ROLE OF CONSERVED AMINO-ACID-RESIDUES IN THE COMPLEMENTARITY-DETERMINING REGIONS ON HAPTEN-ANTIBODY INTERACTION OF ANTI-(4-HYDROXY-3-NITROPHENYL)ACETYL ANTIBODIES

Monoclonal antibodies (mAbs) specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) were prepared at various times after immunization and the amino acid sequences of VH and V lambda 1 in these mAbs were deduced from cDNA nucleotide sequences. Replacements due to somatic mutation were not found in day 7 mAbs but were found in those of days 14, 84 and 294. The affinity of day 7 mAbs to NP-glycine(NP-Gly) was in the order of 10(4) M(-1) and it increased about 8000-fold with time after immunization. The extrinsic circular dichroism (CD) spectrum of the NP-epsilon-aminocaproic acid (NP-Cap)/Ab complex was unique for each mAb, although the spectra were grouped into two types, which tended to shift from one type to another with time, suggesting a variation in the micro-environments around NP-Cap in the combining sites. All these data indicate that the structure of the combining site was altered by somatic mutation; however, the fine-specificity measured by cross-reactivity with hapten analogues did not change significantly with time. We examined the amino acid residues in CDRs responsible for recognition of NP-haptens by comparing the amino acid sequences of anti-NP mAbs. Analyses revealed the presence of several conserved amino acid residues in CDRs of VH and V lambda 1, such as Tyr-32H, and Tyr-60H, in addition to a core segment involving Arg-50H. It was predicted that these conserved residues determine Ab-specificity and that the amino acid residues introduced by somatic mutation may act by refining the orientation of specificity determining residues and resulting in an increase in affinity.