	US 12,275,782 B2
	Means and methods for the determination of the biological activity of neurotoxin polypeptides in cells
Cornelia Brünn, Frankfurt am Main (DE)
Assigned to MERZ PHARMA GmbH & CO. KGaA, Frankfurt am Main (DE)
Filed by MERZ PHARMA Gmbh & CO. KGaA, Frankfurt am Main (DE)
Filed on Mar. 29, 2024, as Appl. No. 18/621,418.
Application 18/621,418 is a division of application No. 14/901,123, granted, now 11,976,110, previously published as PCT/EP2014/063531, filed on Jun. 26, 2014.
Claims priority of application No. 13174176 (EP), filed on Jun. 28, 2013.
Prior Publication US 2024/0270833 A1, Aug. 15, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/53 (2006.01); C07K 16/18 (2006.01); G01N 21/64 (2006.01); G01N 33/50 (2006.01)

CPC C07K 16/18 (2013.01) [G01N 21/6428 (2013.01); G01N 33/5014 (2013.01); G01N 33/5058 (2013.01); G01N 33/5073 (2013.01); C07K 2317/34 (2013.01); G01N 2021/6441 (2013.01); G01N 2333/33 (2013.01); G01N 2333/952 (2013.01)]

11 Claims

1. A kit for directly determining the biological activity of a Clostridium botulinum toxin serotype A (BoNT/A) Neurotoxin polypeptide in cells, comprising

i) a first isolated capture antibody which specifically binds to a non-cleaved BoNT/A Neurotoxin substrate SNAP-25 and a BoNT/A Neurotoxin-cleaved substrate SNAP-25;

ii) a second isolated capture antibody which specifically binds to the cleavage site of the BoNT/A Neurotoxin-cleaved substrate SNAP-25 and does not bind non-cleaved BoNT/A Neurotoxin substrate SNAP-25, under conditions which allow for binding of the first and second isolated capture antibodies to the substrates, and wherein the second isolated capture antibody is a mouse monoclonal antibody clone 20-2-5 comprising a complementarity determining region (CDR) heavy chain variable region 1 having the sequence of SEQ ID NO:20; a CDR heavy chain variable region 2 having the sequence of SEQ ID NO:21; a CDR heavy chain variable region 3 having the sequence of SEQ ID NO:22; a CDR light chain variable region 1 having the sequence of SEQ ID NO:23; a CDR light chain variable region 2 having the sequence of SEQ ID NO:24; and a CDR light chain variable region 3 having the sequence of SEQ ID NO: 25;

iii) a first isolated detection antibody which specifically binds to the first isolated capture antibody under conditions which allow for binding of the first isolated detection antibody to the first isolated capture antibody, thus capable of forming first detection complexes; and

iv) a second isolated detection antibody which specifically binds to the second isolated capture antibody under conditions which allow for binding of the second isolated detection antibody to the second isolated capture antibody, thus capable of forming second detection complexes; and

v) instructions for directly determining the biological activity of the BoNT/A Neurotoxin polypeptide in cells based on the amount of SNAP-25 substrate cleaved by the BoNT/A Neurotoxin polypeptide as determined based on the amounts of the first and second detection complexes.