Synthesis and Identification of Hapten and Artificial Antigen of Free Thyroxine
Carboxyl group of Free Thyroxine(FT4) was selected as the modified site in this study,Firstly,the amino group was protected with di-t-butyl dicarbonate(Boc2O),Then FT4,modified by terminal-alkyne,was prepared with the amidation reaction between Boc-FT4 and Propargylamine catalyzed by HATU.Finally,the FT4H hapten,which contained a "spacer arm" of triazole ring conjugated to carboxyl site of FT4,was synthesized via trace cuprous salt catalyzed "Click Chemistry" between terminal alkyne and 5-azide valeric acid.Immunogen and coating antigen of FT4H were respectively prepared by coupling reaction between hapten and bovine serum albumin(BSA) or keyhole limpet hemocyanin(KLH) using the N-hydroxysuccinimide active ester method.UV spectrum of the artificial antigen(FT4H-BSA) differed significantly from corresponding spectrum of hapten(FT4H) and BSA,the result also showed that FT4H was conjugated to BSA successfully,the coupling ratio of hapten to BSA was 12 ∶ 1.The 6-8 weeks old Balb/c mice were immunized with FT4H(2 g/L,0.1 mg per rat).Antiserum was collected from tails of mice after immunized five times.The antiserum titer could be up to 1.28×105 through indirect ELISA determination.The result showed that the space arm containing triazole ring as the strong determinant couldn’t interfere the combination between antiserum and FT4,and provided a new way to prepare artificial antigen of the small molecule.