Abaza M S, Atassi M Z
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
J Protein Chem. 1992 Oct;11(5):433-44. doi: 10.1007/BF01025020.
Amino acid substitutions outside protein antigenic sites are very frequently assumed to exert no effect on binding to antiprotein antibodies, especially if these are monoclonal antibodies (mAbs). In fact, a very popular method for localization of residues in protein antigenic sites is based on the interpretation that whenever a replacement causes a change in binding to antibody, then that residue will be located in the antigenic site. To test this assumption, mAbs of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any carrier) synthetic peptide representing region 94-100 of sperm whale myoglobin (Mb). The cross-reactivities and relative affinities of three mAbs with eight Mb variants were studied. Five Mb variants which had no substitutions within the boundaries of the designed antigenic site exhibited remarkable, and in two cases almost complete, loss in cross-reactivity relative to the reference antigen, sperm whale Mb. Two myoglobins, each of which had one substitution within region 94-100, showed little or no reactivity with the three mAbs. It is concluded that substitutions outside an antigenic site can exert drastic effects on the reactivity of a protein with mAbs against the site and that caution should be exercised in interpreting cross-reactivity data of proteins to implicate residues directly in an antigenic site.
人们常常认为蛋白质抗原位点之外的氨基酸替换对与抗蛋白质抗体的结合没有影响，尤其是当这些抗体为单克隆抗体（mAb）时。实际上，一种非常流行的确定蛋白质抗原位点中氨基酸残基位置的方法是基于这样一种解释：只要某个替换导致与抗体的结合发生变化，那么该残基就位于抗原位点。为了验证这一假设，通过用代表抹香鲸肌红蛋白（Mb）第94 - 100区域的游离（即未偶联任何载体）合成肽进行免疫，制备了具有预定特异性的单克隆抗体。研究了三种单克隆抗体与八种肌红蛋白变体的交叉反应性和相对亲和力。在设计的抗原位点边界内没有替换的五种肌红蛋白变体，相对于参考抗原抹香鲸肌红蛋白，其交叉反应性显著降低，在两种情况下几乎完全丧失。两种肌红蛋白在第94 - 100区域各有一个替换，与这三种单克隆抗体几乎没有反应。得出的结论是，抗原位点之外的替换可对蛋白质与针对该位点的单克隆抗体的反应性产生巨大影响，在解释蛋白质的交叉反应性数据以直接将残基与抗原位点联系起来时应谨慎行事。
