Pinilla C, Appel J R, Houghten R A
Torrey Pines Institute for Molecular Studies, San Diego, CA 92121.
Mol Immunol. 1993 Apr;30(6):577-85. doi: 10.1016/0161-5890(93)90032-7.
The functional importance of each amino acid residue making up the antigenic determinants of three different peptide-mAb interactions was determined using complete series of substitution analogs of the three immunizing synthetic peptides. Fingerprint substitution profiles for the three different antigenic determinants were obtained separately by direct and competitive ELISA. Competitive ELISA was found to offer the advantage of being able to measure the concn of each peptide substitution analog necessary to inhibit antibody binding to the original peptide. In this manner, the relative functional contribution to antibody binding of each amino acid residue making up the antigenic determinant was determined and termed the relative positional importance factor (RPIF). Each antigenic determinant was found to contain one very highly specific residue (i.e., highest RPIF) that was, on average, the least replaceable with any of the natural L-amino acids (the average decrease in recognition ranged 250- to 28,000-fold). At the other extreme, two or three positions in each antigenic determinant were found to be only weakly involved in recognition. These positions were considered redundant since the average decrease in recognition of the substitution analogs for these positions was found to be 20-fold or less. The remaining antigenic determinant residues exhibited the fine specificity common to antigen-antibody interactions in that only relatively conservative substitutions for these residues were recognized by their respective antibodies. It is of interest that the positional arrangement of specific and nonspecific residues were different for each of the three antigenic determinants examined.
利用三种免疫合成肽的完整系列取代类似物，确定了构成三种不同肽 - 单克隆抗体相互作用抗原决定簇的每个氨基酸残基的功能重要性。通过直接和竞争性ELISA分别获得了三种不同抗原决定簇的指纹取代图谱。发现竞争性ELISA的优势在于能够测量抑制抗体与原始肽结合所需的每种肽取代类似物的浓度。通过这种方式，确定了构成抗原决定簇的每个氨基酸残基对抗体结合的相对功能贡献，并将其称为相对位置重要性因子（RPIF）。发现每个抗原决定簇都包含一个非常高度特异性的残基（即最高的RPIF），平均而言，该残基用任何天然L - 氨基酸替代的可能性最小（识别平均降低范围为250至28,000倍）。在另一个极端，发现每个抗原决定簇中的两三个位置仅微弱地参与识别。这些位置被认为是多余的，因为发现这些位置的取代类似物的识别平均降低为20倍或更低。其余的抗原决定簇残基表现出抗原 - 抗体相互作用共有的精细特异性，即它们各自的抗体仅识别这些残基的相对保守取代。有趣的是，所研究的三个抗原决定簇中每个的特异性和非特异性残基的位置排列都不同。
