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.
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.
Ma
J
J
Centre for Immunology, St. Vincent's Hospital, Sydney, Australia.
Walsh
B
B
Chen
S L
SL
Penny
R
R
Breit
S N
SN
D000818:Animals; D000974:Antibodies, Antinuclear / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D055517:snRNP Core Proteins
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001324:Autoantigens; D007074:Immunoglobulin G; D055517:snRNP Core Proteins