TY - JOUR
T1 - 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
AU - Ma, J.
AU - Walsh, B.
AU - Chen, S. L.
AU - Penny, R.
AU - Breit, S. N.
PY - 1992/10/19
Y1 - 1992/10/19
N2 - 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.
AB - 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.
KW - Autoantigen blockage
KW - Chromatography, high performance liquid
KW - Loss of antibody activity
KW - Monoclonal antibody preparation
KW - Restoring antibody activity
UR - http://www.scopus.com/inward/record.url?scp=0026664673&partnerID=8YFLogxK
U2 - 10.1016/0022-1759(92)90278-2
DO - 10.1016/0022-1759(92)90278-2
M3 - Article
C2 - 1401961
AN - SCOPUS:0026664673
SN - 0022-1759
VL - 155
SP - 121
EP - 127
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1
ER -