Immunocytochemical studies of an antigen in a human T-cell lymphoma line (Jurkat) recognized by certain monoclonal antibodies to CD-13 (aminopeptidase-N).
The immunofluorescent staining of Jurkat cells by a panel of eight CD-13 monoclonal antibodies has been investigated. With unfixed cells all antibodies were negative, in contrast to the CD-13-positive cell line, HL60, which gave staining in each case. These results were in line with our previous enzymological studies in which we showed that the aminopeptidase activity observed with intact Jurkat cells was located in the cytoplasm and was distinct in properties from aminopeptidase-N (CD-13), which is expressed by HL60 cells. After fixation of Jurkat cells, four antibodies, 22A5; MCS-2, 72a and WM-15, gave consistent positive staining, while the other four, WM-47, RMAG6, MY7 and CLB-mon-gran/2 were consistently negative. Prior fixation was an absolute requirement for staining, but was observed over a range of concentrations of paraformaldehyde from 0.4% to 4%, as well as with acetone or methanol/acetic acid. Confocal fluorescence microscopy confirmed that the positive fluorescence given by 22A5 was located in the cytoplasm, contrasting with that given by an antibody to a cell-surface marker, HLA-DR, which was associated with the plasma membrane. The fixatives thus appeared to have rendered the cells permeable to the antibodies, but the molecular size of the antibodies could not explain the different behaviour of the two classes of antibodies, since IgG1 isotypes were present in both positive and negative groups. We exclude the possibility that Jurkat cells express an intracellular form of aminopeptidase-N; the identity of the protein(s) bearing the epitope(s) to which four of the antibodies bound has yet to be determined.
Murray
H
H
Department of Biochemistry and Molecular Biology, University of Leeds, England, UK.
McClaren
H
H
Turner
A J
AJ
Kenny
A J
AJ
D000911:Antibodies, Monoclonal / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000276:immunology; D018826:CD13 Antigens / Q000276:immunology*; D003593:Cytoplasm / Q000276:immunology; D018604:Epitope Mapping; D005455:Fluorescent Antibody Technique; D006801:Humans; D016399:Lymphoma, T-Cell / Q000276:immunology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016707:Tissue Fixation / Q000379:methods; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D018826:CD13 Antigens