Antibody immunodiversity: A study on the marked specificity difference between two anti-yeast iso-1 cytochrome c monoclonal antibodies whose epitopesare closely related

Anti-yeast iso-1 cytochrome c (cyt. c) monoclonal antibodies 2-96-12 and 4- 74-6 have closely related epitopes (antigenic determinants). However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evo lutionarily related cytochromes c. Such a marked difference in specificity of antibodies with overlapping epitopes may represent unique antibody immun o-diversity. Thus, we constructed Fv fragment models consisting of the vari able domains of the heavy and light chains of 2-96-12 and 4-74-6 and that o f another anti-iso-1 cyt. c as a control to gain insight into the origin of this difference in specificity. Our models show that 4-74-6 and 2-96-12 co ntain five and two aromatic side chains, respectively, in or near the centr al area of the antigen-combining site. The side chains of Arg95H (heavy cha in) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the centra l area of the combining site in our model. Antigen docking to our Fv models , combined with previous immunological studies, suggests that iso-1 cyt. c Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6 and that bot h epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt. c Leu58, Asp60, Asn 62, and Asn63. The effect of the Arg95H to Lys mutation on the antigen bind ing is also in accord with our model. The difference in specificity may be partly explained by a greater degree of conformational flexibility in and a round the central area of the: combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing.