25-羟基维生素D_3人工完全抗原的合成与鉴定

Synthesis and identification of artificial complete antigen 25-hydroxyvitamin D_3

目的:合成25-羟基维生素D3人工完全抗原,并制备抗25-羟基维生素D3的特异性抗体。方法:将25-羟基维生素D3进行化学修饰加入羧基活性基团,合成具有半抗原结构特征的25-羟基维生素D3-半琥珀酸酯。采用碳二亚胺法,将25-羟基维生素D3-半琥珀酸酯,分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联,合成人工完全抗原25-羟基维生素D3-半琥珀酸酯-BSA和25-羟基维生素D3-半琥珀酸酯-OVA。通过紫外吸收光谱,SDS-PAGE和MALDI-TOF进行偶联鉴定。用25-羟基维生素D3-半琥珀酸酯-BSA免疫小鼠,获得抗25-羟基维生素D3抗体免疫血清。结果:25-羟基维生素D3-半琥珀酸酯与BSA的偶联比为(12±0.16)∶1,免疫小鼠后获得高效价(效价为6.25×10-4)的抗体,且标准品浓度在37.5～600 ng/mL范围具有显著的竞争性线性关系,检测的灵敏度为37.5 ng/mL。结论:成功合成了25-羟基维生素D3人工完全抗原,制备出25-羟基维生素D3的抗体且其线性关系显著,灵敏度较高,为进一步研制检测25-羟基维生素D3的试剂盒奠定了基础。

AIM： To synthesize the 25-hydroxyvitamin D3 artificial complete antigen and to prepare the specific antibody against 25-hydroxyvitamin D3. METHODS： The active group carboxyl was introduced into 25-hydroxyvitamin D3 and formed 25-hydroxyvitamin D3-hemisuccinate which pos- sessed the structure of the hapten by chemical modification. The EDC method was applied to conjugate 25-hydroxyvitamin D3-hemisuccinate to bovine serum albumin as an artifi- cial immunogen. The coating antigen 25-hydroxyvitamin D3- hemisuccinate-OVA was obtained in the same way. Ultraviolet, SDS-PAGE and MALDI-TOF were used to identify 25- hydroxyvitamin D3-hemisuccinate-BSA. RESULTS： BALB/c mice were immunized with 25-hydroxyvitamin D3-hemisuccinate-BSA to generate the polyclonal antibody of the 25- hydroxyvitamin D3 worth high titer and the immunogen, 25- hydroxyvitamin D3-hemisuccinate-BSA, was successfully prepared with coupling ratio （12 ±0.16）：1（ N =3） coupling. CONCLUSION： The high titer and good sensitivity of anti- 25-hydroxyvitamin D3 antibody are produced in sera immunized BALB/c mice, which made it possible to develop a clinical diagnostics for illness.