Karlsen A, Lernmark A, Kofod H, Dyrberg T
Hagedorn Research Laboratory, Niels Steensensvej, Gentofte, Denmark.
J Immunol Methods. 1990 Apr 17;128(2):151-7. doi: 10.1016/0022-1759(90)90205-a.
Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides.
通过用合成肽免疫兔子可有效且成功地产生位点特异性、高亲和力的多克隆抗血清。由于非特异性结合，这些抗血清在后续免疫分析中的应用常常受到限制。我们描述了一种新的简单方法来有效亲和纯化抗肽抗体。为测试我们的系统，用代表假定的兔生长激素受体序列和几种HLA - DQβ链分子序列的模型肽免疫兔子。将肽包被在聚苯乙烯塑料珠上。将免疫血清与珠子孵育，经过洗涤步骤后，结合的抗体用1 M乙酸洗脱。SDS - PAGE中重链和轻链条带的存在证明，洗脱的物质主要由完整的免疫球蛋白组成。洗脱的抗体在ELISA中具有肽特异性，并且在免疫印迹分析中仅与完整的抗原性蛋白结合。洗脱抗体的序列特异性得到证实，因为与抗原性蛋白的结合可被免疫肽取代，但不能被无关肽取代。
