Monoclonal antibodies in analysis of cathepsin G-digested proteolytic fragments of human plasma fibronectin

Tapio Vartio, Luciano Zardi, Enrica Balza, Harry Towbin, Antti Vaheri

Research output: Contribution to journalArticlepeer-review

Abstract

Proteolytic fragments of fibronectin, obtained by digestion with cathepsin G, were transferred electrophoretically from sodium dodecyl sulphate (NaDodSO4) polyacrylamide gels to nitrocellulose sheets and used as antigens for monoclonal antibodies. All 9 monoclonal antibodies tested reacted with undenatured intact fibronectin or its fragments applied directly to nitrocellulose sheets. Two of the clones did not react with the NaDodSO4-treated transferred material suggesting reactivity with conformational determinants. Distinct fragments of fibronectin could be detected by several of the antibodies. None of the monoclonal or the polyclonal antibodies used reacted with the Mr = 40,000 or Mr = 30,000 gelatin-binding fragments of fibronectin. However, one of the monoclonal antibodies reacted specifically with their precursor Mr = 64,000 fragment, but apparently with its gelatin-nonbinding segment. The apparent non-immunogenicity of the gelatin-binding domain is conspicuous, suggesting that it may be highly conserved in evolution. The present method, combination of controlled proteolytic cleavage with electrophoretic transfer, provides an effective means for characterization of monoclonal antibodies raised against proteins.

Original languageEnglish
Pages (from-to)309-318
Number of pages10
JournalJournal of Immunological Methods
Volume55
Issue number3
DOIs
Publication statusPublished - Dec 30 1982

Keywords

  • fibronectin
  • immunoblotting
  • monoclonal antibody

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

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