Monoclonal antibodies isolated by large-scale screening are suitable for labeling adult zebrafish (Danio rerio) tissues and cell structures

J Immunol Methods. 2009 Jul 31;346(1-2):9-17. doi: 10.1016/j.jim.2009.04.012. Epub 2009 May 3.

Abstract

The lack of a sufficient number of antibodies represents an obstacle in the research performed using the zebrafish (Danio rerio) as a model organism. On the other hand, high-throughput generation of antibodies, especially those suitable for immunohistochemistry, is not an established methodology. Here we present the results of an immunization experiment with a zebrafish tissue lysate that allowed for the isolation of hundreds of monoclonal antibodies suitable for labeling of a large variety of zebrafish tissue and cell structures. Some of them were further characterized in terms of detailed localization and age-dependent expression. In addition, the antigen recognized by one of them was first immunoprecipitated and then identified by mass spectrometry. Furthermore, immunofluorescence-competent recombinant antibodies were also isolated by panning large repertoire phage display libraries, in both single-chain (scFv) and single-domain (VHH) format. Such selection alternative is simpler to organize and could contribute to limit the costs of antibody screening and production.

MeSH terms

  • Age Factors
  • Animals
  • Antibodies, Monoclonal / biosynthesis
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / isolation & purification*
  • Blotting, Western
  • Epitope Mapping
  • Hybridomas / metabolism
  • Immunization*
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Heavy Chains / isolation & purification
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / isolation & purification
  • Immunohistochemistry
  • Immunoprecipitation
  • Mice
  • Mice, Inbred BALB C
  • Microscopy, Fluorescence
  • Peptide Library*
  • Recombinant Proteins / isolation & purification
  • Zebrafish
  • Zebrafish Proteins / analysis
  • Zebrafish Proteins / immunology*

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Variable Region
  • Peptide Library
  • Recombinant Proteins
  • Zebrafish Proteins