Benecke S, Ostermann-Latif C, Mäder M, Schmidt B, Unger J W, Westarp M E, Felgenhauer K
Neurologische Klinik, Universität Göttingen, F.R.G.
J Neurochem. 1993 Sep;61(3):804-11. doi: 10.1111/j.1471-4159.1993.tb03590.x.
Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168-189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.
用五只合成肽对兔子进行免疫，制备抗血清。通过一级和二级结构分析，这些肽已被鉴定为猪胆碱乙酰转移酶（ChAT）序列中潜在的抗原表位。在酶联免疫吸附测定（ELISA）和蛋白质免疫印迹法中，所有五只抗血清均能识别来自猪脑的免疫亲和纯化抗原。四只抗血清在斑点印迹法中能识别ChAT，另外四只抗血清在以单克隆抗体作为捕获抗体的夹心ELISA中，能与天然和降解的酶发生反应。在这种夹心ELISA中，一种肽抗血清的亲和力与针对免疫亲和纯化ChAT产生的多克隆抗体相似。在ELISA、蛋白质免疫印迹法和斑点印迹法中，同一抗血清能与人胎盘的酶发生反应，并能识别大鼠、灵长类动物和人类神经元中的ChAT。因此，单一肽（氨基酸168 - 189）为简便、可靠且可重复地生成抗ChAT抗体提供了方法，这些抗体适合替代传统的多克隆和单克隆抗体。
