MEANS AND METHODS FOR THE DETERMINATION OF THE BIOLOGICAL ACTIVITY OF BONT/E IN CELLS

Abstract

The present invention pertains to a polyclonal or monoclonal antibody specifically binding to BoNT/E-cleaved SNAP-25. Further, the invention provides a method for directly determining the biological activity of BoNT/E in cells, comprising the steps of: a) incubating cells susceptible to BoNT/E intoxication with BoNT/E for a time and under conditions which allow for the BoNT/E to exert its biological activity; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to non-cleaved and BoNT/E-cleaved SNAP-25, and with at least a second capture antibody specifically binding to BoNT/E-cleaved SNAP-25, wherein the second capture antibody is an antibody of the invention, under conditions which allow for binding of said capture antibodies to the indicated substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of said first detection antibody to said first capture antibody, thus forming first detection complexes, and with at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of said second detection antibody to said second capture antibody, thus forming second detection complexes, and wherein the first detection antibody is different from the second detection antibody; e) determining the amount of the first and second detection complexes of step d); and f) calculating the amount of SNAP-25 cleaved by BoNT/E in said cells by means of the second detection complexes, thereby determining the biological activity of BoNT/E in said cells. Furthermore, the invention relates to a kit for carrying out the method of the invention.

Claims

1. A polyclonal or monoclonal antibody specifically binding to BoNT/E-cleaved SNAP-25.

2. The polyclonal or monoclonal antibody of claim 1, wherein the antibody specifically binds to an epitope consisting of a peptide having an amino acid sequence as shown in SEQ ID NO: 1 (“C-NEIDTQNRQIDR”) or SEQ ID NO: 2 (“NEIDTQNRQIDR”).

3. The polyclonal or monoclonal antibody of claim 1, wherein the antibody comprises at least one of the complementarity determining regions (CDRs) selected from the group consisting of CDR-L1 (SEQ ID NO. 15), CDR-L2 (SEQ ID NO. 16), CDR-L3 (SEQ ID NO. 17), CDR-H1 (SEQ ID NO. 18), CDR-H2 (SEQ ID NO. 19) and CDR-H3 (SEQ ID NO. 20), comprised by the monoclonal antibody produced by hybridoma cell line pCNEI 32-7-1, 3614-000 deposited on Dec. 17, 2014, at DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany, under the accession number DSM ACC3261.

4. The polyclonal or monoclonal antibody of claim 1, wherein the antibody comprises the VH region (SEQ ID NO. 22) and/or the VL region (SEQ ID NO. 21) comprised by the monoclonal antibody produced by hybridoma cell line pCNEI 32-7-1, 3614-000 deposited on Dec. 17, 2014, at DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany, under the accession number DSM ACC3261.

Description

[0094] The FIGURE shows:

[0095] FIG. 1: Diagram representing the mode of action of the cell-based assay of the invention. Cells susceptible to BoNT/E intoxication are seeded in multiwell plates, Thereafter, the cells are intoxicated with BoNT/E and after a given intoxication period the cells are fixated. The specific antibody for BoNT/E-cleaved SNAP-25 and the specific antibody for uncleaved SNAP-25 bind to the specific binding sites on SNAP-25. Using enzyme-coupled anti-host specific secondary antibodies, these binding events can be used to generate measurable signals which correlate with the concentration of BoNT/E-cleaved SNAP-25 and the total amount of SNAP-25 within the well. With increasing BoNT/E concentration, the amount of measured cleaved SNAP-25 increases resulting in a gain of signal.

[0096] The invention will now be illustrated by the following Examples which shall, however, not be construed as limiting the scope of the present invention.

EXAMPLE 1: GENERATION OF MONOCLONAL ANTIBODIES SPECIFICALLY BINDING TO THE CLEAVAGE SITE OF THE BONT/E-CLEAVED SUBSTRATE SNAP-25

[0097] Mouse monoclonal antibodies specifically binding to the cleavage site of the BoNT/E-cleaved substrate SNAP-25 have been generated using the hybridoma standard technique. To this end, Balb/c mice (female, 8 weeks) have been immunized with the peptide “C-NEIDTQNRQIDR-OH” (SEQ ID NO: 1). The N-terminal Cysteine residue is not derived from the SNAP-25 amino acid sequence but has been introduced for linking the peptide to the keyhole limpet hemocyanin (KLH). Hybridoma cells have been obtained by the fusion of mouse spleen cells with the myeloma cell line SP2/0-Ag14 (SP2/0) purchased from the German Collection of Microorganisms and Cell Culture (DSMZ GmbH, Braunschweig, ACC 146); see also Hemmerlein et al., Molecular Cancer 2006, 5, 41. Antibodies specifically binding to the cleavage site of the BoNT/E-cleaved substrate SNAP-25 were screened in ELISA. The obtained clones have been selected with respect to their specificity and affinity to BoNT/E-cleaved SNAP-25. As a negative control, the clones have been tested for their non-binding to non-cleaved SNAP-25.sub.206. As a result, the mouse monoclonal antibody produced by hybridoma pCNEI 32-7-1, 3614-000 was found to be highly specific for BoNT/E-cleaved SNAP-25, with no detectable cross-reactivity to SNAP25.sub.206 in ELISA and Western blots.

[0098] The hybridoma cell line pCNEI 32-7-1, 3614-000 producing the monoclonal antibody of the invention specifically binding to BoNT/E-cleaved SNAP-25 has been deposited by the Applicant under the Budapest Treaty on Dec. 17, 2014, at DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany under accession number DSM ACC3261. The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of this monoclonal antibody are shown in SEQ ID NOs. 18, 19 and 20, respectively. The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of this monoclonal antibody are shown in SEQ ID Nos. 15, 16 and 17, respectively. The amino acid sequence of the VH region of this monoclonal antibody is depicted in SEQ ID NO. 22, and the amino acid sequence of the VL region of this monoclonal antibody is shown in SEQ ID NO. 21.

EXAMPLE 2: DOUBLE-FLUORESCENCE-CB-BONT/E ACTIVITY ELISA

[0099] Fixation of Cells

[0100] 1. Remove the media/toxin solution. Add 100 μl/well ice-cold methanol (−20° C.) and incubate for 20 min at −20° C.

[0101] Note: Perform all subsequent steps at room temperature.

[0102] After Cell Fixation:

[0103] 1. Remove the methanol solution and add 100 μl/well PBS buffer. For longer storage (>1 day) one should add 300 μl/well PBS buffer and seal the plates with parafilm. The plates should be stored in the refrigerator.

[0104] 2. Remove the PBS Buffer and wash the cells 3 times with 300 μl/well of PBS buffer. Each step should be performed for 1 minute with gentle shaking.

[0105] 3. Remove the PBS buffer and add 100 μl/well of quenching buffer and incubate for 20 minutes with gentle shaking.

[0106] 4. Remove the quenching buffer and wash the cells once with 300 μl/well of PBS buffer for 3 minutes under gentle shaking.

[0107] 5. Remove the PBS buffer, and add 200 μl/well of blocking buffer and incubate for 1 hour with gentle shaking.

[0108] 6. Remove the blocking buffer and add 100 μl of the primary antibody mixture (antibody dilution in blocking buffer) to each well. Incubate overnight (16-18 h) with gentle shaking. The cells are simultaneously incubated with two primary antibodies: a mouse antibody specific for the BoNT/E-cleaved SNAP25 and a polyclonal rabbit antibody that recognizes SNAP-25 (antibody for determining the total amount of SNAP-25 for normalization).

[0109] 7. Remove the primary antibody mixture and wash the cells 4 times with 300 μl of PBS buffer. Each step should be performed for 3 minutes with gentle shaking.

[0110] 9. Remove the PBS buffer, and add 100 μl of the secondary antibody mixture: HRP-conjugated anti-mouse and AP-conjugated anti-rabbit secondary antibodies (antibody dilution in blocking buffer) to each well and incubate for 2.5-3 hours with gentle shaking.

[0111] 10. Remove the secondary antibody mixture and wash the cells 6 times with 300 μl/well of HEPES buffer. Each wash step should be performed for 3 minutes with gentle shaking.

[0112] 11. Remove the HEPES buffer from the plate and add 75 μl of a fluorogenic substrate for horseradish-peroxidase (HRP substrate) to each well. Incubate for 50 minutes with gentle shaking. Protect the plates from direct light.

[0113] 12. Add 75 μl of a fluorogenic substrate for alkaline phosphatase (AP substrate) to each well and incubate for an additional 50 minutes at with gentle shaking. Protect the plates from direct light.

[0114] 13. Read the plates using a fluorescence plate reader:

[0115] excitation at 540 nm; emission at 600 nm.

[0116] excitation at 360 nm; emission at 450 nm.

[0117] 15. Calculation

[0118] For normalization, the RFU value for BoNT/E-cleaved SNAP-25 (fluorescence at 600 nm) is normalized to RFU of total SNAP-25 (450 nm) in each well. For better illustration of RFUs in a diagram all values are multiplied with a factor 1000 using the following equation:

[00001]$\frac{\mathrm{RFU}.Math..Math.\left(600.Math..Math.\mathrm{nm}\right)}{\mathrm{RFU}.Math..Math.\left(450.Math..Math.\mathrm{nm}\right)}\times 1000$

[0119] Subsequently the resulting RFU values are averaged for each standard or sample.

[0120] Reagent Preparation

[0121] PBS Buffer (10 mM):

[0122] Phosphate buffered saline (Sigma, #P5368) (pH 7.4)

[0123] Quenching buffer:

[0124] 0.6% H.sub.2O.sub.2 in 10 mM PBS buffer (pH 7.4)

[0125] Blocking buffer:

[0126] 2% BSA in 10 mM PBS buffer (pH 7.4)

[0127] HEPES buffer:

[0128] 50 mM HEPES (pH 7.4)

[0129] HRP substrate:

[0130] 50 mM HEPES (pH 7.4)

[0131] 0.007% H.sub.2O.sub.2

[0132] 150 μM Amplex UltraRed

[0133] AP substrate:

[0134] 25 mM Diethanolamine (pH 9.8)

[0135] 2 mM MgCl.sub.2

[0136] 100 μM DiFMUP