Enhancement of ELISAs for screening peptides in epitope phage display libraries

Philippe Valadon, Matthew D. Scharff

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

The complete analysis of epitope phage display libraries requires sensitive assays capable of detecting peptides expressed on phage that have a wide range of affinities for antibody. We have compared two ELISAs, a 'direct' assay where the phage is captured by an anti-phage antibody and the peptide detected by the antibody used for screening, and a 'reverse' assay where the antibody used for screening is first coated on the well and the binding of phage detected by the anti-phage antibody. We demonstrate, by comparing two fUSE5 derived phage bearing five peptides reacting with the anti-cryptococcal polysaccharide antibody 2H1, that the reverse ELISA is the more sensitive assay. Further, there is a limit in affinity, here around 1 μM, above which phage clones are negative by the direct ELISA. We describe an enhancement of the direct assay by mixing 2H1 with 3-fold excess of anti-heavy or anti-light chain antibody. The resulting polymerization of 2H1 induces an increase in antibody avidity that is responsible for the enhancement. The enhanced direct ELISA allowed rapid and sensitive detection of positive clones and is easily inhibited by free peptide, while the reverse ELISA is not. The enhanced ELISA has also been used successfully for immunological screening of intermediate libraries, allowing detection of rare positive clones that would otherwise be lost. The combination of the three ELISAs, reverse, direct, and enhanced direct, should provide a way to rank phage clones into three classes: very low, low, and high affinity, providing information previously obtained only by the synthesis and testing of many peptides.

Original languageEnglish (US)
Pages (from-to)171-179
Number of pages9
JournalJournal of Immunological Methods
Volume197
Issue number1-2
DOIs
StatePublished - Oct 16 1996

Keywords

  • ELISA
  • immunological screening
  • peptide library
  • phage display

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Fingerprint Dive into the research topics of 'Enhancement of ELISAs for screening peptides in epitope phage display libraries'. Together they form a unique fingerprint.

  • Cite this