Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity.

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the "3R" principles of replacing, reducing and refining animal tests, the "binding and cleavage" (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This in vitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics. Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated in vivo detection limit. In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids.

Development, standardization and assessment of PCR systems for purity testing of avian viral vaccines.

The European Pharmacopoeia (Ph. Eur.) requires avian viral vaccines to be free of adventitious agents. Purity testing is an essential quality requirement of immunological veterinary medicinal products (IVMPs) and testing for extraneous agents includes monitoring for many different viruses. Conventional virus detection methods include serology or virus culture, however, molecular tests have become a valid alternative testing method. Nucleic acid testing (NAT) is fast, highly sensitive and has a higher degree of discrimination than conventional approaches. These advantages have led to the development and standardization of polymerase chain reaction (PCR) assays for the detection of avian leucosis virus, avian orthoreovirus, infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, infectious laryngotracheitis virus, influenza A virus, Marek's disease virus, turkey rhinotracheitis virus, egg drop syndrome virus, chicken anaemia virus, avian adenovirus and avian encephalomyelitis virus. This paper reviews the development, standardization and assessment of PCR for extraneous agent testing in IVMPs with examples from an Official Medicines Control Laboratory (OMCL).

Application of RT-PCR for the detection of avian reovirus contamination in avian viral vaccines.

An efficient procedure for the detection of avian reovirus (ARV)-specific RNA sequences in veterinary immunological medicinal products using reverse transcriptase polymerase chain reaction (RT-PCR) is described. Four ARV vaccine strains (1133, 1733, 2408 and Olson WVU2937), two ATCC strains (VR826 and VR856) as well as several ARV field isolates obtained from domestic, wild and pet birds could be easily detected with S2- and S4-specific primers. The identity of the amplified fragments was confirmed by restriction endonuclease analysis and sequence analysis. Furthermore, not only the starting substrates used for the production of a vaccine, but also additives and the manufacturing process (matrix effects) can influence the results. These RT-PCRs have been useful for screening poultry vaccines for extraneous ARV in the quality control of biologicals used in veterinary medicine.

[Vaccine testing without animal experiments?]. / Impfstoffkontrolle ohne Tierversuche?

Vaccines must provide effective protection against disease without any harmful side effects. Antigen purity is a crucial factor which until now has had to be tested on animals. However, recent scientific developments allow the use of alternatives to animal experiments in many cases, e.g. poultry vaccine purity testing. The in vitro Polymerase Chain Reaction method (PCR) is one advance which could reduce the number of animal experiments.