Yao Z J, Kao M C, Chung M C
Bioprocessing Technology Centre, National University of Singapore.
J Protein Chem. 1995 Apr;14(3):161-6. doi: 10.1007/BF01980328.
Screening of bioactive peptides from random peptide libraries using monoclonal antibodies as ligates is an effective method to define epitopes of protein antigens. However, it is thought that polyclonal antibodies might also serve as promising ligates for screening. We illustrate this approach by using recombinant human lymphotoxin (rhLT) polyclonal antibody as a model. The procedure consists in (a) affinity purification of polyclonal antibody to obtain the "monospecific" antibody, (b) screening against a phage-displayed random peptide library using the affinity-purified antibody, (c) plating the enriched phage on agar plates, randomly picking clones, and selecting the positive ones by dot blotting, (d) DNA sequencing of the positive clones and conducting a homology search against the protein sequence databank, and (e) confirming the epitopes by chemical peptide synthesis. By employing this procedure, we identified a dominant epitope RQHPKM, located at residues 15-20 of the human lymphotoxin amino acid sequence. The usefulness of this general procedure is discussed.
使用单克隆抗体作为连接物从随机肽库中筛选生物活性肽是确定蛋白质抗原表位的有效方法。然而，人们认为多克隆抗体也可能作为有前景的连接物用于筛选。我们以重组人淋巴毒素（rhLT）多克隆抗体为模型来说明这种方法。该过程包括：（a）亲和纯化多克隆抗体以获得“单特异性”抗体；（b）使用亲和纯化的抗体筛选噬菌体展示的随机肽库；（c）将富集的噬菌体铺在琼脂平板上，随机挑选克隆，并通过斑点印迹法选择阳性克隆；（d）对阳性克隆进行DNA测序，并针对蛋白质序列数据库进行同源性搜索；（e）通过化学肽合成确认表位。通过采用该过程，我们确定了一个主要表位RQHPKM，位于人淋巴毒素氨基酸序列的第15 - 20位残基处。讨论了该通用过程的实用性。
