Seligman S J, Binley J M, Gorny M K, Burton D R, Zolla-Pazner S, Sokolowski K A
Department of Medicine, SUNY Health Science Center, Brooklyn 11203, USA.
Mol Immunol. 1996 Jun;33(9):737-45. doi: 10.1016/0161-5890(96)00044-2.
Characterization of the sites recognized by antibody on the V3 loop of the envelope glycoprotein gp120 of HIV-1 was done by competition ELISAs on a series of four mouse mAbs, a human mAb and a human Fab. The solid-phase antigen consisted of biotin-YNKRK-RIHIGPGRAFYTTKN, a sequence from the center of the V3 loop of gp120MN, applied to streptavidin-coated wells. Competing antigens were two series of peptides with the HIV-1MN sequence each serially deleted at either the N or C terminus but kept constant at the other terminus. For each series, the amino acid at the deleting end needed to give a minimum KD was identified. The epitope was defined as the sequence including both of the identified amino acids as terminal amino acids. For the six antibodies reported, the epitope length ranged from seven to 14 amino acids. Use of a cyclic peptide as competing fluid-phase antigen suggested the influence of conformational constraints on presumed "linear" epitopes. The operationally-defined epitope was longer than the contact residues in one of two instances in which the X-ray crystallographic structure had been determined. The longer estimates of epitope length in the current study based on competition ELISAs with serial deletions suggest that non-contact residues are significant both in epitope definition and in functional applications including immunogen design.
通过对一系列四种小鼠单克隆抗体、一种人单克隆抗体和一种人Fab进行竞争ELISA，对HIV-1包膜糖蛋白gp120的V3环上抗体识别位点进行了表征。固相抗原由生物素-YNKRK-RIHIGPGRAFYTTKN组成，这是gp120MN的V3环中心的一个序列，应用于链霉亲和素包被的孔中。竞争抗原是两个系列的肽，每个肽都具有HIV-1MN序列，在N端或C端依次缺失，但在另一端保持恒定。对于每个系列，确定了在缺失端产生最小KD所需的氨基酸。表位被定义为包括两个确定的氨基酸作为末端氨基酸的序列。对于所报道的六种抗体，表位长度范围为7至14个氨基酸。使用环状肽作为竞争液相抗原表明构象限制对假定的“线性”表位有影响。在已确定X射线晶体结构的两种情况中的一种情况下，操作定义的表位比接触残基长。在当前研究中，基于连续缺失的竞争ELISA对表位长度的估计更长，这表明非接触残基在表位定义以及包括免疫原设计在内的功能应用中都很重要。
