Potency testing of anti-lymphocyte globulins: in vitro alternatives for the monkey skin graft assay.
Anti-lymphocyte globulins (ALG) are immunosuppressive agents of animal origin currently used in clinical organ transplantation and for the treatment of severe aplastic anaemia. The potency of each batch is tested in vivo using primates as recipients for allogeneic skin grafts. The two in vitro methods commonly used are (i) a cytotoxic assay and (ii) the rosette inhibition assay, both of which are evaluated by microscopy. In addition to animal welfare aspects, these methods require considerable experience, are difficult to validate, and the information as to the biological potency of the sera is questionable. The aim of our study is a better characterization of the biological properties of ALG in order to subsequently define an in vitro alternative for the potency test in monkeys. Several antibody specificities directed against functional molecules on T-cells, B-cells, NK-cells, macrophages as well as non-lineage specificities can be identified in competition assays with monoclonal antibodies. The cytotoxic capacity of ALG in the presence or absence of complement as well as DNA-fragmentation characteristic for apoptosis can be analysed by flow cytometry using propidiumiodide- (PI) incorporation. Immunoprecipitation of cell lysate with ALG and subsequent incubation with radioactive ATP (kinase-assay) shows specific bands which seem to be identical between different batches of one product.
Conrad
C
C
Paul-Ehrlich-Institute, Department of Immunology, Langen, Germany.
Janssen
O
O
Schäffner
G
G
Kabelitz
D
D
D000826:Animal Testing Alternatives; D000818:Animals; D000961:Antilymphocyte Serum / Q000494:pharmacology* / Q000633:toxicity; D002460:Cell Line; D002478:Cells, Cultured; D003165:Complement System Proteins; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D000882:Haplorhini; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D011817:Rabbits; D016038:Skin Transplantation / Q000276:immunology*
D000961:Antilymphocyte Serum; D003165:Complement System Proteins