Immuno-based botulinum toxin serotype A activity assays
AssigneeAllergan, Inc.
InventorsEster Fernandez-Salas, Joanne Wang, Patton Garay, Lina S. Wong, D. Dianne Hodges, Kei Rogers Aoki
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Abstract
The present specification discloses SNAP-25 compositions, methods of making α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P1 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 P1 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.
Claims4
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 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 with a disassociation constant of less than 8.82×10−4 s−1;
- wherein the anti-SNAP-25 antibody has an association rate constant for an epitope of intact SNAP-25 polypeptide comprising SEQ ID NO:
- 5 of less than 1×101M−1 s−1;
- and wherein
- (i) the anti-SNAP-25 antibody comprises a heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of SEQ ID NOs:
- 95, 99, and 101 and a light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 103, 108, and 113,
- (ii) the anti-SNAP-25 antibody comprises a heavy chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 93, 96, and 100 and a light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 105, 110, and 115,
- (iii) the anti-SNAP-25 antibody comprises a heavy chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 94, 98, and 102 and a light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 106, 111, and 116, or
- (iv) the anti-SNAP-25 antibody comprises a heavy chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 93, 97, and 101 and a light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 104, 109, and 114;
- 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.
Dependencies are inferred from the claim text.