Mahmood Rubab
a Interdisciplinary Biotechnology Unit , Aligarh Muslim University , Aligarh , India.
Prep Biochem Biotechnol. 2016;46(2):161-4. doi: 10.1080/10826068.2014.996232.
Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)-bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.
亲和沉淀已成为一种非常有用的蛋白质纯化技术。在此，它被用于从兔血清中纯化抗菠萝蛋白酶抗体。已开发出一种用于可逆结合和热沉淀抗体的系统。通过用菠萝蛋白酶免疫兔子来产生抗菠萝蛋白酶抗体。制备聚-N-异丙基丙烯酰胺（pNIPAm）-菠萝蛋白酶偶联物，并与兔血清一起孵育。之后升高温度以使聚合物热沉淀。然后通过将复合物与少量pH 3.0的缓冲液孵育从复合物中洗脱抗体。该方法在浓缩抗体方面非常有效。分别通过凝胶电泳和酶联免疫吸附测定（ELISA）检查抗体的纯度和特异性。对pH和温度对抗体与偶联物结合的影响的研究表明，最佳结合发生在pH 8.0和25°C。聚合物酶偶联物进一步用于另一个循环。
