A novel affinity purification method to isolate peptide specific antibodies

Research output: Contribution to journalArticle

Abstract

Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ β-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displayed by the immunizing but not by unrelated peptides.

Details

Authors
External organisations
  • Hagedorn Research Institute
Research areas and keywords

Keywords

  • Affinity purification, Anti-peptide antibody, Growth hormone receptor, HLA class II β chain molecule
Original languageEnglish
Pages (from-to)151-157
JournalJournal of Immunological Methods
Volume128
Issue number2
Publication statusPublished - 1990 Apr 17
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes