Monoclonal antibodies detecting discrete epitopes of human perforin.
Perforin is a cytolytic protein of natural killer (NK) cells and cytotoxic T cells (CTL). Purified perforin has been shown to cause cell lysis and to form stable pores in the target cell membrane, but its relevance to cytolysis in vivo is not clear. The gene for human perforin has been cloned, but monoclonal antibodies (mabs) have not been available. In order to study further its role in cytotoxicity, we have generated mabs to different regions of human perforin. Four mabs were produced from mice immunized with hybrid proteins comprising E. coli TrpE protein at the N-terminus and different regions of human perforin at the C-terminus. These proteins were made using the pATH expression plasmids into which fragments of perforin cDNA were subcloned. Monoclonal antibody PA1 was made from a mouse immunized with a hybrid protein containing the C-terminal 240 amino acids (AA) of perforin, PE1 - the N-terminal 118 AA, and PB1 and PB2 - the central 199 AA. The three plasmid constructs contained non-overlapping cDNA segments which covered the entire sequence of perforin. All mabs reacted with the immunizing hybrid protein, but not with the other hybrid proteins, indicating that at least three epitopes are recognized by this set of mabs. All mabs immunoprecipitated a molecule of about 68 kd from lysates of metabolically-labelled cytolytic large granular lymphocytic leukemia cells, but not from control lysates of non-cytolytic promyelocytic U937 cells. These mabs should be of use in determining structure-function relationships for perforin.
Laboratory of Human Immunogenetics, Memorial Sloan-Kettering Cancer Center, New York, New York.
M
M
Geisberg
J A
JA
Trapani
B
B
Dupont
D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007694:Killer Cells, Natural / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D054353:Perforin; D010957:Plasmids; D052899:Pore Forming Cytotoxic Proteins; D011233:Precipitin Tests; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin