Immuno-based botulinum toxin serotype A activity assays
The present specification discloses SNAP-25 compositions, methods of making α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P 1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P 1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, methods of detecting BoNT/A activity, and methods of detecting neutralizing α-BoNT/A antibodies.
1. A method of detecting anti-BoNT/A enzymatic activity neutralizing antibodies in a mammal comprising the steps of:
a. obtaining a test sample from a mammal, wherein the mammal is being tested for the presence or absence of an anti-BoNT/A neutralizing antibodies and wherein the test sample is a blood or serum sample from the mammal;
b. adding a known quantity of BoNT/A to the test sample;
c. contacting a cell from an established cell line expressing SNAP-25 with the test sample, wherein the cell from the established cell line is susceptible to BoNT/A intoxication;
d. isolating from the cell a SNAP-25 cleavage product by BoNT/A having a carboxyl terminus glutamine from the BoNT/A cleavage site scissile bond;
e. contacting the SNAP-25 cleavage product or fragment with an anti-SNAP-25 antibody linked to a solid phase support, wherein the anti-SNAP-25 antibody specifically binds to an epitope of the BoNT/A cleavage product of SNAP-25 consisting of the amino acid sequence of SEQ ID NO:38, and wherein the anti-SNAP-25antibody comprises a heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of at least one of SEQ ID NOs: 93, 96, and 100 and a light chain variable region comprising CDRs comprising the amino acid sequences of at least one of SEQ ID NOs: 105, 110 and 115; or a heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of at least one of SEQ ID NOs: 95, 99, and 101 and a light chain variable region comprising CDRs comprising the amino acid sequences of at least one of SEQ ID NOs: 103, 108 and 113;
f. detecting the presence of an antibody-antigen complex comprising the anti-SNAP-25 antibody and the SNAP-25 cleavage product;
g. performing steps b-f with a negative control sample instead of a test sample, wherein the negative control sample comprises the known quantity of BoNT/A and a serum known not to contain anti-BoNT/A enzymatic activity neutralizing antibodies;
h. comparing the amount of antibody-antigen complex detected in step f relative to the amount of antibody-antigen complex detected in the negative control sample; and
i. determining the presence of anti-BoNT/A enzymatic activity neutralizing antibodies in the test sample when the amount of antibody-antigen complex detected in step f is less than the amount of antibody-antigen complex detected in the negative control sample.
2. The method of claim 1 , wherein the quantity of known BoNT/A is 10 pM.
3. The method of claim 1 , wherein the detecting the presence of an antibody-antigen complex is through the use of a sandwich immunoassay.
4. The method of claim 3 , wherein the sandwich immunoassay comprises an electrochemiluniescense or chemiluminescense substrate.