Tacrolimus immunogen, anti-tacrolimus specific antigen and tacrolimus detection reagent
A tacrolimus, immunogenic technology, applied in immunoglobulins, specific peptides, measuring devices, etc., can solve the problems of complex processing, high cost, radiation hazards for operators, etc., and achieve high immunogenicity.
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Embodiment 1
[0066] Tacrolimus immunogen, wherein R is N-O-(CH 2 ) 3 -COO-, the carrier is bovine serum albumin (Bovine Serum Albumin, BSA).
[0067] The preparation method is as follows:
[0068] Step 1. Synthesis of Tacrolimus Derivatives with Formula (III) Structure
[0069] (1) Preparation of compound 4-phthalimide oxybutyrate ethyl ester with formula (IV) structure:
[0070] First dissolve 15.0g, 93mmol N-hydroxy-phthalimide in 200mL dimethylformamide (DMF) to obtain N-hydroxy-phthalimide solution, then add 4.97g, 92mmol sodium methoxide (NaOMe) and 17.9g, 92mmol ethyl 5-bromovalerate, obtain the first mixed solution, the first mixed solution was stirred at 100 DEG C for 8-16 hours, add water after cooling, obtain the second mixed solution, The second mixed solution was extracted with diethyl ether, washed with concentrated sodium carbonate after extraction to obtain the first extract, and the first extract was dried over sodium sulfate to obtain 20 g, 78% yellow oil.
[0071] Br...
Embodiment 2
[0086] The preparation of anti-tacrolimus specific antibody, the preparation method is as follows:
[0087] (1) Dilute the tacrolimus immunogen synthesized in Example 1 to 1.5 mg / ml with phosphate buffered saline (PBS) to obtain an antigen solution, then mix the antigen solution with Freund's complete adjuvant, and conduct injection;
[0088] (2) After 2 to 3 weeks, inject the rabbit once with 1.0ml of the same antigen solution mixed with Freund's incomplete adjuvant, and then once every four weeks, twice in total, and extract the antiserum of the rabbit to obtain effective antibodies.
Embodiment 3
[0090] Preparation of tacrolimus detection reagent,
[0091] Raw materials: the specific anti-tacrolimus antibody in the above example 2, the tacrolimus enzyme-labeled conjugate and 3,3',5,5'-tetramethylbenzidine for conventional ELISA detection (3,3'5,5'-Tetramethylbenzidine, TMB) substrate solution.
[0092] The preparation method of tacrolimus enzyme-labeled conjugate is as follows:
[0093] Weigh 20mg of HRP and dissolve it in 5ml of 0.2M, pH 8.5 phosphate buffer at room temperature; weigh 5mg of tacrolimus derivative with the structure of formula (III) in a small beaker, and add 350μL of DMF, 350 μL of absolute ethanol, 700 μL of 10 mM potassium phosphate buffer at pH 5.0, 20 mg of EDAC and 3 mg of Sulfo-NHS were stirred and reacted at room temperature for 30 min; then the activated tacrolimus derivative was added dropwise to the HRP solution, and the Stir at -8°C for 8-16 hours, and dialyze and purify the coupled antigen to obtain a HRP-tacrolimus conjugate.
[0094] ...
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