In vitro determination of specific toxicity in tetanus vaccines.
Tetanus vaccine is prepared from detoxified tetanus neurotoxin. To ensure the absence of residual toxin activity or to exclude the reversion to toxicity reliable control testing is based on in vivo methods, because no in vitro assay provides the required specificity and sensitivity. Tetanus neurotoxin is a 150 kDa protein produced by Clostridium tetani. The 50 kDa light chain of this neurotoxin belongs to the family of zinc metalloproteases. It cleaves synaptobrevin, a small synaptic vesicle protein, which is involved in neuroexocytosis, at the single Q76-F77 peptide bond. To develop a sensitive in vitro assay capable of quantifying the proteolytic activity of this toxin, we used as substrate a recombinant fragment of synaptobrevin2 (1-97). For detecting the cleavage products a peptide antibody raised against the N-terminal cleavage site was used. In Western Blot analysis only the cleaved substrate was detected while the uncleaved substrate showed no signal. In different approaches, recombinant synaptobrevin was either (i) bound to a microtitre plate, reduced toxin was added and the N-terminal cleavage product was detected by a specific antibody or (ii) the cleavage was performed in test tubes, the samples were transferred to a microtitre plate and immobilised cleavage products were detected. When toxoid or crude toxin is used, non-specific cleavage of synaptobrevin substrate occurs. Depending on the toxoid used different patterns of degradation of substrate are visible in Western Blots. Different protease inhibitors and reaction conditions seem to have an effect on the inhibition of this non-specific cleavage.
Kegel
B
B
Paul-Ehrlich-Institute, Federal Agency for Sera and Vaccines, Langen, Germany.
Bonifas
U
U
Silberbach
K
K
Krämer
B
B
Weisser
K
K
D000595:Amino Acid Sequence; D000818:Animals; D003017:Clostridium tetani / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D050683:R-SNARE Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013744:Tetanus Toxin / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D013745:Tetanus Toxoid / Q000378:metabolism / Q000633:toxicity*
D008565:Membrane Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; D013744:Tetanus Toxin; D013745:Tetanus Toxoid; D010450:Endopeptidases