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In this study a test system was developed for quantitative detection of protective antibodies against alphatoxin of Clostridium novyi type B in rabbit serum. Here we tested the suitability of this in vitro-Methode for replacing the currently used mouse neutralisation test including prevalidation.The toxin inhibition test which was developed had good reproducibility (coefficient of variation: 10,5%) and high specificity. Correlation studies between the result of the animal experiment and the toxin inhibition test revealed a coefficient of correlation of 84%. This test may therefore be a suitable replacement for the currently equired mouse neutralisation assay.The necessary reference materials (monoclonal antibody, antigen, reference serum) are produced and a standard operating procedure established. The monoclonal antibodies are produced through a fusion of a spleenocyt of a alphatoxin-immunisated Balb/c mice with a myeloma cell. It was possible to propagare 5 stabil hybridoma ceff lines (41F91 3A7/ 1 E10/ 1 G4/ 1 E4). Monoclonal antibodies of the hybridoma cell line 4F9 neutralised the alphatoxin and protected the cell culture and the animal against the toxic effect. The 1:10 diluted miniPerm® supernatant neutralised the 1:1000 diluted alphatoxin (Hauer) in the cell culture assay. The undiluted miniPerm® supernatant protected against the 1Ofold LD50 of the alphatoxin.