[Potency testing of anti-lymphocyte Globulins: In vitro alternatives for the monkey skin-graft assay]

Antilymphocyte globulins (ALG) are immunosuppressive agents of animal origin currently used in clinical transplantation medicine and for the treatment of severe aplastic anemia. The potency of each batch is tested in vivo using primates as hosts for allogeneic skin transplantation. The test is done with a maximum of three animals, one as a control and two after the treatment with ALG. The two in vitro methods in use are a cytotoxic assay and the rosette inhibition assay. These methods are evaluated with the microscope. Besides wellfare aspects these methods require a lot of experience, are subjective, difficult to validate and the information about the biological potency of the sera is questionable. The aim of our study is a better biological characterisation as a prerequisite to subsequently define an in vitro alternative for the potency test in monkeys. Using a competition assay with monoclonal antibodies we can identify several specificities directed against functional molecules on T cells (e.g., CD2, CD3, CD5, CD28), B Cells (CD19), macrophages and natural killer cells (CD16) and nonlineage specificities such as CD18, CD25, CD29, CD95. This method could describe a part of the biological potency and control homogeneity of batches. The cytotoxic capacity of ALG either with or without complement as well as DNA-fragmentation characteristic for apoptosis can be analysed by flowcytometry using propidiumiodide- (PI) incorporation. Immunoprecipitation of cell-lysate with ALG<

[Optimisation and establishing of serological methods for the potency testing of immunglobulins against Clostridium tetani-toxin]

The quality control of human tetanus immunglobulin requires animal experiments according to European Pharmacopoeia monograph 398. The potency estimation has to be done in a toxin neutralisation test in mice (MNT) or guinea pigs. Immunoassays could also be used if they show a suitable sensitivity and specificity. The first results of our study verify that an indirect enzyme linked immunosorbent assay (ELISA), a rocket immunelectrophoresis (RIE) and a toxin binding inhibition test (ToBI) could be used as serological alternativ methods to the MNT. Studies on the reproducibility of the in vitro methods resulted inter-assay coefficients of variation between 2 and 27%. The ELISA is more sensitive (limit of detectability: 0,005 IE/ml) than the ToBI (0,04 IE/ml) and the RIE (5 IE/ml). The transferability of the ELISA to other labs is proofed. The transferability of the RIE and the ToBI will be tested in the near future.