SPECIFICITY AND V-H SEQUENCE OF 2 MONOCLONAL-ANTIBODIES AGAINST THE N-TERMINUS OF DYSTROPHIN

We have used a random library of 15-mer peptides expressed on phage to show that two monoclonal antibodies (mAbs) require only the first thr ee amino acids of dystrophin (Leu-Trp-Trp) for binding. Since the mAbs recognize dystrophin in frozen muscle sections, the results suggest t hat this hydrophobic N-terminus of dystrophin is accessible to antibod y in situ. Quantitative binding studies suggested minor differences in specificity between the two mAbs, so the Ig heavy-chain variable regi on (V-H) sequences of the two hybridomas were determined by RT-PCR and cDNA sequencing. After elimination of PCR errors, the two cDNA. seque nces were found to be identical except for five somatic mutations whic h resulted in three amino acid changes in the second hypervariable reg ion (CDR2). The results suggest that the two hybridomas originated fro m the same lymphocyte clone in a germinal centre of the spleen, but un derwent different point mutations and subtype switches during clonal e xpansion to form blast cells.