The use of unideterminant fragments of ferredoxin in the genetic mapping of determinant specificity of the immune response.
The ferredoxin (Fd) molecule is a small non-mammalian immunogenic protein containing 55 amino acid residues with only two major antigenic determinants located with the NH2-terminal heptapeptide and the COOH-terminal pentapeptide. Selective enzyme cleavages of Fd with either trypsin or carboxypeptidase A result in the inactivation of the antigenic determinants by the removal of a tripeptide at the NH2-terminal and two amino acid residues at the COOH-terminal, effectively leaving 52 and 53 amino acid fragments respectively, each containing a single antigenic determinant. Fd digested with both enzymes yielded a 50 amino acid peptide with both determinants inactivated. Purity of these digests was assessed using monoclonal antibodies in standard and antigen-blocking ELISAs. The doubly digested peptide had virtually no reactivity with anti-Fd sera, reconfirming that the central cysteine-rich region is serologically silent. It was found that the sum of the reactivities of the N- and C-determinant-bearing peptides as equal to that of the native Fd and that the ratio of the reactivities could be used to assess determinant selectivity in the response to Fd in congenic recombinant strains of mice. This method was used in mapping the determinant selectivity in the antibody response to the MHC of mice to the left of the I-B subregion. Use of the B10.HTT strain indicated that separate genes mapping to the same subregion code for the magnitude of the antibody response and the determinant selectivity of the response.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002874:Chromosome Mapping; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005260:Female; D005288:Ferredoxins / Q000276:immunology*; D005802:Genes, MHC Class II; D008297:Male; D051379:Mice; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005288:Ferredoxins; D010446:Peptide Fragments