Di Modugno F, Castelli M, Chersi A
Laboratory of Biochemistry, Regina Elena Institute for Cancer Research, Roma, Italy.
Z Naturforsch C J Biosci. 1998 Sep-Oct;53(9-10):902-10. doi: 10.1515/znc-1998-9-1020.
The main linear epitopes of pi-glutathione transferase (pi-GST, EC 2.5.1.18), an enzyme related to cancer progression in a restricted number of tumours, were identified by testing in ELISA the reactivities of polyclonal anti-pi-GST rabbit sera against a panel of 51 overlapping decapeptides, covering the whole 216-residue sequence of the protein. Several major reactivity peaks were detected, each covering two or three adjacent peptides. The most active fragments were then reconstructed by conventional solid-phase synthesis, linked to Sepharose, and used as affinity ligands for isolating specific anti-pi-GST antibody subsets. A second group of antisera was then prepared in rabbits by using as immunogens some of the above described synthetic fragments, linked to a carrier protein, and antipeptide antibodies purified by affinity chromatography. An ELISA test was then performed, using as antigens a panel of peptides and different isoforms of GST, in order to establish whether antibodies isolated from total anti-pi-GST sera would display higher reactivity and specificity, as compared to traditional antipeptide antibodies. Binding data clearly confirm that the formers might be indeed better reagents for the detection and possibly quantitation of pi-GST.
π-谷胱甘肽转移酶（π-GST，EC 2.5.1.18）是一种与少数肿瘤的癌症进展相关的酶，通过在ELISA中检测多克隆抗π-GST兔血清与一组51个重叠十肽的反应性，确定了该酶的主要线性表位，这些十肽覆盖了该蛋白质完整的216个残基序列。检测到几个主要的反应性峰，每个峰覆盖两个或三个相邻的肽段。然后通过常规固相合成重建最具活性的片段，将其与琼脂糖连接，并用作亲和配体来分离特定的抗π-GST抗体亚群。接着，用上述一些与载体蛋白连接的合成片段作为免疫原在兔中制备第二批抗血清，并通过亲和层析纯化抗肽抗体。然后进行ELISA试验，使用一组肽段和不同同工型的GST作为抗原，以确定从总抗π-GST血清中分离的抗体与传统抗肽抗体相比是否具有更高的反应性和特异性。结合数据清楚地证实，前者可能确实是用于检测和可能定量π-GST的更好试剂。
