Rucklidge G J, Milne G, Chaudhry S M, Robins S P
Bone Research Group, Rowett Research Institute, Bucksburn, Aberdeen, Scotland.
Anal Biochem. 1996 Dec 1;243(1):158-64. doi: 10.1006/abio.1996.0495.
A method combining the affinity purification and biotinylation of antibodies has been developed utilizing the high-capacity binding of blotting membranes and glutaraldehyde treatment to immobilize antigen. Following reaction of the antisera with the membrane-bound antigen, the biotinylation of the antibodies is performed in situ, before release of the IgG-biotin complex with low pH buffer. The antibody is able to recognize antigen in subsequent reactions in enzyme-linked immunosorbent assays, and preliminary results have shown that the antibody can also be used in immunocytochemical localization of antigen in frozen sections. Biotinylation in situ may protect the variable region of the IgG compared to antibodies biotinylated in solution, thus increasing their antigen recognition. The method has the advantage of being suitable for multiple use of the antigen-coated membrane, making it particularly attractive where only small amounts of antigen may be available.
利用印迹膜的高容量结合和戊二醛处理来固定抗原，已开发出一种将抗体亲和纯化与生物素化相结合的方法。抗血清与膜结合抗原反应后，在使用低pH缓冲液释放IgG-生物素复合物之前，原位进行抗体的生物素化。该抗体能够在酶联免疫吸附测定的后续反应中识别抗原，初步结果表明该抗体也可用于冷冻切片中抗原的免疫细胞化学定位。与在溶液中生物素化的抗体相比，原位生物素化可能会保护IgG的可变区，从而提高其抗原识别能力。该方法的优点是适用于抗原包被膜的多次使用，在只有少量抗原可用的情况下尤其具有吸引力。
