Dehydroepiandrosterone hapten, artificial antigen and antibody as well as preparation method and application thereof
A technology of dehydroepiandrosterone and artificial antigen, which is applied in the field of artificial antigen and antibody preparation, can solve the problem that there is no immunological detection method for dehydroepiandrosterone, and achieves high sensitivity, high affinity and fast speed. Effect
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Embodiment 1
[0062] (1) The preparation of dehydroepiandrosterone hapten (n=3), as shown in the following reaction formula:
[0063]
[0064] dehydroepiandrosterone hapten
[0065] 250mg (0.827mmol) of DHAE, 511mg (4.48mmol) of glutaric anhydride and 11mg (90μmol) of DMAP were dissolved in 1ml of anhydrous pyridine, and the reaction was stirred at room temperature for 23 hours. Add 10ml of saturated sodium carbonate solution after the reaction, and continue to stir for 10 minutes; then use ethyl acetate to extract, take the water layer, and repeat the extraction 3 times; cool the water layer, and acidify the water layer with 2mol / L hydrochloric acid to make its pH 3, Filter and collect the precipitate; wash the precipitate with saturated sodium carbonate solution to neutrality, and then vacuum dry the precipitate to obtain the dehydroepiandrosterone hapten.
[0066] (2) The preparation of DHEA artificial antigen, the immune antigen was prepared by the active ester method, and the coate...
Embodiment 2
[0076] (1) The preparation of dehydroepiandrosterone hapten (n=2), as shown in the following reaction formula:
[0077]
[0078] 250 mg (0.827 mmol) of DHAE, 120 mg (0.827 mmol) of succinic anhydride and 20 mg (165 μmol) of DMAP were dissolved in 2 ml of anhydrous pyridine, and the reaction was stirred at room temperature for 23 hours. After the reaction, add 5ml of saturated sodium chloride solution and continue to stir for 20min; extract with dichloromethane, take the water layer, and repeat the extraction 3 times; cool the water layer, and acidify the water layer with 2mol / L hydrochloric acid to make its pH 2, Filter and collect the precipitate; wash the precipitate with saturated sodium chloride solution to neutrality, and then dry the precipitate to obtain the dehydroepiandrosterone hapten.
[0079] (2) The preparation of DHEA artificial antigen, the immune antigen was prepared by the active ester method, and the coated antigen was prepared by the mixed acid anhydride ...
Embodiment 3
[0085](1) The preparation of dehydroepiandrosterone hapten (n=4), as shown in the following reaction formula:
[0086]
[0087] 250 mg (0.827 mmol) of DHAE, 680 mg (6.62 mmol) of adipic anhydride and 6.7 mg (55 μmol) of DMAP were dissolved in 1 ml of anhydrous pyridine, and the reaction was stirred at room temperature for 23 hours. After the reaction, add 15ml of saturated sodium carbonate and continue to stir for 5 minutes; extract with petroleum ether, take the water layer, and repeat the extraction 3 times; cool the water layer, and acidify the water layer with 2mol / L hydrochloric acid to make the pH 4, filter and collect Precipitation; the precipitate was washed with saturated saline until neutral, and then dried to obtain the dehydroepiandrosterone hapten.
[0088] (2) The preparation of DHEA artificial antigen, the immune antigen was prepared by the active ester method, and the coated antigen was prepared by the mixed acid anhydride method:
[0089] Preparation of im...
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