Cellular vamp cleavage assay
A VAMP epitope suitable for generating an antibody against a VAMP C-terminal neurotoxin cleavage product. Method of using such an epitope to generate an antibody against cleaved VAMP. Method of using such an antibody to assay for cleavage of a VAMP by clostridial neurotoxin.
1. An antigenic polypeptide consisting of 16 to 65 amino acid residues, the polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 22-3o and 48-78, wherein the antigenic polypeptide shares no more than 15 consecutive amino acid residues in common with a naturally-occurring VAMP amino acid sequence.
2. The antigenic polypeptide of claim 1 , wherein the polypeptide consists of 16 or 17 amino acid residues.
3. An antigenic protein comprising a polypeptide covalently linked to a carrier, wherein the polypeptide:
consists of 16 to 65 amino acid residues;
comprises a VAMP epitope comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEO ID NOs: 15-34 and 48-78; and shares no more than 15 consecutive amino acid residues in common with a naturally-occurring VAMP amino acid sequence.
4. A method of generating an antibody against a C-terminal VAMP cleavage product, the method comprising using a polypeptide that:
consists of 16 to 65 amino acid residues;
comprises a VAMP epitope comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 15-34 and 48-78; and shares no more than 15 consecutive amino acid residues in common with a naturally-occurring VAMP amino acid sequence;
the method comprising immunizing an animal or an egg with the polypeptide; or
the method comprising immunizing a lymphocyte in vitro with the polypeptide.
5. An antigenic protein comprising an antigenic polypeptide covalently linked to a carrier, the antigenic polypeptide consisting of 10 to 3o amino acid residues and comprising a VAMP epitope comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1S-24 and 48-78.
6. The polypeptide of claim 1 , comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 53-78.
7. The polypeptide of claim 1 , comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 62, 66, and 75.
8. The antigenic protein of claim 3 , wherein the VAMP is VAMP 1 , VAMP 2 , VAMP 3 , VAMP 4 , VAMP 5 or YKT6.
9. The antigenic protein of claim 3 , wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 22, 27, 32, 49, and 53.
10. The antigenic protein of claim 3 , wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 22-30 and 48-78.
11. The antigenic protein of claim 3 , wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 53-78.
12. The antigenic protein of claim 3 , wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 62, 66, and 75.
13. The method of claim 4 , wherein the VAMP is VAMP 1 , VAMP 2 , VAMP 3 , VAMP 4 , VAMP 5 or YKT6.
14. The method of claim 4 , wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 15, 22, 27, 32, 49, and 53.
15. The method of claim 4 , wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 22-30 and 48-78.
16. The method of claim 4 , wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 53-78.
17. The method of claim 4 , wherein the VAMP epitope comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 62, 66, and 75.