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 method for directly determining the biological activity of Botulinum neurotoxin E (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 upon cells; 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 Synaptosomal Nerve Associated Protein-25 (SNAP-25), and with at least a second capture antibody specifically binding to BoNT/E-cleaved SNAP-25, under conditions which allow for binding of the first capture antibody to non-cleaved and BoNT/E-cleaved SNAP-25 and for binding of the second capture antibody to BoNT/E-cleaved SNAP-25, wherein the second capture antibody is a polyclonal or monoclonal antibody specifically binding to BoNT/E-cleaved SNAP-25; 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 amounts 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 dividing the amount of the second detection complex by the amount of the first detection complex to normalize the amount of the second detection complex, thereby determining the proteolytic activity of BoNT/E, wherein proteolytic activity is a characteristic of biological activity of said BoNT/E in said cells.

2. The method of claim 1, wherein the BoNT/E comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO. 12 or SEQ ID NO: 14; and b) an amino acid sequence which has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO. 12 or SEQ ID NO: 14.

3. The method of claim 1, wherein the cells are neuronal cells or neuronal differentiated cells selected from the group consisting of: primary neuronal cells, tumor cells which are capable of differentiating to neuronal cells such as neuroblastoma cells, P19 cells or induced pluripotent stem cell (IPS)-derived neurons.

4. The method of claim 1, wherein fixing the cells is carried out by the addition of a fixation agent selected from the group consisting of: methanol, ethanol, acetone, formaldehyde or mixtures thereof.

5. The method of claim 1, wherein said first capture antibody specifically binding to the non-cleaved and BoNT/E-cleaved SNAP-25 is the rabbit polyclonal anti-SNAP-25 antibody S9684, the rabbit polyclonal anti-SNAP25 antibody PA5-19708, the rabbit monoclonal anti-SNAP25 antibody ab108990, or the rabbit polyclonal anti-SNAP25 antibody PA5-19701.

6. The method of claim 1, wherein the first detection antibody is an alkaline phosphatase (AP)-conjugated antibody, a horseradish-peroxidase (HRP)-conjugated antibody or an antibody conjugated to a fluorescence dye.

7. The method of claim 1, wherein the second detection antibody is an alkaline phosphatase (AP)-conjugated antibody, a horseradish-peroxidase (HRP)-conjugated antibody, a glucose oxidase-conjugated antibody, a tyrosinase-conjugated antibody or a R-Galactosidase antibody.

8. The method of claim 1, wherein the HRP substrate is Amplex UltraRed, 10-Acetyl-3,7-Dihydroxyphenoxazine (ADHP) or 3-(4-Hydroxyphenyl) propionic acid (HPPA).

9. The method of claim 1, wherein the AP substrate is a 4-methylumbelliferryl phosphate derivative.

10. A kit for carrying out the method of claim 1, comprising: a) reagents comprising a first capture antibody, a second capture antibody which is a polyclonal or monoclonal antibody specifically binding to BoNT/E-cleaved SNAP-25, a first detection antibody and a second detection antibody, wherein said reagents are used for carrying out the method of claim 1; b) instructions for carrying out said method.

11. The method of claim 9, wherein the 4-methylumbelliferryl phosphate derivative is 6,8-Difluoro-4-methylumbelliferyl phosphate.

Description

(1) The FIGURE shows:

(2) 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.

(3) 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

(4) 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 5P2/0-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.

(5) 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 DSMZDeutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstrae 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

(6) Fixation of Cells

(7) 1. Remove the media/toxin solution. Add 100 l/well ice-cold methanol (20 C.) and incubate for 20 min at 20 C.

(8) Note: Perform all subsequent steps at room temperature.

(9) After Cell Fixation:

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

(11) 2. Remove the PBS Buffer and wash the cells 3 times with 300 l/well of PBS buffer. Each step should be performed for 1 minute with gentle shaking.

(12) 3. Remove the PBS buffer and add 100 l/well of quenching buffer and incubate for 20 minutes with gentle shaking.

(13) 4. Remove the quenching buffer and wash the cells once with 300 l/well of PBS buffer for 3 minutes under gentle shaking.

(14) 5. Remove the PBS buffer, and add 200 l/well of blocking buffer and incubate for 1 hour with gentle shaking.

(15) 6. Remove the blocking buffer and add 100 l 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).

(16) 7. Remove the primary antibody mixture and wash the cells 4 times with 300 l of PBS buffer. Each step should be performed for 3 minutes with gentle shaking.

(17) 9. Remove the PBS buffer, and add 100 l 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.

(18) 10. Remove the secondary antibody mixture and wash the cells 6 times with 300 l/well of HEPES buffer. Each wash step should be performed for 3 minutes with gentle shaking.

(19) 11. Remove the HEPES buffer from the plate and add 75 l 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.

(20) 12. Add 75 l 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.

(21) 13. Read the plates using a fluorescence plate reader:

(22) excitation at 540 nm; emission at 600 nm.

(23) excitation at 360 nm; emission at 450 nm.

(24) 15. Calculation

(25) 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:

(26) $\frac{\mathrm{RFU}\left(600\mathrm{nm}\right)}{\mathrm{FRU}\left(450\mathrm{nm}\right)}1000$

(27) Subsequently the resulting RFU values are averaged for each standard or sample.

(28) Reagent Preparation

(29) PBS Buffer (10 mM):

(30) Phosphate buffered saline (Sigma, #P5368) (pH 7.4)

(31) Quenching buffer:

(32) 0.6% H.sub.2O.sub.2 in 10 mM PBS buffer (pH 7.4)

(33) Blocking buffer:

(34) 2% BSA in 10 mM PBS buffer (pH 7.4)

(35) HEPES buffer:

(36) 50 mM HEPES (pH 7.4)

(37) HRP substrate:

(38) 50 mM HEPES (pH 7.4)

(39) 0.007% H.sub.2O.sub.2

(40) 150 M Amplex UltraRed

(41) AP substrate:

(42) 25 mM Diethanolamine (pH 9.8)

(43) mM MgCl.sub.2

(44) 100 M DiFMUP