PMID: 7515258Feb 1, 1994Paper

Serial deletion mapping by competition ELISA assay: characterization of a linear epitope in the V3 loop of HIV-1

AIDS Research and Human Retroviruses
S J Seligman

Abstract

Precise epitope mapping and characterization is important for development of a subunit vaccine. To identify epitopes in the principal neutralizing determinant (PND) within the V3 loop of human immunodeficiency virus type 1 (HIV-1), sera were screened in a direct ELISA assay with a coating peptide consisting of IHIGPGRAF, a specific sequence commonly found in the loop, linked at the C terminus to GAGAAK, a nonspecific hexapeptide. Epitope mapping experiments revealed that a competition ELISA assay using IGPGRAFGAGAAK as coating peptide was superior to a direct ELISA assay for epitope definition and characterization. The competing peptides contained only specific sequences and were serially deleted of single amino acids first at the N terminus and then at the C terminus. Study of the most highly reactive serum identified in the initial screening identified the epitope (the shortest peptide with the most potent inhibitory activity) as IGPGRAF. Deletion of a single amino acid from the C terminus of the epitope resulted in complete loss of activity as competing peptide. In contrast, single amino acid deletions of three N-terminal amino acids resulted in a stepwise 2700-fold reduction in affinity. RAF was the shortest peptide with in...Continue Reading

References

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