Studies of hyperimmune restricted and partially restricted anti-pneumococcal polysaccharide antibodies from allotype-defined pedigreed rabbits—V variable region heavy chain sequence analysis of the cyanogen bromide C1 fragment obtained from an unusual restricted anti-SVIII antibody from A homozygous a1 partially inbred rabbit

Abstract

Rabbit 723569, a male strain III partially inbred (coefficient of inbreeding was 0.92) rabbit of a 1 a 1, b 4 b 4, c 7 c 7, d 12 d 12 allotype, was hyperimmunized with type VIII capsular pneumococcal vaccine for two courses of immunization. During the first course of immunization, the rabbit antisera contained an acidic antibody with highly restricted structural and electrophoretic properties. The rabbit yielded ≈25 g of precipitable antibody (≈89 ml/m of precipitable antibody at its peak response). Later, during the first course of immunization, the acidic antibody gradually disappeared and was replaced by a second more basic antibody with partially restricted heterogeneity. The amino-terminal residues from both the b 4 kappa light chain and the a 1 heavy chain from the acidic and basic antibodies were all blocked. The restricted acidic antibody was purified by affinity chromatography. Approximately 2.8 μmoles of purified antibody was used to obtain the variable region heavy chain sequence. The purified antibody was mildly reduced and alkylated to obtain pure heavy chain, which was then treated with CNBr. The C1 fragment was isolated by gel filtration in 8 M urea-0.2 M formic acid, pH 2.2. The C1 fragment was completely reduced and alkylated with 2- 14C-labelle iodoacetic acid, and the N1-34, N35-82 and N83-258 peptides were separated by gel filtration. The N1-34 peptide was digested with trypsin, and the N1-10 and N11-34 peptides were purified by gel filtration. The amino-terminal sequences of the N35-82 and N83-258 peptides were obtained by automatic Edman degradation using a standard protein/polypeptide programme. The entire amino-terminal sequence of the N11-34 peptide was achieved using a ‘cytochrome C’ procedure developed to completely sequence small peptides (≈7–25 residues long, using only 100–250 nmoles of peptide) using the automatic Sequencer. Sequences obtained for all purified peptides were as homogeneous as a human kappa light chain, from an IgG (γ1) myeloma protein, sequenced by the same methods. Variable region residues not obtained with the automatic Sequencer were obtained by digestion with carboxypeptidase A and/or B followed by identification by amino acid analysis. Using these procedures, the amino-terminal N1-123 sequence was obtained for this anti-SVIII antibody a 1 heavy chain. The sequence was compared and contrasted with other published anti-SIII and a 1, a 2, and a 3 rabbit allotype variable region sequences. The variable region sequence was similar to other a 1 rabbit sequences. Most differences were confined mainly to the hypervariable regions (especially the N101-110 hypervariable region). The 723569 sequence showed an unusual His-His sequence at positions 61 and 62. The amino-terminal 123 residues of 723569 heavy chain required the insertion of 12 deletions to obtain maximum homology with human, mouse, guinea pig and rabbit variable region heavy chain sequences. The largest number of deletions was required in the N101-110 hypervariable region.