Microcytotoxicity assay using mouse L cells transfected with human MHC class II genes. A method and its application.
Modifications of the standard microcytotoxicity assay make it possible to use this technique to screen both alloantisera and monoclonal antibodies with mouse L cells transfected with Class II genes. It is necessary to maintain a protein-rich environment in order to prevent nonspecific complement lysis. Selection of the complement itself is also an important factor, the best results being achieved using a commercially available complement that had previously been absorbed with mouse cells and used at a dilution of 1/8. Using this modified method with transfectants of DW2 origin we could show that alloantisera against DRw15 recognize the DRB1*1501 gene product, whereas broad DR2 sera react only with the DRB5*0101 product. This technique can be applied successfully to study the fine specificity of polymorphic monoclonal antibodies, as shown by the reactivity of HU-30 which binds to the LDR2b transfectant and not to the LDR2a, indicating that the antibody recognizes an epitope present on the DRB1 chain and not the DRB5 chain of DR2 cell lines.
Schreuder
G M
GM
Tissue Antigen Laboratory, Imperial Cancer Research Fund, London, United Kingdom.
Marsh
S G
SG
Heyes
J M
JM
Moses
J H
JH
Krausa
P
P
Bodmer
J G
JG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D003601:Cytotoxicity Tests, Immunologic; D005821:Genetic Techniques; D006681:HLA-D Antigens / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000032:analysis*; D007739:L Cells; D051379:Mice; D014162:Transfection
D000911:Antibodies, Monoclonal; D006681:HLA-D Antigens; D007518:Isoantibodies