The first surfactant is 0.5-4g / L; Second surfactant 0-4g / L; Stabilizer 1-2 g / L; First biological enzyme 2-4kU / L; Second biological enzyme 1-2kU / L; The third biological enzyme 1-2kU / L; Enzyme substrate 50-100mmol / L; The R2 reagent includes the following components at the following concentrations: Second buffer 7-8 g / L; pH 7.0-8.0; Catalytic enzyme 1-5 kU / L; Chromogenic substrate 0.5-1mmol / L; Color developer 0.5-1g / L.

[0008] Preferably, the R1 reagent comprises the following components at the following concentrations: First buffer 7-7.5 g / L; pH 7.0-7.5; First surfactant 1-2g / L; Second surfactant 1-2 g / L; Stabilizer 1-2 g / L; First biological enzyme 2-3kU / L; Second biological enzyme 1-2kU / L; The third biological enzyme 1-2kU / L; Enzyme substrate 50-80mmol / L; The R2 reagent includes the following components at the following concentrations: Second buffer 7-7.5 g / L; pH 7.0-7.5; Catalytic enzyme 3-5 kU / L; Chromogenic substrate 0.5-1mmol / L; Color developer 0.5-1g / L.

[0009] In the triglyceride kit for eliminating cross contamination between reagents as described above, the first buffer and the second buffer are each selected from any one of MOPS buffer, TRIS buffer, HEPES buffer, MES buffer, and PB buffer. Preferably, the first buffer and the second buffer are each selected from MOPS buffer.

[0010] In the triglyceride kit for eliminating cross-contamination between reagents as described above, the first surfactant is selected from any one of sodium tripolyphosphate, polyoxyethylene lauryl ether, cetyltrimethylammonium chloride, PEG6000, and CHAPS. Preferably, the first surfactant is selected from any one of sodium tripolyphosphate, PEG6000, and CHAPS. Most preferably, the first surfactant is selected from sodium tripolyphosphate.

[0011] In the triglyceride test kit for eliminating cross-contamination between reagents as described above, the second surfactant is selected from any one of dioctyl sodium sulfosuccinate, Tween-20, Triton X-100, and PEG 6000. Preferably, the second surfactant is selected from dioctyl sodium sulfosuccinate or PEG 6000. Most preferably, the second surfactant is selected from dioctyl sodium sulfosuccinate.

[0012] In the triglyceride test kit described above, which eliminates cross-contamination between reagents, the first surfactant is sodium tripolyphosphate, and the second surfactant is sodium dioctyl sulfosuccinate. The mass ratio of sodium tripolyphosphate to sodium dioctyl sulfosuccinate is (1:2) to (2:1). Under these conditions, since CHAPS is a bile acid analog, carryover contamination occurs during triglyceride testing, leading to a spike in bile acid values ​​during subsequent testing, significantly increasing the false positive rate. However, after replacing the two surfactants, sodium tripolyphosphate and sodium dioctyl sulfosuccinate, the prepared triglyceride test kit has a minimal effect on bile acid values, with deviations within 10%, demonstrating excellent results. Most preferably, the mass ratio of sodium tripolyphosphate to sodium dioctyl sulfosuccinate is 2:1. Under these conditions, the prepared triglyceride test kit achieves optimal linearity, analytical sensitivity, repeatability, and accuracy, while maintaining stable and accelerated stability.

[0013] The principles of the present invention are as follows: The triglyceride test kit for eliminating cross contamination between reagents as described above, preferably the first surfactant is sodium tripolyphosphate, and preferably the second surfactant is diisooctyl sulfosuccinate sodium.Under these conditions, the surfactant sodium tripolyphosphate added in the present invention is compounded diisooctyl sulfosuccinate sodium, which is played a role by the mechanisms such as metal chelation, pH buffering, solubilization dispersion, etc., and reagent stability, sensitivity and anti-interference ability can be improved by synergistic effect after mixing. Relative to the surfactant using CHAPS or bile salts in reagent R1, compounded sodium tripolyphosphate and diisooctyl sulfosuccinate are added, the stability of the reaction system can be improved, the cross contamination between bile acid detection kit is eliminated, the present invention provides a kind of metal ion chelation and dispersion effect enhanced by surfactant, the degradation of biological enzyme in the formula is synergistically delayed, the shelf life of the test kit is extended, and reagent stability is optimized.

[0014] In the triglyceride kit for eliminating cross contamination between reagents as described above, the stabilizer is selected from any one of EDTA Na2, trehalose, NaCl, and KCl. Preferably, the stabilizer is selected from EDTA Na2.

[0015] In the triglyceride kit for eliminating cross contamination between reagents as described above, the first biological enzyme is lipoprotein lipase, the second biological enzyme is glycerol phosphate oxidase, and the third biological enzyme is peroxidase.

[0016] In the triglyceride kit for eliminating cross-contamination between reagents described above, the chromogenic substrate is 4-aminoantipyrine (hereinafter referred to as "4-AAP"); the chromogenic agent is any one of sodium 3,5-dichloro-2-hydroxybenzenesulfonate (hereinafter referred to as "DHBS"), N-ethyl-N-(3-sulfopropyl)-3-methylaniline, and 3-methyl-2-benzothiazolinonehydrazone. Preferably, the chromogenic agent is sodium 3,5-dichloro-2-hydroxybenzenesulfonate.

[0017] As some preferred embodiments of the present invention, in the triglyceride kit for eliminating cross contamination between reagents as described above, the R1 reagent includes the following components in the following concentrations: MOPS7-8 g / L; pH 7.0-8.0; Sodium tripolyphosphate 0.5-4g / L; Sodium dioctyl sulfosuccinate 0.5-4g / L; EDTA Na21-2 g / L; Lipoprotein lipase 2-4 kU / L; Glycerol phosphate oxidase 1-2 kU / L; Peroxidase 1-2 kU / L; Adenosine triphosphate 50-100mmol / L; The R2 reagent includes the following components at the following concentrations: MOPS7-8 g / L; pH 7.0-8.0; Glycerol kinase 1-5 kU / L; 4-AAP0.5-1mmol / L; DHBS0.5-1g / L.

[0018] Based on the same inventive concept, the present invention provides a method for preparing the triglyceride kit for eliminating cross contamination between reagents as described above, comprising the following steps: (1) Preparation of R1 reagent According to the component content of R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 7.0-8.0 using a first pH adjusting solution. Then, the first surfactant, the second surfactant, the stabilizer, the first biological enzyme, the second biological enzyme, the third biological enzyme and the enzymatic substrate are added in sequence and stirred evenly to obtain R1 reagent; (2) Preparation of R2 reagent According to the component content of R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.0-8.0 using a second pH adjusting solution. Then, the catalytic enzyme, the chromogenic substrate, and the chromogenic agent are added in sequence and stirred evenly to obtain R2 reagent.

[0019] Compared with the existing technology, the effects and advantages of the present invention are: 1. The present invention provides a triglyceride test kit that eliminates cross-contamination between reagents. Since CHAPS is a bile acid analog, there is carryover contamination when detecting triglycerides, resulting in a surge in the measured value when subsequently detecting bile acid, thereby causing a significant increase in the false positive rate of the sample. By adding a compounded sodium tripolyphosphate and sodium dioctyl sulfosuccinate to replace the bile salt or CHAPS in the R1 reagent, the cross-contamination between the bile acid detection kit can be eliminated. The prepared triglyceride test kit has little effect on the bile acid measurement value, with a deviation within 10%, and a better effect.

[0020] 2. The present invention provides a triglyceride test kit that eliminates cross-contamination between reagents and can significantly improve the stability of the reaction system and enzyme, especially the opening stability and accelerated stability. It also has good sensitivity, high test accuracy, high consistency with the measured values ​​of commercially available reagents, and a correlation coefficient greater than 0.99, effectively extending the real-time stability of the serum triglyceride detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the linear curve graph of experimental group 5 of the present invention; Figure 2This is a correlation diagram between the experimental group 5 of the present invention and the measured values ​​of commercially available reagents. DETAILED DESCRIPTION

[0022] The following will be combined with the contents of the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are intended solely for the purpose of describing specific embodiments and are not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] Example 1 This embodiment provides a triglyceride kit that eliminates cross contamination between reagents. The preparation method of the kit comprises the following steps: (1) Preparation of R1 reagent: According to the component content of R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 7.4 using a first pH adjusting solution. Subsequently, the first surfactant, the second surfactant, the stabilizer, the first biological enzyme, the second biological enzyme, the third biological enzyme and the enzymatic substrate are added in sequence and stirred evenly to obtain R1 reagent.

[0027] In this embodiment, the first buffer solution is a MOPS buffer solution prepared by dissolving MOPS in water, the first pH adjusting solution is an 8 mol / L sodium hydroxide solution adjusted to a pH of 7.4, the first surfactant is 3-[(3-cholamidopropyl)dimethylaminopropyl]-1-propanesulfonic acid inner salt (CHAPS), the stabilizer is EDTA Na2, the first biological enzyme is lipoprotein lipase, the second biological enzyme is glycerol phosphate oxidase, the third biological enzyme is peroxidase, and the enzymatic substrate is adenosine triphosphate.

[0028] After preparation, the final concentrations of the components of R1 reagent are: MOPS 7.45 g / L pH 7.4 CHAPS1.0g / L EDTA Na21.54g / L Lipoprotein lipase 2.5 kU / L Glycerol phosphate oxidase 1.5 kU / L Peroxidase 1.2 kU / L Adenosine triphosphate 50mmol / L (2) Preparation of R2 reagent: According to the component content of R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.4 using a second pH adjusting solution. Then, the catalytic enzyme, the chromogenic substrate, and the chromogenic agent are added in sequence and stirred evenly to obtain R2 reagent.

[0029] In this embodiment, the second buffer solution is a MOPS buffer solution prepared by dissolving MOPS in water, the second pH adjusting solution is an 8 mol / L sodium hydroxide solution, the catalytic enzyme is glycerol kinase, the chromogenic substrate is 4-aminoantipyrine (4-AAP), and the chromogenic agent is sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS).

[0030] After preparation, the final concentrations of the components of R2 reagent are: MOPS 7.48 g / L pH 7.4 Glycerol kinase 5kU / L 4-AAP0.5mmol / L DHBS0.5g / L Example 2 Referring to the preparation method of the triglyceride detection kit of Example 1, the kits of the following multiple experimental groups were prepared according to the table below, wherein Experimental Group 1 is the technical solution of Example 1.

[0031]

[0032] Among them, the kits of experimental groups 1 to experimental groups 5 were subjected to subsequent kit linearity, analytical sensitivity, repeatability, and accuracy experiments together; the kits of experimental groups 1, experimental groups 5, and Example 5 were subjected to subsequent kit cross-contamination experiments together; and the kits of Example 1, Example 3, Example 4, and Example 5 were subjected to subsequent kit stability experiments together.

[0033] Example 3 This embodiment provides a triglyceride kit that eliminates cross contamination between reagents. The preparation method of the kit comprises the following steps: (1) Preparation of R1 reagent: According to the component content of R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 7.4 using a first pH adjusting solution. Subsequently, the first surfactant, the second surfactant, the stabilizer, the first biological enzyme, the second biological enzyme, the third biological enzyme and the enzymatic substrate are added in sequence and stirred evenly to obtain R1 reagent.

[0034] In this embodiment, the first buffer solution is a MOPS buffer solution prepared by dissolving MOPS in water, the first pH adjusting solution is an 8 mol / L sodium hydroxide solution adjusted to a pH of 7.4, the first surfactant is sodium tripolyphosphate, the second surfactant is PEG6000, the stabilizer is EDTA Na2, the first biological enzyme is lipoprotein lipase, the second biological enzyme is glycerol phosphate oxidase, the third biological enzyme is peroxidase, and the enzymatic substrate is adenosine triphosphate.

[0035] After preparation, the final concentrations of the components of R1 reagent are: MOPS 7.45 g / L pH 7.4 Sodium tripolyphosphate 2g / L PEG60005g / L EDTA Na21.54g / L Lipoprotein lipase 2.5 kU / L Glycerol phosphate oxidase 1.5 kU / L Peroxidase 1.2 kU / L Adenosine triphosphate 50mmol / L (2) Preparation of R2 reagent: According to the component content of R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.4 using a pH adjusting solution. Then, the catalytic enzyme, the chromogenic substrate, and the chromogenic agent are added in sequence and stirred evenly to obtain R2 reagent.

[0036] In this embodiment, the second buffer solution is a MOPS buffer solution prepared by dissolving MOPS in water, the second pH adjusting solution is an 8 mol / L sodium hydroxide solution, the catalytic enzyme is glycerol kinase, the chromogenic substrate is 4-aminoantipyrine (4-AAP), and the chromogenic agent is sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS).

[0037] After preparation, the final concentrations of the components of R2 reagent are: MOPS 7.48 g / L pH 7.4 Glycerol kinase 5kU / L 4-AAP0.5mmol / L DHBS0.5g / L Example 4 This embodiment provides a triglyceride kit that eliminates cross contamination between reagents. The preparation method of the kit comprises the following steps: (1) Preparation of R1 reagent: According to the component content of R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 7.4 using a first pH adjusting solution. Subsequently, the first surfactant, the second surfactant, the stabilizer, the first biological enzyme, the second biological enzyme, the third biological enzyme and the enzymatic substrate are added in sequence and stirred evenly to obtain R1 reagent.

[0038] In this embodiment, the first buffer solution is a MOPS buffer solution prepared by dissolving MOPS in water, the first pH adjusting solution is an 8 mol / L sodium hydroxide solution adjusted to pH 7.4, the first surfactant is PEG6000, the second surfactant is AOT, the stabilizer is EDTA Na2, the first biological enzyme is lipoprotein lipase, the second biological enzyme is glycerol phosphate oxidase, the third biological enzyme is peroxidase, and the enzymatic substrate is adenosine triphosphate.

[0039] After preparation, the final concentrations of the components of R1 reagent are: MOPS 7.45 g / L pH 7.4 PEG60005g / L AOT1g / L EDTA Na21.54g / L Lipoprotein lipase 2.5 kU / L Glycerol phosphate oxidase 1.5 kU / L Peroxidase 1.2 kU / L Adenosine triphosphate 50mmol / L (2) Preparation of R2 reagent: According to the component content of R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.4 using a second pH adjusting solution. Then, the catalytic enzyme, the chromogenic substrate, and the chromogenic agent are added in sequence and stirred evenly to obtain R2 reagent.

[0040] In this embodiment, the second buffer solution is a MOPS buffer solution prepared by dissolving MOPS in water, the second pH adjusting solution is an 8 mol / L sodium hydroxide solution, the catalytic enzyme is glycerol kinase, the chromogenic substrate is 4-aminoantipyrine (4-AAP), and the chromogenic agent is sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS).

[0041] After preparation, the final concentrations of the components of R2 reagent are: MOPS 7.48 g / L pH 7.4 Glycerol kinase 5kU / L 4-AAP0.5mmol / L DHBS0.5g / L Example 5 This embodiment provides a triglyceride kit that eliminates cross contamination between reagents. The preparation method of the kit comprises the following steps: (1) Preparation of R1 reagent: According to the component content of R1 reagent, a buffer solution is prepared, and the pH is adjusted to 7.2 using a pH adjusting solution. Subsequently, the first surfactant, the second surfactant, the stabilizer, the first biological enzyme, the second biological enzyme, the third biological enzyme and the enzymatic substrate are added in sequence and stirred evenly to obtain R1 reagent.

[0042] In this embodiment, the first buffer solution is a MOPS buffer solution prepared by dissolving MOPS in water, the first pH adjusting solution is an 8 mol / L sodium hydroxide solution, the first surfactant is sodium tripolyphosphate, the second surfactant is AOT, the stabilizer is EDTA Na2, the first biological enzyme is lipoprotein lipase, the second biological enzyme is glycerol phosphate oxidase, the third biological enzyme is peroxidase, and the enzymatic substrate is adenosine triphosphate.

[0043] After preparation, the final concentrations of the components of R1 reagent are: MOPS 7.45 g / L pH 7.2 Sodium tripolyphosphate 2g / L AOT1g / L EDTA Na21.54g / L Lipoprotein lipase 2.5 kU / L Glycerol phosphate oxidase 1.5 kU / L Peroxidase 1.2 kU / L Adenosine triphosphate 50mmol / L (2) Preparation of R2 reagent: According to the component content of R2 reagent, a buffer solution is prepared, and the pH is adjusted to 7.2 using a pH adjusting solution. Then, the catalytic enzyme, the chromogenic substrate, and the chromogenic agent are added in sequence and stirred evenly to obtain R2 reagent.

[0044] In this embodiment, the second buffer solution is a MOPS buffer solution prepared by dissolving MOPS in water, the second pH adjusting solution is an 8 mol / L sodium hydroxide solution, the catalytic enzyme is glycerol kinase, the chromogenic substrate is 4-aminoantipyrine (4-AAP), and the chromogenic agent is sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS).

[0045] After preparation, the final concentrations of the components of R2 reagent are: MOPS 7.48g / L pH 7.2 Glycerol kinase 5kU / L 4-AAP0.5mmol / L DHBS0.5g / L Example 6 Application of a triglyceride kit to eliminate cross contamination between reagents This embodiment provides a method for detecting triglycerides using a kit that eliminates cross contamination between reagents, and the steps are as follows: Using the Hitachi 7600 as an example, the primary and secondary wavelengths are 520 / 660 nm. For a calibrator volume of 2 μL, add 160 μL of R1 reagent, mix thoroughly, incubate at 37°C for 5 minutes, and read the absorbance value A1. Add 40 μL of R2 reagent, mix thoroughly, incubate at 37°C for 5 minutes, and read the absorbance value A2. Calculate the absorbance difference ΔA = A2 - A1. Repeat the measurement twice for each tube. Plot a "concentration-absorbance difference" calibration curve using the average of the two absorbance differences ΔA for each calibration tube as the ordinate and the corresponding calibrator concentration as the abscissa. For serum or plasma samples to be tested, measure the absorbance difference using the same method. Substituting the difference into the calibration curve allows the triglyceride (TG) content in the sample to be calculated. If the TG concentration in the sample exceeds the range of the calibration curve, dilute the sample before retesting to ensure accuracy. This test kit is not only suitable for Hitachi 7600, but also for other brands and models of semi-automatic and fully automatic biochemical analyzers. The specific parameters can be adjusted according to the instrument.

[0046] Example 7 Quality Evaluation of a Triglyceride Kit for Eliminating Cross-Contamination Between Reagents (1) Linear range A. Experimental Methods The linear range is 0-11.4 mmol / L. A concentrated sample near the high end of the linear range (11.21 mmol / L) and a sample near the low end of the linear range (0.01 mmol / L) were mixed in ratios of 1:0, 5:1, 1:1, 1:5, and 0:1. Concentrations were determined using the above procedure. Two to three measurements were performed for each sample, and the average was taken. A linear plot was plotted based on the average and theoretical values. The correlation coefficient, r, and linear deviation were calculated. The correlation coefficient, r, should be ≥ 0.9900. Within the range [0, 0.6] mmol / L, the absolute linear deviation should not exceed ±0.2 mmol / L. Within the range [0.6, 11.4] mmol / L, the relative linear deviation should not exceed ±10%.

[0047] B. Experimental results and analysis The linearity test results of the kit are shown in Table 1.

[0048] Table 1 Linearity test results

[0049] As shown in Table 1, the linear correlation coefficients and deviations of experimental groups 1-5 are all within the acceptable range, and the linearity meets the requirements. The linear curve of experimental group 5 is shown in Figure 1 The results of experimental group 5 were the best.

[0050] (2) Analytical sensitivity A. Experimental Methods The concentration of the tested triglyceride sample was 3.04 mmol / L, and the absorbance change per unit concentration was ≥0.050.

[0051] B. Experimental results and analysis Table 2 Analytical sensitivity test results

[0052] As shown in the results of Table 2, the analytical sensitivities of experimental groups 1-5 all met the requirements, among which the sensitivity of experimental group 5 was closest to that of the control group (experimental group 1).

[0053] (3) Repeatability testing A. Experimental Methods The Landau composite quality control (lot 1285UN, concentration 1.20 mmol / L) was tested 10 times. The mean (mean) and standard deviation (SD) were calculated, along with the coefficient of variation (CV). Repeatability was assessed for the kits in experimental groups 1-5 according to the biochemical analyzer assay method. The acceptable range for CV was ±5%.

[0054] B. Experimental results and analysis Table 3 Repeatability test results

[0055] As shown in the results of Table 3, the coefficients of variation (CV) of experimental groups 1-5 were all within an acceptable range, indicating that the components and their concentration ranges involved in the present invention all had good in-bottle uniformity, and the CV result of the kit of experimental group 5 was the best.

[0056] (4) Accuracy test A. Experimental Methods Using a commercially available reagent as a reference reagent, 40 human samples were tested at varying concentrations within the assay range. Each sample was tested individually using the aforementioned protocol and comparison method. Linear regression was used to calculate the correlation coefficient (r) and the relative or absolute deviation at each concentration point. Within the range of 0-11.4 mmol / L, the correlation coefficient r was ≥ 0.9900.

[0057] B. Experimental results and analysis Table 4 Accuracy test results

[0058] The correlation between experimental group 5 and the measured values ​​of commercial reagents is as follows Figure 2 As shown, the measured values ​​have good linear correlation within the test interval.

[0059] As shown in the results of Table 4, comparing the linear correlation coefficients of experimental groups 1-5 with the measured values ​​of commercially available reagent samples, the result of experimental group 5 is closest to the control group (experimental group 1).

[0060] (5) Cross-contamination experiment A. Experimental methods: Serum bile acid was first tested separately, and then serum triglyceride and serum bile acid were tested simultaneously. The reagent preparation process used was the same as that of Experimental Group 1, Experimental Group 5, and Example 5 in Example 2. 20 clinical serum samples were tested, and the results of TBA testing alone were used as the reference standard for subsequent relative deviation calculations. The test results are shown in Table 5.

[0061] B. Experimental results and analysis Table 5 Cross contamination test results

[0062] As can be seen from Table 5, since the CHAPS in experimental group 1 is a bile acid analog, there is carryover contamination when detecting triglycerides, which leads to a surge in the measured value when the bile acid project is subsequently detected, thereby causing a significant increase in the false positive rate of the sample. After the surfactant is replaced, the test kits in experimental group 5 and Example 5 have little effect on the bile acid measurement value, with the deviation within 10%, and the effect is better.

[0063] (6) Open bottle stability test A. Experimental Methods Reagents were prepared according to Examples 1, 3, 4, and 5. After opening the R1 and R2 reagents, they were placed in the corresponding reagent positions of the fully automatic biochemical analyzer. Landau composite quality control level 2 was tested at 1.20 mmol / L and quality control level 3 at 2.91 mmol / L 1, 6, 12, 18, 24, and 30 days after opening. The average value was calculated for each test, and the results of the test on the first day of opening the bottle were used for comparison. The relative deviation of each test was calculated. The test results are shown in Table 6.

[0064] B. Experimental results and analysis Table 6 Results of the open bottle stability test of the triglyceride kit to eliminate cross contamination between reagents

[0065] As can be seen from Table 6, during the 30-day opening process, the unsealing effect of Example 5 is close to that of Example 1, the test values ​​are relatively stable, and the relative deviation of the measured values ​​is very small, indicating that replacing CHAPS with sodium tripolyphosphate and AOT does not affect the unsealing performance of the kit.

[0066] (7) Accelerated thermal stability test A. Experimental Methods Reagents were prepared according to Examples 1, 3, 4, and 5. Reagents R1 and R2 were placed in a 37°C waterbath with the lids closed. The reagents were removed from the waterbath at 3, 5, 7, and 14 days, respectively, and tested on an automatic biochemical analyzer. Unaccelerated, normal reagents were used for comparison. The Landau composite quality control level 2 (1.20 mmol / L) and the quality control level 3 (2.91 mmol / L) were tested three times each, and the relative deviations were calculated. The test results are shown in Table 7.

[0067] B. Experimental results and analysis Table 7 Accelerated thermal stability test results of triglyceride kit to eliminate cross contamination between reagents

[0068] As can be seen from Table 7, the reagent of Example 5 was continuously destroyed at 37°C for 14 days, and the relative deviations were all within an acceptable range. The results of Example 5 were the best, and the acceleration effect was consistent with that of Example 1, indicating that replacing CHAPS with sodium tripolyphosphate and AOT did not affect the accelerated destruction performance of the kit, and replacing it with other surfactants was not conducive to the accelerated thermal stability of the kit.

[0069] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solutions and concepts of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A triglyceride kit for eliminating cross contamination between reagents, characterized in that: Comprising R1 reagent and R2 reagent; wherein: The R1 reagent includes the following components: a first buffer, a first surfactant, a second surfactant, a stabilizer, a first biological enzyme, a second biological enzyme, a third biological enzyme and an enzymatic substrate; The R2 reagent includes the following components: a second buffer, a catalytic enzyme, a color-developing substrate, and a color-developing agent.

2. The triglyceride kit for eliminating cross contamination between reagents according to claim 1, characterized in that: The R1 reagent includes the following components at the following concentrations: First buffer 7-8 g / L; pH 7.0-8.0; The first surfactant is 0.5-4g / L; Second surfactant 0-4g / L; Stabilizer 1-2 g / L; First biological enzyme 2-4kU / L; Second biological enzyme 1-2kU / L; The third biological enzyme 1-2kU / L; Enzyme substrate 50-100mmol / L; The R2 reagent includes the following components at the following concentrations: Second buffer 7-8 g / L; pH 7.0-8.0; Catalytic enzyme 1-5 kU / L; Chromogenic substrate 0.5-1mmol / L; Color developer 0.5-1g / L.

3. The triglyceride kit for eliminating cross contamination between reagents according to claim 2, characterized in that: The first buffer and the second buffer are respectively selected from any one of MOPS buffer, TRIS buffer, HEPES buffer, MES buffer, and PB buffer.

4. The triglyceride kit for eliminating cross contamination between reagents according to claim 3, characterized in that: The first surfactant is selected from any one of sodium tripolyphosphate, oxyethylene lauryl ether, cetyltrimethylammonium chloride, PEG6000, and CHAPS.

5. The triglyceride kit for eliminating cross contamination between reagents according to claim 4, characterized in that The second surfactant is selected from any one of sodium dioctyl sulfosuccinate, Tween-20, Triton X-100, and PEG6000.

6. The triglyceride kit for eliminating cross contamination between reagents according to claim 2, characterized in that: The first surfactant is sodium tripolyphosphate, the second surfactant is sodium dioctyl sulfosuccinate, and the mass ratio of the sodium tripolyphosphate to the sodium dioctyl sulfosuccinate is (1:2) to (2:1).

7. The triglyceride kit for eliminating cross contamination between reagents according to claim 2, characterized in that: The stabilizer is selected from any one of EDTA Na2, trehalose, NaCl, and KCl.

8. The triglyceride kit for eliminating cross contamination between reagents according to claim 2, characterized in that: The first biological enzyme is lipoprotein lipase, the second biological enzyme is glycerol phosphate oxidase, and the third biological enzyme is peroxidase.

9. The triglyceride kit for eliminating cross contamination between reagents according to claim 2, characterized in that: The color developing substrate is 4-aminoantipyrine; the color developing agent is any one of 3,5-dichloro-2-hydroxybenzenesulfonate sodium, N-ethyl-N-(3-sulfopropyl)-3-methylaniline, and 3-methyl-2-benzothiazolinone hydrazone.

10. The method for preparing the triglyceride kit for eliminating cross contamination between reagents according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) Preparation of R1 reagent According to the component content of R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 7.0-8.

0. Then, the first surfactant, the second surfactant, the stabilizer, the first biological enzyme, the second biological enzyme, the third biological enzyme and the enzymatic substrate are added in sequence, and stirred evenly to obtain R1 reagent; (2) Preparation of R2 reagent According to the component content of R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.0-8.

0. Then, the catalytic enzyme, the chromogenic substrate and the chromogenic agent are added in sequence and stirred evenly to obtain R2 reagent.

Images