Pinilla C, Buencamino J, Appel J R, Hopp T P, Houghten R A
Torrey Pines Institute for Molecular Studies, San Diego, CA 92121, USA.
Mol Divers. 1995 Sep;1(1):21-8. doi: 10.1007/BF01715806.
The detailed specificity of monoclonal antibody M1, which has been reported to bind in a calcium-dependent manner to the 'FLAG' sequence DYKDDDDK-NH2, was examined using soluble hexa- and decapeptide positional scanning synthetic combinatorial libraries (PS-SCLs) made up of 52 x 10(6) and 4 x 10(12) different sequences, respectively. To study the influence of calcium on the specificity of this antigen-antibody interaction, each PS-SCL was screened in the presence and absence of calcium using a competitive ELISA. Overall, peptide mixtures had greater inhibitory activity against mAb M1 binding to FLAG in the absence of calcium. A total of 16 individual hexapeptides were identified, all of which contained the motif-DYK_K_(-), and were recognized by mAb M1 in the absence of calcium with 50- to 100-fold higher affinity than the FLAG octapeptide (IC50 = 273 nM). On average, the same set of peptides bound 10-fold less effectively in the presence of calcium. Upon screening the decapeptide PS-SCL in the absence of calcium, lysine was also more active in the fifth position than the original aspartic acid. Based on the screening results, 24 individual decapeptides were prepared and were found to have activities 10- to 100-fold higher than the FLAG octapeptide in the absence of calcium. The specificity of lysine at the fifth position in the antigen-antibody interaction was further examined by synthesizing and assaying substitution analogs at this position for the octapeptide and hexapeptide forms of the FLAG sequence, as well as for two hexapeptides identified from the PS-SCL. Truncation analog analysis was also carried out on the FLAG octapeptide to determine optimal antigen length for antibody binding. Overall, lysine at the fifth position could be substituted with ornithine with no significant loss in activity, and peptide length was not a critical factor for antibody binding in the absence of calcium. Also, the octapeptide having lysine at the fifth position in place of the aspartic acid had the same activity in the presence or absence of calcium. This study demonstrates the ease and effectiveness of PS-SCLs over individual peptide analogs for the examination of the degree of cross-reactivity for a given monoclonal antibody as well as for the identification of novel, high-affinity peptides.
据报道，单克隆抗体M1以钙依赖的方式与“FLAG”序列DYKDDDDK-NH2结合，使用分别由52×10⁶和4×10¹²个不同序列组成的可溶性六肽和十肽位置扫描合成组合文库（PS-SCLs），研究了该单克隆抗体M1的详细特异性。为了研究钙对这种抗原-抗体相互作用特异性的影响，使用竞争性酶联免疫吸附测定法（ELISA），在有钙和无钙的情况下对每个PS-SCL进行筛选。总体而言，在无钙的情况下，肽混合物对单克隆抗体M1与FLAG结合的抑制活性更强。总共鉴定出16种单个六肽，所有这些六肽都含有基序-DYK_K_(-)，并且在无钙的情况下被单克隆抗体M1识别，其亲和力比FLAG八肽高50至100倍（IC50 = 273 nM）。平均而言，在有钙的情况下，同一组肽的结合效率低10倍。在无钙的情况下筛选十肽PS-SCL时，赖氨酸在第五位的活性也比原来的天冬氨酸更高。基于筛选结果，制备了24种单个十肽，发现在无钙的情况下，它们的活性比FLAG八肽高10至100倍。通过合成并检测FLAG序列的八肽和六肽形式以及从PS-SCL中鉴定出的两种六肽在该位置的取代类似物，进一步研究了抗原-抗体相互作用中赖氨酸在第五位的特异性。还对FLAG八肽进行了截短类似物分析，以确定抗体结合的最佳抗原长度。总体而言，第五位的赖氨酸可用鸟氨酸替代，活性无明显损失，并且在无钙的情况下，肽长度不是抗体结合的关键因素。此外，在第五位用赖氨酸替代天冬氨酸的八肽在有钙或无钙的情况下具有相同的活性。这项研究证明了PS-SCLs相对于单个肽类似物在检查给定单克隆抗体的交叉反应程度以及鉴定新型高亲和力肽方面的简便性和有效性。
