Estriol homogeneous phase enzyme immunoassay reagent as well as preparation method and detection method thereof
A technology of homogeneous enzyme immunization and detection reagents, which is applied in measurement devices, instruments, scientific instruments, etc., to achieve the effect of being beneficial to clinical popularization, high immunogenicity, and saving operation time.
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Embodiment 1
[0051] Example 1: Synthesis of Estriol Immunogen
[0052] The estriol immunogen is formed by linking bovine serum albumin (Bovine Serum Albumin, BSA) to the terminal carboxyl group of the estriol derivative represented by formula (II), and the specific steps are as follows:
[0053] 1. Dissolve 200 mg bovine serum albumin in 50 mL 0.2 M, pH 8.5 phosphate buffer;
[0054] 2. Add the following chemicals into a small beaker and stir to dissolve: 200 mg estriol derivatives, 3.5 mL dimethylformamide, 3.5 mL ethanol, 7.0 mL 10mM potassium phosphate buffer, pH 5.0, 200 mg 1- Ethyl-3-(-3-dimethylaminopropyl) carbodiimide, these chemicals were stirred and dissolved at room temperature for 30 min;
[0055] 3. Add the dissolved solution dropwise to the BSA solution, and stir overnight at 2-8°C to obtain the antigen; purify the synthesized antigen by dialysis to obtain the estriol immunogen.
Embodiment 2
[0056] Example 2: Preparation of anti-estriol specific antibody
[0057] The estriol immunogen prepared in Example 1 was inoculated into experimental animal rabbits by conventional methods, and antiserum was taken after booster immunization. The specific steps were as follows:
[0058] 1. Dilute the above synthesized estriol immunogen to 1.0 mg / mL with PBS to obtain an antigen solution, then mix 1.0 mL of the antigen solution with an equal amount of Freund's complete adjuvant, and inject the experimental animal rabbit.
[0059] 2. After 2 to 3 weeks, inject 1.0 mL of the same antigen solution and an equal amount of Freund's incomplete adjuvant to the above-mentioned experimental rabbit once, and then inject once every four weeks, for a total of 4 injections.
[0060] 3. Take blood from the experimental animal rabbit in step 2, separate and purify to obtain anti-estriol specific antibody with a titer of 1:30000~1:50000.
Embodiment 3
[0061] Embodiment three: the ELISA test of estriol
[0062] 1. Establishment of ELISA detection standard curve of estriol
[0063] (1) Preparation of standard products
[0064] Estriol powder (purchased from Sigma) was dissolved in methanol solution to prepare a 1 mg / mL stock solution. The stock solution was sequentially diluted with ELISA buffer to 16.00ng / mL, 8.00ng / mL, 4.00ng / mL, 2.00ng / mL, 1.00ng / mL and 0.00ng / mL standard solutions. Among them, the ELISA buffer contains 50.0 mM Tris, 145 mM NaCl and 0.25% BSA.
[0065] (2) Utilize the ELISA test method of estriol to prepare standard curve
[0066] The anti-estriol specific antibody prepared in Example 2 was diluted with PBS to a final concentration solution of 1:7500, and 100 μL / hole was coated on a 96-well enzyme-linked plate, and placed at 4°C for 12-24h; After the 96-well enzyme-linked plate coated with anti-estriol antibody was washed three times, 200 μL / well of 0.5% BSA solution was added, and sealed at 4° C. for ...
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