Combined assay for the detection of tetanus toxicity in tetanus toxoids

This functional assay is designed to detect active tetanus neurotoxin (TeNT) in vaccines containing a tetanus toxoid component. Only active TeNT would inhibit neurotransmitter release from neuronal cells leading to symptoms of tetanus disease detected with the in vivo assay.

TeNT molecules consist of two subunits: a heavy chain which is responsible for the binding to target cells, and a light chain (L-chain) with a proteolytic domain which specifically cleaves the neuronal protein synaptobrevin-2, and thus inhibits neurotransmitter release.

These two functional properties of the active TeNT molecules are measured in a stepwise approach: First, the test sample is incubated on a microtiter plate coated with the TeNT receptor (i.e. ganglioside GT1b). Active TeNT binds to the receptor, while molecules lacking a functional receptor domain are removed by washing. The bound TeNT molecules are treated with a reducing agent to release and activate the L-chains. The supernatant containing the activated L-chains is then transferred to a second microtiter plate containing recombinant synaptobrevin-2 (rSyb2) which is specifically cleaved by the active L-chains. The resulting cleavage fragment is detected using a cleavage-site specific antibody. Signal amplification is obtained by employing a biotinylated secondary antibody followed by peroxidase-coupled streptavidin, and tetramethylbenzidine is used as colorimetric substrate for quantification of the peroxidase. The absorbance is measured at 450 nm against 620 nm as reference wavelength. Absorption values of the test sample are determined and the decision on toxicity is based on statistical comparison to (historical) control samples.

The European Pharmacopoeia monographs for tetanus vaccines for human or veterinary use stipulate that vaccine manufacturer demonstrate the absence of active TeNT during quality control of their products. The tests described are based on guinea pigs.

The in vitro assay has the potential to replace the test in guinea pigs.