Fachbereich Veterinärmedizin



    Entwicklung und Prävalidierung eines Toxininhibitionstestes als Tierversuchsersatz in Rahmen der Prüfung von Immunpräparaten gegen das Alphatoxin von Clostridium novyi Typ B,Develpoment and prevalidation of an toxin inhibition test for potency testing of (2001)

    Belinga, Kristin
    Berlin, 2001 — 80 Seiten
    Institut für Mikrobiologie und Tierseuchen

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35
    14163 Berlin
    Tel.+49 30 83 8-518 40/518 43 Fax.+49 30 838 45 18 51

    Abstract / Zusammenfassung

    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.