Means and Methods for the Determination of the Biological Activity of Neurotoxin Polypeptides in Cells

Abstract

The invention pertains to a method for directly determining the biological activity of a Neurotoxin polypeptide in cells, comprising the steps of: a) incubating cells susceptible to Neurotoxin intoxication with a Neurotoxin polypeptide for a time and under conditions which allow for the Neurotoxin polypeptide 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 the non-cleaved and Neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the Neurotoxin-cleaved substrate, under conditions which allow for binding of the capture antibodies to the 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 the first detection antibody to the first capture antibody, thus forming first detection complexes, and at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of the second detection antibody to the second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d), and f) calculating the amount of substrate cleaved by the Neurotoxin polypeptide in the cells by means of the second detection complexes, thereby determining the biological activity of the Neurotoxin polypeptide in the cells. The invention further provides for a kit for carrying out the method of the invention.

Claims

1. A kit for directly determining the biological activity of a Neurotoxin polypeptide in cells, comprising a) an arrangement of: i) a first capture antibody which specifically binds to a non-cleaved Neurotoxin substrate SNAP-25 and a Neurotoxin-cleaved substrate SNAP-25; ii) a second capture antibody which specifically binds to the cleavage site of the Neurotoxin-cleaved substrate SNAP-25 and does not bind non-cleaved Neurotoxin substrate SNAP-25, under conditions which allow for binding of the first and second capture antibodies to the substrates, and wherein the second 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, or a mouse monoclonal antibody MC-6053; iii) a first detection antibody which specifically binds to the first capture antibody under conditions which allow for binding of the first detection antibody to the first capture antibody, thus forming first detection complexes; and iv) a second detection antibody which specifically binds to the second capture antibody under conditions which allow for binding of the second detection antibody to the second capture antibody, thus forming second detection complexes; b) means for calculating the amount of SNAP-25 substrate cleaved by the Neurotoxin based on the amounts of the first and second detection complexes determined by the arrangement according to a); and c) instructions for directly determining the biological activity of a Neurotoxin polypeptide in cells.

2. The kit of claim 1, wherein the Neurotoxin polypeptide is Clostridium botulinum toxin serotype A (BoNT/A), Clostridium botulinum toxin serotype C1 (BoNT/C1), or Clostridium botulinum toxin serotype E (BoNT/E).

3. The kit of claim 1, wherein the cells susceptible to Neurotoxin intoxication 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, neuroblastoma cells, P19 cells and induced pluripotent stem (iPS) cell-derived neurons.

4. The kit of claim 1, wherein the first capture antibody specifically binds to the non-cleaved Neurotoxin substrate SNAP-25 and the Neurotoxin-cleaved substrate SNAP-25 and allows for the determination of the total amount of the Neurotoxin substrate in the cells.

5. The kit of claim 1, wherein the first capture antibody which specifically binds to the non-cleaved Neurotoxin substrate SNAP-25 and the Neurotoxin-cleaved substrate SNAP-25 is a rabbit polyclonal anti-SNAP-25 antibody S9684, a rabbit polyclonal anit-SNAP25 antibody PA5-19708, or a rabbit polyclonal anti-SNAP25 antibody PA5-19701.

6. The kit of claim 1, wherein the second capture antibody is a mouse monoclonal antibody clone 20-2-5 or a mouse monoclonal antibody MC-6053.

7. The kit of claim 1, wherein the first and/or second capture antibody is immobilized.

8. The kit 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 fluorescent dye.

9. The kit of claim 1, wherein a substrate for the HRP-conjugated antibody is selected from the group consisting of Amplex UltraRed, 10-Acetyl-3,7-Dihydroxyphenoxazine (ADHP) and 3-(4-Hydroxyphenyl) propionic acid (HPPA).

10. The kit of claim 1, wherein a substrate for the AP-conjugated antibody is selected from the group consisting of 4-methylumbelliferryl phosphate derivative selected from 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP) and fluorescein diphosphate (FDP).

11. The kit 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 ?-Galactosidase-conjugated antibody.

12. The kit of claim 1, wherein a substrate for the HRP-conjugated antibody is selected from the group consisting of Amplex UltraRed, 10-Acetyl-3,7-Dihydroxyphenoxazine (ADHP) and 3-(4-Hydroxyphenyl) propionic acid (HPPA).

13. The kit of claim 1, wherein a substrate for the AP-conjugated antibody is selected from the group consisting of 4-methylumbelliferryl phosphate derivative selected from 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP) and fluorescein diphosphate (FDP).

Description

[0090] The figures show:

[0091] FIG. 1: Diagram representing the mode of action of the cell-based assay of the invention. Cells susceptible to Neurotoxin intoxication are seeded in multiwell plates, Thereafter, the cells are intoxicated with Neurotoxin polypeptide and after a given intoxication period the cells are fixated. The specific antibody for Neurotoxin-cleaved SNAP-25 and the specific antibody for un-cleaved 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 neurotoxin cleaved SNAP-25 and the total amount of SNAP-25 within the well. With increasing BoNT/A concentration the amount of measured cleaved SNAP-25 increases resulting in a gain of signal.

[0092] FIG. 2: The two graphs represent the resulting BoNT/A calibration curves for iPS-derived neurons and SiMa cells according to Example 2. They show the dependency between respectively the concentration and activity of BoNT/A and the determined fluorescence signal (RFU) for the HRP substrate and the content of BoNT/A-cleaved SNAP-25 normalized to the total amount of SNAP-25 within the well. Upon increasing concentration and activity, respectively, of BoNT/A, more SNAP-25 is converted by the Neurotoxin, resulting in an increase in the content of cleaved SNAP-25.

[0093] FIG. 3: The graph represents the resulting BoNT/A calibration curve for iPS derived neurons according to Example 4. It shows the dependency between respectively the concentration and activity of BoNT/A and the determined fluorescence signal (RFU) for the HRP substrate and the content of BoNT/A-cleaved SNAP-25 and the content of BoNT/A-cleaved SNAP-25 normalized to the total amount of SNAP-25 within the well. Upon increasing concentration and activity, respectively, of BoNT/A, more SNAP-25 is converted by the Neurotoxin, resulting in an increase in the content of cleaved SNAP-25.

[0094] 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 NEUROTOXIN-CLEAVED SUBSTRATE SNAP-25

[0095] Mouse monoclonal antibodies specifically binding to the cleavage site of the Neurotoxin-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 SNAP-25.sub.190-197 with a Cysteine residue at the N-terminus, C-TRIDEANQ (SEQ ID NO: 17). Said N-terminal Cysteine residue is not derived from the SNAP-25 amino acid sequence but has been introduced for linking the SNAP-25.sub.190-197 peptide (SEQ ID NO: 74) 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 Neurotoxin-cleaved substrate SNAP-25 were screened in ELISA. The obtained clones have been selected with respect to their specificity and affinity to BoNT/A-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 antibodies 20-2-5, 5-10-5, 1-10-4, 16-5-4, 6-3-8, 18-3-3, and 14-12-1 were found to be highly specific for BoNT/A-cleaved SNAP-25.sub.197, with no detectable cross-reactivity to SNAP25.sub.206 in ELISA and Western blots. Isotyping of said monoclonal antibodies has been carried out using the mouse monoclonal antibody isotyping test kit (Serotec). As a result it has been found that mAb 20-2-5, 14-12-1, 6-3-8, and 5-10-5 are IgG1 antibodies, whereas mAb 18-3-3, 16-5-4, and 1-10-4 are IgG2a antibodies.

The corresponding amino acid sequences of the VH and VL chains and the corresponding CDR (complementarity determining region) sequences of the mentioned mouse monoclonal antibodies are indicated in the sequence listing.

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

Fixation of Cells

[0096] 1. Remove the media/toxin solution. Add 100 ?l/well ice-cold methanol (?20? C.) and incubate for 20 min at ?20? C.
Note: Perform all subsequent steps at room temperature.

After Cell Fixation:

[0097] 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. [0098] 2. Remove the PBS buffer and wash the cells 3 times with 200 ?l/well of wash buffer. Each step should be performed for 1 minute with gentle shaking. [0099] 3. Remove the wash buffer and add 100 ?l/well of quenching buffer and incubate for 20 minutes with gentle shaking. [0100] 4. Remove the quenching buffer and wash the cells once with 300 ?l/well of wash buffer for 5 minutes under gentle shaking. [0101] 5. Remove the wash buffer, and add 200 ?l/well of blocking buffer and incubate for 1 hour with gentle shaking. [0102] 6. Remove the blocking buffer, and add 100 ?l/well permeabilization buffer and incubate for 15 minutes with gentle shaking. [0103] 7. Remove the permeabilization buffer and wash the cells once with 300 ?l/well of PBS buffer. This step should be performed for 1 minute with gentle shaking. [0104] 8. Remove the PBS 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/A-cleaved SNAP-25 and a polyclonal rabbit antibody that recognizes SNAP-25 (antibody for determining the total amount of SNAP-25 for normalization). [0105] 9. Remove the primary antibody mixture and wash the cells 4 times with 200 ?l of wash buffer. Each step should be performed for 5 minutes with gentle shaking. [0106] 10. Remove the wash 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. [0107] 11. Remove the secondary antibody mixture and wash the cells 5 times with 200 ?l/well of wash buffer, followed by 1 washing step with 300 ?l/well of HEPES buffer. Each wash step should be performed for 5 minutes with gentle shaking. [0108] 12. Remove the PBS 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. [0109] 13. 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. [0110] 14. Read the plates using a fluorescence plate reader:
excitation at 540 nm; emission at 600
nm.excitation at 360 nm; emission at 450 nm. [0111] 15. Calculation
For normalization, the RFU value for 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]$\mathrm{RFU}\left(600\mathrm{nm}\right)\mathrm{RFU}\left(450\mathrm{nm}\right)?1000$

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

Reagent Preparation

Wash Buffer:

[0112] 0.1% Triton X-100 in 10 mM PBS buffer (pH 7.4)

PBS Buffer (10 mM):

[0113] Phosphate buffered saline (Sigma, #P5368) (pH 7.4)

Quenching Buffer:

[0114] 0.6% H.sub.2O.sub.2 in 10 mM PBS buffer (pH 7.4)

Blocking Buffer:

[0115] 2% BSA in 10 mM PBS buffer (pH 7.4)+0.05% Triton X-100

Permeabilization Buffer:

[0116] 0.5% Triton X-100 in 10 mM PBS buffer

HEPES Buffer:

[0117] 50 mM HEPES (pH 7.4)

HRP Substrate:

[0118] 50 mM HEPES (pH 7.4) [0119] 0.007% H.sub.2O.sub.2 [0120] 150 pM Amplex UltraRed

AP Substrate:

[0121] 25 mM Diethanolamine (pH 9.8) [0122] 2 mM MgCl.sub.2 [0123] 100 ?l M DiFMUP

EXAMPLE 3: ILLUSTRATION OF BONT/A CALIBRATION CURVES IN THE CBA-ELISA ACCORDING TO EXAMPLE 2 OF THE PRESENT INVENTION

[0124] Cell culture and intoxication with BoNT/A of parental SiMa cells has been carried out according to the provider's manual. Similarly, cell culture and intoxication with BoNT/A of human induced pluripotent stem (iPS)cell-derived neurons (Cellular Dynamics) has been carried out according to the protocol by the manufacturer.

[0125] The ELISA has been carried out according to Example 2. As first capture antibody specifically binding to the non-cleaved and BoNT/A-cleaved SNAP-25, the rabbit polyclonal anti-SNAP-25 antibody S9684 (Sigma) has been used. This antibody allows for the detection of the total amount of SNAP-25 within the cells. As a second capture antibody specifically binding to the cleavage site of the BoNT/A-cleaved SNAP-25, the monoclonal antibody clone 20-2-5 of the invention (see Example 1) has been utilized.

[0126] The two graphs in FIG. 2 show the obtained BoNT/A calibration curves. They demonstrate the dependency between respectively the concentration and activity of BoNT/A and the determined fluorescence signal (RFU) for the HRP substrate and the content of BoNT/A-cleaved SNAP-25 (RFU values are not blank-corrected in order to illustrate the errors of the single BoNT/A standards). Upon increasing concentration and activity, respectively, of BoNT/A, more SNAP-25 is converted by the Neurotoxin resulting in an increase in the content of cleaved SNAP-25. The dependency of the signal of the BoNT/A concentration/activity of BoNT/A is illustrated by using a 4-parameter equation.

EXAMPLE 4: DOUBLE-FLUORESCENCE-CB-BONT/A ACTIVITY ELISA

Fixation of Cells

[0127] 1. Remove the media/toxin solution. Add 100 ?l/well ice-cold methanol (?20? C.) and incubate for 20 min at ?20? C.
Note: Perform all subsequent steps at room temperature.

After Cell Fixation:

[0128] 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. [0129] 2. Remove the PBS buffer and wash the cells 3 times with 200 ?l/well of PBS buffer. Each step should be performed for 1 minute with gentle shaking. [0130] 3. Remove the PBS buffer and add 100 ?l/well of quenching buffer and incubate for 20 minutes with gentle shaking. [0131] 4. Remove the quenching buffer and wash the cells once with 300 ?l/well of PBS buffer for 3 minutes under gentle shaking. [0132] 5. Remove the PBS buffer, and add 200 ?l/well of blocking buffer and incubate for 1 hour with gentle shaking. [0133] 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/A-cleaved SNAP-25 and a polyclonal rabbit antibody that recognizes SNAP-25 (antibody for determining the total amount of SNAP-25 for normalization). [0134] 7. Remove the primary antibody mixture and wash the cells 4 times with 200 ?l of PBS buffer. Each step should be performed for 3 minutes with gentle shaking. [0135] 8. 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. [0136] 9. Remove the secondary antibody mixture and wash the cells 5 times with 200 ?l/well of PBS buffer, followed by 1 washing step with 300 ?l/well of HEPES buffer. Each wash step should be performed for 3 minutes with gentle shaking. [0137] 10. 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. [0138] 11. 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. [0139] 12. Read the plates using a fluorescence plate reader:
excitation at 540 nm; emission at 600 nm.
excitation at 360 nm; emission at 450 nm. [0140] 13. Calculation
For normalization, the RFU value for 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:

[00002]$\mathrm{RFU}\left(600\mathrm{nm}\right)\mathrm{RFU}\left(450\mathrm{nm}\right)?1000$

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

Reagent Preparation

PBS Buffer (10 mM):

[0141] Phosphate buffered saline (Sigma, #P5368) (pH 7.4)

Quenching Buffer:

[0142] 1.6% H.sub.2O.sub.2 in 10 mM PBS buffer (pH 7.4)

Blocking Buffer:

[0143] 2% BSA in 10 mM PBS buffer (pH 7.4)+0.05% Triton X-100

HEPES Buffer:

[0144] 50 mM HEPES (pH 7.4)

HRP Substrate:

[0145] 50 mM HEPES (pH 7.4) [0146] 1.7 7% H.sub.2O.sub.2 [0147] 150 pM Amplex UltraRed

AP Substrate:

[0148] 25 mM Diethanolamine (pH 9.8) [0149] 2 mM MgCl.sub.2 [0150] 100 ?l M DiFMUP

EXAMPLE 5: ILLUSTRATION OF BONT/A CALIBRATION CURVES IN THE CBA-ELISA ACCORDING TO EXAMPLE 4 OF THE PRESENT INVENTION

[0151] Cell culture and intoxication with BoNT/A of human induced pluripotent stem (iPS) cell-derived neurons (Cellular Dynamics) has been carried out according to the protocol by the manufacturer.

[0152] The ELISA has been carried out according to Example 4. As first capture antibody specifically binding to the non-cleaved and BoNT/A-cleaved SNAP-25, the rabbit polyclonal anti-SNAP-25 antibody S9684 (Sigma) has been used. This antibody allows for the detection of the total amount of SNAP-25 within the cells. As a second capture antibody specifically binding to the cleavage site of the BoNT/A-cleaved SNAP-25, the monoclonal antibody clone 20-2-5 of the invention (see Example 1) has been utilized.

[0153] The graph shown in FIG. 3 represents the obtained BoNT/A calibration curve. It shows the dependency between respectively the concentration and activity of BoNT/A and the determined fluorescence signal (RFU) for the HRP substrate and the content of BoNT/A-cleaved SNAP-25. Upon increasing concentration and activity, respectively, of BoNT/A, more SNAP-25 is converted by the Neurotoxin resulting in an increase in the content of cleaved SNAP-25. The dependency of the signal of the BoNT/A concentration/activity of BoNT/A is illustrated by using a 4-parameter equation.