人血管能抑素免疫兔抗血清制备及其特性测定

Preparation and Characteristic Test of the Rabbit Antiserum Against Human Canstatin

纯化的人血管能抑素包涵体进行SDS＿PAGE分离,切胶回收目的蛋白,与弗氏不完全佐剂充分乳化后,进行多点皮下注射免疫新西兰白兔,制备兔抗人血管能抑素抗血清,抗血清去除交叉反应抗体后,进行WesternBlotting分析抗血清与人血管能抑素及其N端和C端片段的抗原抗体反应特性。结果表明,经过4次加强免疫后,获得了高效价的兔抗人血管能抑素抗血清。抗血清去除交叉反应抗体后,WesternBlotting分析表明,它能与人血管能抑素发生特异的抗原抗体反应,同时还能与人血管能抑素的N端片段和C端片段发生特异性较弱的抗原抗体反应。证明制备的兔抗人血管能抑素抗血清能应用于人血管能抑素及其N端和C端片段免疫学研究中。

The purified human canstatin was isolated by recovering the target band on the SDS-PAGE gel and used to immunize the New Zealand white rabbits by subcutaneous injection after emulsified with Freund's incomplete adjuvant. After removing its cross reacting antibodies, the antiserum against human canstatin was used to test its characteristic reaction with human canstatin and its N-terminal and C-terminal domains by Western blotting analyses. The results showed that high titer antiserum against human canstatin was obtained in immunized rabbits after four reinforced immunizations. The results of Western blotting analyses after removing the cross reacting antibodies showed that the rabbit antiserum against human canstatin specifically intensively reacted with human canstatin and faintly with its N-terminal and C-terminal domains. These results demonstrated the prepared rabbit antiserum against human canstatin can specifically react with human canstatin and its N-terminal and C-terminal domains, and can be applied in immunological stndy of N-terminal and C-terminal domains.