Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies directed against intracellular antigens.
For investigations involving monoclonal antibodies (mAbs) against cellular antigens cell binding assays are routinely used to determine the immunoreactive fraction after radiolabeling. In general, surface antigens are targets for radioimmunodetection, but recent studies indicate that intracellular determinants may also prove useful for this purpose. Thus, there is a need to adapt the regular cell binding assay for use with antibodies directed against cytoplasmic antigens. Here we describe a fixation method which permits such mAbs to bind to cell surfaces as well as to intracellular determinants. Moreover, the procedure may be used for antigens that are sensitive to the commonly used aldehyde fixatives. The method is illustrated with two human IgM mAbs 16.88 and 28A32, which recognize cytoplasmic antigens. Human colon cancer cells in suspension were fixed with either acetone or glutaraldehyde. Intracellular antigens appeared to be best exposed for antibody binding after fixation with acetone as determined by immunofluorescent staining and flow cytometry. An antibody directed against the cell surface antigen HLA class I showed similar binding with both live cells and acetone-fixed cells. Double-inverse plots of the binding of radiolabeled 16.88 or 28A32 antibody with acetone-fixed cells gave reliable immunoreactive fraction values. Acetone-fixed cells stored at 4 degrees C could be used for immunoreactivity assays for at least 6 months without loss of performance.
Haisma
H J
HJ
Department of Oncology, Free University Hospital, Amsterdam, Netherlands.
Pinedo
H M
HM
Silva
C A
CA
Boven
E
E
D000096:Acetone; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000378:metabolism*; D001666:Binding Sites, Antibody; D003110:Colonic Neoplasms / Q000276:immunology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005976:Glutaral; D006801:Humans; D007075:Immunoglobulin M; D011863:Radioimmunoassay / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007075:Immunoglobulin M; D000096:Acetone; D005976:Glutaral