Magnetic bead enzyme-linked immunosorbent assay (ELISA) detects antigen-specific binding by phage-displayed scFv antibodies that are not detected with conventional ELISA

Anal Biochem. 1997 Dec 15;254(2):263-6. doi: 10.1006/abio.1997.2378.

Abstract

An efficient means for the detection of antigen-specific binding by phage-displayed antibodies would facilitate the selection of such phage, especially from libraries with large repertoires of V-genes. We report the development and characterization of a magnetic bead phage ELISA which detects antigen binding phage which could not be detected by conventional ELISA. We were attempting to select phage binding to the oncodevelopmental antigen, heat-stable alkaline phosphatase (HSAP). Although there was an obvious enrichment in the phage titers after successive rounds of selection, we were unable to detect antigen-binding phage by ELISA on a plastic surface. However, ELISA with a suspension of superparamagnetic particles covalently conjugated to HSAP effectively identified antigen-binding phage after the fourth round of selection. This method could also detect antigen-specific binding of individual phage clones. Some of the phage clones bound to either amino- or carboxy-terminal-conjugated HSAP, perhaps reflecting the differences in the exposed epitopes. It is suggested that a sensitive method such as magnetic bead phage ELISA be tried before declaring a phage selection as unsuccessful or concluding that a phage clone does not bind antigen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Antigen-Antibody Reactions*
  • Attachment Sites, Microbiological*
  • Bacteriophages / immunology*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Immunomagnetic Separation

Substances

  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Alkaline Phosphatase