RESUMO
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.
RESUMO
The requirements for the quality control of C. perfringens vaccines for veterinary use are described in the monograph 363 of the European Pharmacopoeia (Ph. Eur.). In the current used potency test neutralising antibodies against C. perfringens beta- and epsilontoxin are determined in a mouse neutralisation test (MNT). Two ELISA methods were developed for the replacement of the MNT. Both methods use monoclonal antibodies to determine the quantity of specific antibodies against beta toxin (Capture-ELISA) and epsilon toxin (Competitive-ELISA) in vitro. In parallel to the routine batch potency test in mice, the beta- and epsilonantitoxin levels in 523 samples were estimated in the ELISA procedures. A high specificity and a good reproducibility are evident for both test systems. An interlaboratory prevalidation study was carried out to evaluate the relevance and the transferability of the ELISA procedures. It is concluded that both ELISA systems seems to be suitable alternative methods for assessing the potency of beta- and epsilontoxoid in batches of vaccines for veterinary use.
RESUMO
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.
