Bhardwaj D, Singh S S, Abrol S, Chaudhary V K
Department of Biochemistry, University of Delhi, India.
J Immunol Methods. 1995 Feb 27;179(2):165-75. doi: 10.1016/0022-1759(94)00280-a.
We have produced monoclonal antibodies (MAbs) which react with a minor and the major coat proteins of filamentous phage M13 and have characterised them by combining the techniques of enzyme-linked immunosorbent assay (ELISA) and Western blotting. These coat proteins are the minor coat protein, gIIIp, the product of gene III and the major coat protein, gVIIIp, the product of gene VIII. Both gIIIp and gVIIIp are important in the context of 'phage display' of foreign peptides/proteins as fusions to these proteins. The anti-gIIIp MAbs were able to detect native gIIIp as well as the fusion proteins comprising foreign proteins and gIIIp in ELISA and on Western blot, indicating their utility for studying the expression of foreign proteins in phage display. Similarly anti-gVIIIp MAbs detected gVIIIp both in ELISA and on Western blot. In an 'affinity capture phage ELISA', phages that were captured by virtue of the interaction between the foreign protein (ligand fused to the gIIIp and displayed on the phage surface) and the immobilised counterpart (receptor), could be easily detected using anti-gVIIIp MAbs. Considering the potential of 'phage display' technology in protein engineering, these antibodies should find wide applications.
我们制备了与丝状噬菌体M13的次要和主要外壳蛋白发生反应的单克隆抗体（MAb），并通过结合酶联免疫吸附测定（ELISA）和蛋白质印迹技术对其进行了表征。这些外壳蛋白分别是次要外壳蛋白gIIIp（基因III的产物）和主要外壳蛋白gVIIIp（基因VIII的产物）。在将外源肽/蛋白质作为这些蛋白质的融合物进行“噬菌体展示”的情况下，gIIIp和gVIIIp都很重要。抗gIIIp单克隆抗体能够在ELISA和蛋白质印迹中检测到天然gIIIp以及包含外源蛋白质和gIIIp的融合蛋白，表明它们在研究噬菌体展示中外源蛋白质表达方面的实用性。同样，抗gVIIIp单克隆抗体在ELISA和蛋白质印迹中均能检测到gVIIIp。在“亲和捕获噬菌体ELISA”中，利用外源蛋白质（与gIIIp融合并展示在噬菌体表面的配体）与固定化对应物（受体）之间的相互作用捕获的噬菌体，可以使用抗gVIIIp单克隆抗体轻松检测到。考虑到“噬菌体展示”技术在蛋白质工程中的潜力，这些抗体应具有广泛的应用。
