乳牛孕酮人工抗原的制备与鉴定

Preparation and identification of progesterone antigen for dairy cow

采用二环己基碳化二亚胺偶联法将孕酮-11α-半琥珀酸酯(11-αOH-P4-HS)分别与牛血清白蛋白(BSA)、卵清白蛋白(OVA)载体偶联,制备乳牛孕酮人工抗原P4-BSA和P4-OVA,并用SDS-PAGE对制备的人工抗原进行测定。再用制备的人工免疫抗原P4-BSA免疫BALB/c小鼠制备免疫血清。用P4-OVA作为ELISA检测抗原,方阵滴定法确定检测抗原的最佳包被浓度,间接ELISA测定免疫血清中抗体的特异性及效价。结果表明,成功制备了乳牛孕酮人工抗原,P4-OVA的最佳包被浓度为5.82×10-5ng/mL,最佳封闭液为50 mL/L的羊血清,免疫血清具有良好的特异性。

llα-hydroxyprogesterone hemisuccinate（llα-OH-P4-HS） was conjugated with carrier proteins BSA and OVA by dicyclohexylcarbodiimide（DCC）, respectively, to obtain immunization antigen（P4- BSA） and detection antigen（P4-OVA）. The 2 antigens were accredited by SDS-PAGE. BALB/c mice were injected with P4-BSA to prepare antiserum against P4. Optimal coating concentration of P4-OVA was determined by ELISA, and the specificity and titer of antiserum were detected by indirect ELISA. The results showed that P4-BSA and P4-OVA were conjugated successfully, the optimal coating concentration of P4-OVA was 5.82× 10^-5 ng/mL, the optimal blocking reagent was sheep serum at 50 mL/L, and the antiserum against P4 had high specificity. These results demonstrated that P4-BSA and P4-OVA conjugated by DCC provided a basis for the preparation of monoclonal antibody against P4 for dairy cow.