A kit for determining apolipoprotein A5 by latex immunoturbidimetry

Abstract

The invention discloses a kit for determining apolipoprotein A5 by latex immunoturbidimetry. The kit comprises a reagent R1, a reagent R2 and an apolipoprotein A5 calibrator. The kit is easy to operate and can directly use a full-automatic biochemical analyzer to perform sample detection. The demand for high-throughput rapid detection of samples in a clinical laboratory can be met, and human errors can be reduced to significantly improve the inspection efficiency. The kit adopts the latex immunoturbidimetry, so that the stability of the reagent is further improved. The reagent can usually be stably stored for one year or more under a 2-8°C environment. Therefore, the kit has a large application space in the clinic and can be widely promoted and applied in most clinical laboratories.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnostic reagents, and in particular relates to a kit for determining apolipoprotein A5 by latex immunoturbidimetry. Background Art

[0002] At present, human apolipoprotein A5 (APOA5) is a protein containing 366 amino acids, with an average molecular weight of 38905Da, 4 tryptophan residues, 5 tyrosine residues and 1 cysteine ​​residue. Human APOA5 protein is specifically synthesized mainly in the liver, with a low serum content of about 24-406μg / L, and is mainly distributed in very low-density lipoprotein (VLDL), high-density lipoprotein (HDL) and chylomicrons (CM). Human APOA5 protein contains 1 N-terminal signal peptide and multiple secondary α-helical structures, and has the characteristics of typical lipid-bound apolipoproteins. APOA5 protein is not easily soluble in neutral buffer, and there is no glycosylation site at the N-terminus. Its N-terminal signal peptide may be located between amino acids 23-24. The human APOA5 protein has a high degree of α-helical structure, which not only contains a large number of α-helices but also β-folding structures and β-turn structures, indicating that APOA5 has a certain degree of hydrophobicity. Its hydrophobic residues are distributed asymmetrically. The hydrophilic region is from the N-terminus to residue 85 and 1 / 3 of the C-terminus, while the hydrophobic region is in the middle region of residues 85-220. In addition, the APOA5 protein also contains some discontinuous structural regions, which have different lipid affinities. The APOA5 protein has three different lipid enzymatic particles, namely very low density lipoprotein (VLDL), triglycerides (phospholipid emulsion), and dimyristoylphosphatidylcholine (DMPC). According to the structural characteristics of the APOA5 protein, it is shown that it has low elasticity, strong hydrophobicity, and high lipid affinity. Due to these characteristics of APOA5, the aggregation rate of particles rich in triglycerides (TG) may be slow, and the TG content will be reduced by activating lipoprotein lipase (LPL) and accelerating the catabolism of very low density lipoprotein (VLDL).

[0003] Apolipoprotein plays an important role in binding and transporting lipids, regulating the activity of key lipoprotein enzymes, and recognizing lipoprotein receptors. Its structural changes or abnormal synthesis will affect blood lipid metabolism. Dyslipidemia is an important risk factor for various cardiovascular and cerebrovascular diseases, and is also a susceptibility factor for diseases such as hypertension, diabetes, and obesity. As an independent pathogenic factor of coronary artery atherosclerosis, elevated triglyceride levels are closely related to common diseases such as coronary heart disease, myocardial infarction, and diabetes. Apolipoprotein A5 is a new member of the apolipoprotein family discovered in recent years. Studies have shown that it is closely related to TG metabolism and is one of the factors that most significantly affect plasma TG levels. Therefore, detecting the content of APOA5 in human serum and plasma samples will provide new ideas for the diagnosis and treatment of atherosclerotic cardiovascular and cerebrovascular diseases, and has important preventive significance.

[0004] Existing APOA5 detection technologies mostly use the enzyme-linked immunosorbent assay (ELISA) method. Although the ELISA method is relatively traditional and widely used, the measurement process is cumbersome, the operation time is long, the degree of automation is low, the repeatability and batch difference are relatively large, and it cannot adapt to the screening of large-volume samples. In addition, this method requires the use of a variety of special equipment, which also increases the cost of use to a certain extent.

[0005] In order to solve the above problems existing in the prior art, we propose a kit for determining apolipoprotein A5 using latex immunoturbidimetry. Summary of the invention

[0006] The purpose of the present invention is to provide a kit for determining apolipoprotein A5 by latex immunoturbidimetry, which is simple and quick to operate, has good detection stability and repeatability, can be applied to a fully automatic biochemical analyzer, realizes automated operation and automated analysis, and has a relatively low cost of use, thereby making up for the shortcomings of the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a kit for determining apolipoprotein A5 by latex immunoturbidimetry, the kit comprising reagent R1, reagent R2 and apolipoprotein A5 calibrator, wherein:

[0008] The reagent R1 comprises: a buffer solution 10-500 mmol / L, a stabilizer 0.2-20 g / L, an inorganic salt 1-50 g / L, a surfactant 0.5-50 g / L, a coagulant 10-100 g / L, an antioxidant 0.01-10 g / L, a preservative 1-10 g / L, and a pH value of 6.5-9.0;

[0009] The reagent R2 comprises: a buffer solution of 10 to 500 mmol / L, anti-human apolipoprotein A5 antibody-coated latex particles of 10 to 100 mg / L, a stabilizer of 0.2 to 20 g / L, an inorganic salt of 1 to 50 g / L, an antioxidant of 0.01 to 10 g / L, a preservative of 1 to 10 g / L, and a pH value of 6.5 to 9.0;

[0010] The apolipoprotein A5 calibrator comprises: a buffer solution of 10 to 500 mmol / L, an apolipoprotein A5 antigen of 0.4 to 0.5 mg / L, a stabilizer of 0.2 to 20 g / L, an antioxidant of 0.01 to 10 g / L, and a preservative of 1 to 10 g / L; the pH value is 6.5 to 9.0.

[0011] Preferably, the buffer is selected from one of tris(hydroxymethylaminomethane)-hydrochloric acid buffer, 3-morpholinepropanesulfonic acid buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, piperazine-1,4-diethanesulfonic acid buffer, 2-(N-morpholino)ethanesulfonic acid buffer, 3-(cyclohexylamino)2-hydroxy-1-propanesulfonic acid buffer, glycine buffer, and phosphate buffer;

[0012] The stabilizer is selected from one or more of disodium ethylenediaminetetraacetate, bovine serum albumin, sorbitol, mannose, glucose, trehalose, and gelatin;

[0013] The inorganic salt is selected from one or more of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate;

[0014] The surfactant is selected from one or more of Tween series, Triton series, Span series, lauryl alcohol polyoxyethylene ether, polyoxyethylene lauryl ether, fatty alcohol polyoxyethylene ether, and sophorolipids;

[0015] The coagulant is selected from one or more of polyethylene glycol 6000, polyethylene glycol 8000 and polyvinyl pyrrolidone;

[0016] The preservative is selected from one or more of sodium azide, proclin 300, sodium thimerosal, phenol, ethyl paraben, and hydroxybenzoic acid;

[0017] The anti-human apolipoprotein A5 antibody is selected from one of goat anti-human, mouse anti-human, rabbit anti-human, bovine anti-human or pig anti-human apolipoprotein A5 antibodies;

[0018] The antioxidant is selected from one or more of L-ascorbic acid, dilauryl thiodipropionate, 2,6-di-tert-butyl-p-cresol, butylated hydroxyanisole, butylated hydroxytoluene, and tert-butylhydroquinone.

[0019] Preferably, the kit comprises the following components,

[0020] The reagent R1: pH value is 7 to 9

[0021]

[0022]

[0023] The reagent R2: pH value is 7 to 9

[0024]

[0025] The calibrator: pH 7.4

[0026]

[0027] Preferably, the preparation method of the anti-human apolipoprotein A5 antibody coated latex solution is as follows: 1 ml to 2 ml of a surface carboxylated polystyrene latex solution with a diameter of 200 nm is added to 8 to 9 ml of a PBS buffer solution to make the final concentration of the latex particles 1.0%; then 0.1 to 1 mg of the anti-human apolipoprotein A5 antibody and 20 mg to 40 mg of an activator 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, and the mixture is incubated at 37° C. for 2 to 4 hours to allow the activated carboxyl groups to bind to the latex particle solution. The solution is chemically cross-linked to form a covalent antibody latex complex; then, 10 to 20 ml of 0.1% BSA solution is added to block at 37° C. for 0.5 to 2 hours to block the surface active groups of the latex particles that are not bound to the antibody, and then centrifuged at 12000 rpm for 30 to 50 minutes, the supernatant is removed, and the resulting precipitate is washed and dispersed by adding 10 ml of 30 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 0.1% BSA, and the anti-human apolipoprotein A5 antibody-coated latex solution is obtained.

[0028] Compared with the prior art, the present invention has the following beneficial effects: the kit has simple instructions and is easy to operate; a fully automatic biochemical analyzer can be directly used for sample detection, which can meet the needs of clinical laboratories for high-throughput rapid detection of samples, while reducing human errors and significantly improving the inspection efficiency; the kit adopts latex immunoturbidimetry, which further improves the stability of the reagent, and can usually be stably stored for one year or more at 2-8°C, so it has a large application space in clinical practice and can be widely promoted and applied in most clinical laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a correlation diagram of the test results of Example 2 of the present invention and the comparative kit. DETAILED DESCRIPTION

[0030] Unless otherwise specified, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0031] In the present invention, the same reagent may be expressed by different names or English abbreviations, which are explained here as follows: tris-hydroxymethylaminomethane-hydrochloric acid (Tris-HCL) buffer, 3-morpholinepropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, piperazine-1,4-diethanesulfonic acid (PIPES) buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 3-(cyclohexylamino)2-hydroxy-1-propanesulfonic acid (CAPSO) buffer; polyoxyethylene lauryl alcohol ether (Brij-35), polyoxyethylene lauryl ether (C12E10), fatty alcohol polyoxyethylene ether (AEO); dilauryl thiodipropionate (DLTP), 2,6-di-tert-butyl-p-cresol (BHT), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ).

[0032] The present invention will be described in detail below with reference to the embodiments. 1. Specific embodiments

[0034] Example 1 formulation components:

[0035] Reagent R1: pH 7.5

[0036]

[0037] Reagent R2: pH 7.5

[0038]

[0039] Calibrator: pH 7.4

[0040]

[0041]

[0042] According to the required calibrator concentration, a corresponding amount of apolipoprotein A5 antigen is added to the above calibrator solution to prepare an apolipoprotein A5 calibrator.

[0043] In this embodiment, six calibrators with different concentrations were prepared, namely: 0 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 400 ng / mL.

[0044] Embodiment 2 formula components:

[0045] Reagent R1: pH 8.0

[0046]

[0047] Reagent R2: pH 8.0

[0048]

[0049] Calibrator: pH 7.4

[0050]

[0051] According to the required calibrator concentration, a corresponding amount of apolipoprotein A5 antigen is added to the above calibrator solution to prepare an apolipoprotein A5 calibrator.

[0052] In this embodiment, six calibrators with different concentrations were prepared, namely: 0 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 400 ng / mL.

[0053] The actual delivery can include a high-value calibrator, and the customer can dilute it according to the instructions. Alternatively, multiple calibrators of different concentrations can be prepared and placed directly in the kit, and the six points of the calibrator can be used to make a calibration curve. In the example, the measured value is calculated by the instrument test sample on the calibration curve.

[0054] Example 3 formulation components:

[0055] Reagent R1: pH 7.5

[0056]

[0057] Reagent R2: pH 7.5

[0058]

[0059] Calibrator: pH 7.4

[0060]

[0061] According to the required calibrator concentration, a corresponding amount of apolipoprotein A5 antigen is added to the above calibrator solution to prepare an apolipoprotein A5 calibrator.

[0062] In this embodiment, six calibrators with different concentrations were prepared, namely: 0 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 400 ng / mL.

[0063] Embodiment 4 formula components:

[0064] Reagent R1: pH 7.0

[0065]

[0066]

[0067] Reagent R2: pH 7.0

[0068]

[0069] Calibrator: PH7.4

[0070]

[0071] According to the required calibrator concentration, a corresponding amount of apolipoprotein A5 antigen is added to the above calibrator solution to prepare an apolipoprotein A5 calibrator.

[0072] In this embodiment, six calibrators with different concentrations were prepared, namely: 0 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 400 ng / mL.

[0073] 2. Detection method:

[0074] When the kit described in the above embodiment is used, the determination method is to use Canon TBA-120FR fully automatic biochemical analyzer, use the endpoint method for determination, the main detection wavelength is 600nm, and the operation procedure is as shown in Table 1:

[0075] Table 1 Kit detection method

[0076]

[0077] 3. Calculation of results:

[0078] Apolipoprotein A5 content (ng / mL) = (ΔA sample / ΔA calibrator) × calibrator concentration.

[0079] 4. Performance verification:

[0080] 4.1 Repeatability and accuracy:

[0081] The calibrators of each example and human serum with a labeled value of 46 ng / ml were used as samples to investigate the repeatability of the kit in each example. The determination was repeated 10 times according to the required determination method, and the determination mean value X, standard deviation SD and coefficient of variation CV were calculated respectively to investigate the repeatability of the kit in each example. The determination results are shown in Table 2.

[0082] Table 2 Kit repeatability test results

[0083]

[0084]

[0085] According to the results in Table 2, it can be seen that the CVs of Examples 1, 2, 3, and 4 are all less than 5%, among which the repeatability result of Example 2 is the most ideal.

[0086] 40 normal human serum samples were tested simultaneously with the calibrator and kit of each example and the control kit (enzyme-linked immunosorbent assay apolipoprotein A5 detection kit from a certain manufacturer, referred to as "ELISA kit"), the test results were statistically analyzed, the correlation coefficient between the two was calculated, and linear regression was performed to perform correlation analysis on the test results, so as to investigate the accuracy of the kit in each example. The measurement results are shown in Table 3.

[0087] Table 3 Kit accuracy test results

[0088]

[0089]

[0090]

[0091] According to the results in Table 3, the correlation coefficients of Examples 1, 2, 3 and 4 are all less than 0.975, indicating that the kits of each example of the present invention have high accuracy. The correlation of Example 2 is 0.9987, and the test results of the kit of Example 2 have the best correlation with the ELISA kit. Figure 1 .

[0092] 4.2 Stability:

[0093] After the kits in each example of the present invention were opened and stored at 2-8°C for 28 days, human serum samples with labeled values ​​of 46 ng / ml and 137 ng / ml were measured respectively, and the measurement was repeated 3 times. The average value and the relative deviation from the labeled value were calculated to investigate the stability of the kits in each example after opening the bottle. The measurement results are shown in Tables 4 and 5.

[0094] Table 4 Stability after 28 days of opening - Test value of sample with a labeled value of 46 ng / ml

[0095]

[0096] Table 5 Stability after 28 days of opening - Test value of sample with a labeled value of 137 ng / ml

[0097]

[0098] According to the results in Table 4 and Table 5, it can be seen that the relative deviations between the test results of Example 1, Example 2, Example 3, and Example 4 and the labeled values ​​are all less than 5%, among which the test result of the kit of Example 2 is the most ideal, indicating that the kit of Example 2 of the present invention has good opening stability.

[0099] After the kits in each example of the present invention were stored at 2-8°C for 13 months, the apolipoprotein A5 calibrators with labeled values ​​of 25 ng / ml and 200 ng / ml were measured respectively, and the measurements were repeated 3 times. The average value and the relative deviation from the labeled value were calculated to investigate the shelf life stability of the kits in each example. The measurement results are shown in Tables 6 and 7.

[0100] Table 6: Stability during shelf life - test values ​​with a labeled value of 25 ng / ml

[0101]

[0102] Table 7 Stability during shelf life - Detection value with a labeled value of 200 ng / ml

[0103]

[0104] According to the results in Tables 6 and 7, it can be seen that the relative deviations between the test results of Example 1, Example 2, Example 3, and Example 4 and the labeled values ​​are all less than 10%, among which the test result of the kit of Example 2 is the most ideal, indicating that the shelf life stability of the kit of Example 2 of the present invention can reach at least 13 months, and has good long-term stability.

[0105] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A kit for determining apolipoprotein A5 by latex immunoturbidimetry, characterized in that: The kit comprises reagent R1, reagent R2 and apolipoprotein A5 calibrator, The reagent R1 has a pH value of 7 to 9 and contains the following components: Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer 10~ 500mmol / L Tween 20 0.5 ~ 50g / L Disodium EDTA 0.1~10g / L Bovine serum albumin 0.1~10g / L Sodium chloride 1~50g / L Polyethylene glycol 6000 1~100g / L Dilauryl thiodipropionate 0.1~50g / L Sodium thimerosal 1~10g / L The reagent R2 has a pH value of 7 to 9 and contains the following components: Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer 10~ 500mmol / L Anti-human apolipoprotein A5 antibody coated latex particles 10~100mg / L Disodium EDTA 0.1~10g / L Bovine serum albumin 0.1~10g / L Sodium chloride 1~50g / L Dilauryl thiodipropionate 0.1~50g / L Sodium thimerosal 1~10g / L The calibrator has a pH value of 7.4 and contains the following components: Phosphate buffer 10~ 500mmol / L Disodium EDTA 0.1~10g / L Bovine serum albumin 0.1~10g / L Dilauryl thiodipropionate 0.1~50g / L Sodium thimerosal 1~10g / L Apolipoprotein A5 antigen 0.4~0.5mg / L.

2. The kit for determining apolipoprotein A5 by latex immunoturbidimetry according to claim 1, characterized in that: The preparation method of the anti-human apolipoprotein A5 antibody-coated latex solution is as follows: 1 ml to 2 ml of a surface carboxyl-modified polystyrene latex solution with a diameter of 200 nm is added to 8 to 9 ml of a PBS buffer solution to make the final concentration of the latex particles 1.0%; then 0.1 to 1 mg of the anti-human apolipoprotein A5 antibody and 20 mg to 40 mg of an activator 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, and the mixture is incubated at 37° C. for 2 to 4 hours to chemically crosslink the latex particle solution through the activated carboxyl group to form a covalent antibody latex complex; then 10 to 20 ml of a 0.1% BSA solution is added to block the latex particles at 37° C. for 0.5 to 2 hours to block the surface active groups of the latex particles that are not bound to the antibodies, and then the mixture is centrifuged at 12,000 rpm for 30 to 50 minutes to remove the supernatant, and the resulting precipitate is added with 10 ml of a 30 mmol / L 0.1% BSA solution. The anti-human apolipoprotein A5 antibody-coated latex solution was obtained by washing and dispersing with tris(hydroxymethylaminomethane)-hydrochloric acid buffer.

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