MAPPING THE DETAILED SPECIFICITY OF A CALCIUM-DEPENDENT MONOCLONAL-ANTIBODY THROUGH THE USE OF SOLUBLE POSITIONAL SCANNING COMBINATORIAL LIBRARIES - IDENTIFICATION OF POTENT CALCIUM-INDEPENDENT ANTIGENS

The detailed specificity of monoclonal antibody M1, which has been rep orted to bind in a calcium-dependent manner to the 'FLAG' sequence DYK DDDDK-NH2,, was examined using soluble hexa- and decapeptide positiona l 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 inter action, each PS-SCL was screened in the presence and absence of calciu m using a competitive ELISA. Overall, peptide mixtures had greater inh ibitory activity against mAb M1 binding to FLAG in the absence of calc ium. A total of 16 individual hexapeptides were identified, all of whi ch 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 th e decapeptide PS-SCL in the absence of calcium, lysine was also more a ctive in the fifth position than the original aspartic acid. Based on the screening results, 24 individual decapeptides were prepared and we re found to have activities 10- to 100-fold higher than the FLAG octap eptide in the absence of calcium. The specificity of lysine at the fif th position in the antigen-antibody interaction was further examined b y 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 an alysis was also carried out on the FLAG octapeptide to determine optim al antigen length for antibody binding. Overall, lysine at the fifth p osition could be substituted with ornithine with no significant loss i n 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 acti vity in the presence or absence of calcium. This study demonstrates th e ease and effectiveness of PS-SCLs over individual peptide analogs fo r the examination of the degree of cross-reactivity for a given monocl onal antibody as well as for the identification of novel, high-affinit y peptides.