Thrombin-antithrombin III compound determination kit as well as preparation method and application thereof
An antithrombin and thrombin technology, applied in the field of medical diagnosis, to achieve the effects of high sensitivity, high cost performance and good stability
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Embodiment 1
[0061] This embodiment provides a thrombin-antithrombin III complex assay kit, the kit includes (1) a solution of a magnetic microsphere conjugate coated with a thrombin monoclonal antibody; (2) labeled with a thrombin monoclonal antibody Solution of enzyme-labeled conjugate of antithrombin III monoclonal antibody; (3) substrate solution; (4) calibrator.
[0062] The magnetic microsphere conjugate coated with thrombin monoclonal antibody is a combination of magnetic particle and thrombin monoclonal antibody; the diameter of the magnetic particle is 1000 nanometers to 3000 nanometers; In the solution of the magnetic microsphere conjugate of the monoclonal antibody, the concentration of the magnetic microsphere conjugate coated with the thrombin monoclonal antibody was 0.2 mg / mL. The concentration of thrombin monoclonal antibody in the magnetic microsphere conjugate was 4 μg / mL.
[0063] In the solution of the enzyme-labeled conjugate labeled with antithrombin III monoclonal an...
Embodiment 2
[0084] Example 2 Comparison of antigen coupling conditions
[0085] The changes in this example from Example 1 include the comparison of the preparation conditions of the thrombin-antithrombin III complex:
[0086] (1) Selection of crosslinking agent:
[0087] Three experimental groups were set up, each group took 10 mg of thrombin to replace the stock solution with PBS buffer, added 1 mg of Sulfo-EGS, Sulfo-MBS and DSS respectively, incubated for 1 h at room temperature, and activated to obtain a mixture; The original solution of thrombin III was replaced with PBS (phosphate) buffer, and all of them were immediately added to the above three groups of mixed solutions, and incubated for 1 h at room temperature for cross-linking. After the cross-linking was completed, desalted and purified, and mixed to obtain 10 mL of thrombin-antithrombin III complex solution each.
[0088] (2) Comparison of thrombin and Sulfo-EGS mass ratio conditions:
[0089]Three experimental groups wer...
Embodiment 3
[0097] Example 3: Performance evaluation of the kit of the present invention
[0098] 1. Standard curve
[0099] The thrombin-antithrombin III complex described in Example 1 was diluted with the standard substance diluent to prepare calibrator solutions S0-S9 of different concentrations, the concentrations were 0, 0.5ng / mL, 1.0ng / mL, 2.0 ng / mL, 4.0ng / mL, 9.0ng / mL, 18ng / mL, 36ng / mL, 60ng / mL, 120ng / mL, and stored at -20°C for later use. Then, use the kit provided in Example 1 of the present invention to detect the calibrators, and read the luminescence intensity values corresponding to each calibrator respectively. The standard curve was obtained by fitting the concentration as the abscissa and the luminescence intensity as the ordinate.
[0100] 2. Linear
[0101] The basal serum sample (concentration≤blank limit) or calibrator dilution (as much as possible to use real high-value serum samples) with added antigens is used as the high-concentration sample, and the basal ser...
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