title: [An in vitro alternative to the mouse neutralisation assay for potency testing of betatoxoid containing Clostridium, perfringens type B and type C vaccines for veterinary use]
abstract: The quality control of Clostridium (C.) perfringens type B and type C vaccines requires animal experiments according to European Pharmacopoiea monograph 363. For potency estimation, the vaccine is first administered to rabbits. In a second step antibodies from these rabbits against C. perfringens betatoxin are measured quantitatively in a mouse neutralisation assay using lethal doses of betatoxin for the challenge. We report about the development of an in vitro assay enabling specific and reproducible measurement of antibodies against C. perfringens betatoxin in rabbit sera. A Capure-Enzyme Linked Immuno Sorbent Assay (ELISA) using a monoclonal antibody against betatoxin as catching antibody was used. A rabbit serumpool freeze dried in 3500 aliquots was always used as reference. This reference serum can be supplied for further national or international collaborative studies. The estimation of relative potency of unknown sera in a parallel line assay was calculated with a computer programme provided by the World health organisation (WHO). The capture-ELISA did not show unspecific reactivity with pre-vaccination sera of cross-reactivity with sera from rabbits immunised with other clostridial antigens e.g. C. perfringens type D, C. chavoei or C. tetani. Reproducibility studies focused on the linear parts of the dose-response curves resulted in intra-assay coefficient of variations of less then 10%. The inter-assay coefficient varied between 12-25% depending on the serum dilutions used. Correlation studies between the result of the animal experiment (only one test) and the capture-ELISA (10 repetitions) from four rabbit serum pools revealed a coefficient of correlation of 0.81-0.84 depending on the basis for calculation of r (Mean or Median from ELISA repetitions). Therefore this test may be a suitable alternative for the currently required mouse neutralisation assay. For acceptance of this test by the European Pharmacopoiea further validation studies are necessary.
authors.affiliation: Paul-Ehrlich-Institut, Bundesamt für Sera und Impfstoffe, D-Langen.
authors.forename: Elvira
authors.initials: E
authors.lastname: Ebert
authors.forename: Manuela
authors.initials: M
authors.lastname: Kusch
authors.forename: Volker
authors.lastname: Öppling
authors.forename: Esther
authors.initials: E
authors.lastname: Werner
authors.forename: Klaus
authors.initials: K
authors.lastname: Cussler