Method for the detection of Clostridium neurotoxins using a chimeric substrate fusion protein

A method for detecting and assaying the presence of a Clostridium neurotoxin in a sample comprising:

• a) providing a universal recombinant substrate fusion protein comprising:
• i) a human SNAP-25 amino acid sequence of as set forth in SEQ ID NO:
• 1 from amino acid 141 to 206;
• ii) a human VAMP amino acid sequence of as set forth in SEQ ID NO:
• 4 from amino acid 27 to 93,
• iii) a human syntaxin amino acid sequence as set forth in SEQ ID NO:
• 5 from amino acid 221-264;
• iv) at least one Förster resonance energy transfer (FRET) or a luminescence reporter;
• b) exposing the sample containing a Clostridium neurotoxin to a sample comprising the universal recombinant substrate fusion protein capable of producing a detectable Förster resonance energy transfer (FRET), or a luminescence signal change, following cleavage of the universal recombinant substrate fusion protein by the Clostridium neurotoxin;
• c) detecting and assaying the presence of the Clostridium neurotoxin by measuring a change in the Förster resonance energy transfer or the luminescence signal in the sample;
• and
• d) determining an electrophoretic mobility pattern of one or more cleaved protein bands or a degraded protein, using a high throughput automated system to identify one or more serotypes of the Clostridium neurotoxin and tetanus.