Restoring antibody activity of a monoclonal anti-RNP antibody by dissociative HPLC demonstration of blocking antibody binding sites with antigen released from effete hybridoma cells

J. Ma*, B. Walsh, S. L. Chen, R. Penny, S. N. Breit

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

In biomedical research, monoclonal anti-nuclear antibodies have a number of advantages over polyclonal antibodies in terms of both specificity and reproducibility. However, there are some potential problems in the preparation of monoclonal antibodies. A well characterized mouse monoclonal anti-ribonucleoprotein antibody (anti-RNP antibody, 2.73) known to function in Western blotting was found to lose this activity when produced in vitro from long term hybridoma cell culture. Whilst it could no longer detect RNP antigen by Western blotting, it could still function effectively in affinity purification of RNP antigen. Further studies suggested that this was due to blocking of antibody binding sites by RNP antigen released from effete hybridoma cells in culture. The activity of the antibody in affinity purification was retained because the antigen was stripped away by repeated elutions with 6 M urea. HPLC gel filtration in the presence of 6 M guanidine was able to restore the antibody activity of the protein A purified monoclonal antibody. This finding has important general consequences for the preparation of monoclonal antibodies against antigens present in hybridoma cell culture media.

Original languageEnglish
Pages (from-to)121-127
Number of pages7
JournalJournal of Immunological Methods
Volume155
Issue number1
DOIs
Publication statusPublished - 19 Oct 1992
Externally publishedYes

Keywords

  • Autoantigen blockage
  • Chromatography, high performance liquid
  • Loss of antibody activity
  • Monoclonal antibody preparation
  • Restoring antibody activity

Fingerprint

Dive into the research topics of 'Restoring antibody activity of a monoclonal anti-RNP antibody by dissociative HPLC demonstration of blocking antibody binding sites with antigen released from effete hybridoma cells'. Together they form a unique fingerprint.

Cite this