Methotrexate homogenous enzyme immunoassay reagent as well as preparation method and detection method thereof
A homogeneous enzyme immunoassay and methotrexate technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problem that the number of products cannot meet clinical needs, and achieve high-throughput rapid determination, high potency, and accuracy The effect of increasing
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Embodiment 1
[0065] Example 1: Synthesis and structure confirmation of methotrexate derivatives
[0066] The chemical structure of the methotrexate derivative used in the following examples is shown in formula (II):
[0067]
[0068] The synthetic route of this methotrexate derivative is as follows:
[0069]
[0070] Concrete synthetic steps are as follows:
[0071] Synthesis of compound 2
[0072]
[0073] Weigh 5.0 g of compound 1, dissolve it in 50 mL of MeOH, and add 4.0 g (34.1 mmol) of SOCl dropwise at 0°C 2 , then the reaction mixture was stirred overnight at 70 °C, the solvent was evaporated by reduced pressure, and the residue was passed through 200 mL of DCM and 200 mL of NaHCO 3 Saturated aqueous solution was separated, the organic phase was washed with brine, and then passed through Na 2 SO 4 It was dried and concentrated to obtain 5.1 g of white solid compound 2 with a yield of 96%.
[0074] Synthesis of Compound 3
[0075]
[0076] Weigh 5.1g of compound 2,...
Embodiment 2
[0080] Example 2: Synthesis of BSA-methotrexate immunogen
[0081] The BSA-methotrexate immunogen is formed by linking bovine serum albumin (BSA) to the terminal carboxyl group of methotrexate shown in formula (Ⅲ), and the specific synthesis steps of the immunogen are as follows:
[0082] a. Weigh 2.72g of potassium dihydrogen phosphate, 4.26g of disodium hydrogen phosphate, 8.5g of sodium chloride, and 0.95g of magnesium chloride, dissolve them in 1L of deionized water, adjust the pH to 7.4, and make buffer solution A;
[0083] b. Weigh 3 mg of BSA and dissolve it in 3 mL of the above buffer solution A at room temperature to make a BSA solution;
[0084] c. Weigh 3mg of methotrexate derivatives and dissolve in 300ul of the above buffer solution A to make methotrexate derivatives solution;
[0085] d. When the above-mentioned methotrexate derivative solution just became clear, it was added dropwise to the above-mentioned BSA solution, and then the mixed solution was stirred a...
Embodiment 3
[0087] Example 3: Preparation of Anti-Methotrexate Specific Antibody
[0088] The BSA-methotrexate immunogen prepared above was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:
[0089] The above synthesized BSA-methotrexate immunogen was diluted to 1.0 mg / ml with PBS to obtain an antigen solution, and then 1.0 ml of the antigen solution was mixed with Freund's complete adjuvant, and then injected into experimental animal rabbits.
[0090] After 2 to 3 weeks, the rabbits were injected once with 1.0 ml of the same antigen solution and Freund's incomplete adjuvant, and then injected once every four weeks, for a total of 4 injections.
[0091] Blood was collected from the above-mentioned experimental animal rabbit, separated and purified to obtain anti-methotrexate specific antibody with a titer of 1:30000-1:50000.
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