RESUMO
In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant 'a' region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178-186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing 'a' region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175-189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178-186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.
Assuntos
Epitopos de Linfócito B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Variação Genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Camundongos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , CoelhosRESUMO
Direct adsorption of small peptides to polystyrene surfaces is often not satisfactory. Therefore, a simple and general coating procedure to improve the coating efficiency of small synthetic peptide antigens to polystyrene is described. In this study, the binding capacities of four small synthetic peptides N-terminally linked to various moieties during synthesis were compared to their parent counterparts in terms of the amount of peptide coat concentration required to achieve 50% of the maximum enzyme-linked immunosorbent assay signal. Elongation of a short epitope sequence by an N-terminal acetyl-thio-acetyl (Ata) group or a lysyl moiety resulted in an enormous reduction in peptide coat concentration for all tested peptides of net two to four orders of magnitude when corrected for chain elongation. The optimal length of the lysyl moiety depended on the length of the model peptide. Replacement of both extensions by analogues (i.e., Ata analogues and other basic amino acid residues in the case of the lysyl moiety) was possible without reducing their enhancing properties to a great extent. Additional experiments showed that a lysyl moiety consisting of a linear stretch of seven lysyl moiety consisting of a linear stretch of seven lysyl residues was more effective in comparison to a branched lysyl construct and could easily compete with the multiple antigen peptide approach.
