Means and methods for the determination of the biological activity of neurotoxin polypeptides in cells

Abstract

The present 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 said capture antibodies to said 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 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; e) determining the amount of the first and second detection complexes of step d), and f) calculating the amount of substrate cleaved by said Neurotoxin polypeptide in said cells by means of the second detection complexes, thereby determining the biological activity of said Neurotoxin polypeptide in said cells. The invention further provides for a kit for carrying out the method of the invention.

Claims

1. A method for directly determining the biological activity of a Clostridium botulinum toxin serotype A (BoNT/A) polypeptide in cells, such method comprising the steps of: a) culturing cells susceptible to BoNT/A in wells of a culture dish, incubating the cells susceptible to BoNT/A intoxication with a BoNT/A polypeptide for a period of time and under conditions which allow for the BoNT/A 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 which specifically binds to a non-cleaved Neurotoxin substrate SNAP-25 and a Neurotoxin-cleaved substrate SNAP-25 and contacting the cells with at least 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; d) contacting the cells in step c) with at least 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 at least 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; e) determining the total amount of SNAP-25 by measuring the amount of signal of the first detection complexes in the cells and determining the content of cleaved SNAP-25 in the cells by measuring the amount of signal of the second detection complexes of step d), and normalizing the signal of the second detection complexes to the signal of the first detection complexes, and f) determining a dependency between a concentration of BoNT/A and activity of BoNT/A based on an increase of the normalized signal in step e), and further generating a calibration curve based on concentration and activity of BoNT/A polypeptide in the cells; 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.

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

3. 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 fluorescent dye.

4. The method of claim 3, wherein a substrate for the HRP-conjugated antibody is selected from the group consisting of a fluorogenic substrate for horseradish peroxidase, 10-Acetyl-3,7-Dihydroxyphenoxazine (ADHP) and 3-(4-Hydroxyphenyl) propionic acid (HPPA).

5. The method of claim 3, 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).

6. 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 ?-Galactosidase-conjugated antibody.

7. The method of claim 6, wherein a substrate for the HRP-conjugated antibody is selected from the group consisting of a fluorogenic substrate for horseradish peroxidase, 10-Acetyl-3,7-Dihydroxyphenoxazine (ADHD) and 3-(4-Hydroxyphenyl) propionic acid (HPPA).

8. The method of claim 6, 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).

9. The method of claim 1, wherein the method is a fluorescence method.

10. The method of claim 1, wherein the cells susceptible to BoNT/A 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.

11. 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 and mixtures thereof.

12. The method 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 PAS-19701.

Description

(1) The figures show:

(2) 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.

(3) 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.

(4) 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.

(5) 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

(6) 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.

(7) 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

(8) Fixation of Cells

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

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

(11) After Cell Fixation:

(12) 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.

(13) 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.

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

(15) 4. Remove the quenching buffer and wash the cells once with 300 ?l/well of wash buffer for 5 minutes under gentle shaking.

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

(17) 6. Remove the blocking buffer, and add 100 ?l/well permeabilization buffer and incubate for 15 minutes with gentle shaking.

(18) 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.

(19) 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).

(20) 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.

(21) 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.

(22) 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.

(23) 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.

(24) 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.

(25) 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.

(26) 15. Calculation

(27) 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:

(28) $\frac{\mathrm{RFU}\left(600\mathrm{nm}\right)}{\mathrm{RFU}\left(450\mathrm{nm}\right)}?1000$

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

(30) Reagent Preparation

(31) Wash Buffer: 0.1% Triton X-100 in 10 mM PBS buffer (pH 7.4)

(32) PBS Buffer (10 mM): Phosphate buffered saline (Sigma, #P5368) (pH 7.4)

(33) Quenching Buffer: 0.6% H.sub.2O.sub.2 in 10 mM PBS buffer (pH 7.4)

(34) Blocking Buffer: 2% BSA in 10 mM PBS buffer (pH 7.4)+0.05% Triton X-100

(35) Permeabilization Buffer: 0.5% Triton X-100 in 10 mM PBS buffer

(36) HEPES Buffer: 50 mM HEPES (pH 7.4)

(37) HRP Substrate: 50 mM HEPES (pH 7.4) 0.007% H.sub.2O.sub.2 150 pM Amplex UltraRed

(38) AP Substrate: 25 mM Diethanolamine (pH 9.8) 2 mM MgCl.sub.2 100 ?l M DiFMUP

EXAMPLE 3

Illustration of BoNT/A Calibration Curves in the CBA-ELISA According to Example 2 of the Present Invention

(39) 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.

(40) 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.

(41) 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

(42) Fixation of Cells

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

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

(45) After Cell Fixation:

(46) 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.

(47) 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.

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

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

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

(51) 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).

(52) 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.

(53) 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.

(54) 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.

(55) 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.

(56) 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.

(57) 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.

(58) 13. Calculation

(59) 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:

(60) $\frac{\mathrm{RFU}\left(600\mathrm{nm}\right)}{\mathrm{RFU}\left(450\mathrm{nm}\right)}?1000$

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

(62) Reagent Preparation

(63) PBS Buffer (10 mM): Phosphate buffered saline (Sigma, #P5368) (pH 7.4)

(64) Quenching Buffer: 0.6% H.sub.2O.sub.2 in 10 mM PBS buffer (pH 7.4)

(65) Blocking Buffer: 2% BSA in 10 mM PBS buffer (pH 7.4)+0.05% Triton X-100

(66) HEPES Buffer: 50 mM HEPES (pH 7.4)

(67) HRP Substrate: 50 mM HEPES (pH 7.4) 0.007% H.sub.2O.sub.2 150 pM Amplex UltraRed

(68) AP Substrate: 25 mM Diethanolamine (pH 9.8) 2 mM MgCl.sub.2 100 ?l M DiFMUP

EXAMPLE 5

Illustration of BoNT/A Calibration Curves in the CBA-ELISA According to Example 4 of the Present Invention

(69) 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.

(70) 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.

(71) 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.