CELL-BASED METHOD FOR DETERMINING AN ACTIVITY OF BOTULINUM TOXIN
20220003752 · 2022-01-06
Assignee
Inventors
- Chee Gun Lee (Yongin-si, KR)
- Ji Hyun Oum (Seongnam-si, KR)
- Vijayakumar Ajay (Seoul, KR)
- Xiangai Gui (Suwon-si, KR)
Cpc classification
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
G01N33/50
PHYSICS
Abstract
A new cell line and an antibody for determining the activity of botulinum toxin are disclosed. Also disclosed is a method of determining the activity of botulinum toxin using the cell line and/or the antibody.
Claims
1. A method for determining activity of botulinum toxin in a sample, comprising the steps of: (a) contacting a cell with the sample, wherein the cell is from a cell line clonally derived from parental neuro-2a cell (accession number KCTC AC28106), wherein the clonally derived cell line has homogenous cell population, and wherein the clonal cell line comprises cells susceptible to intoxication by botulinum toxin type A (BoNT/A) by about 25 pM or less of BoNT/A, and the cell shows higher sensitivity to BoNT/A, BoNT/B, BoNT/C, and BoNT/F compared to the parental neuro-2a cell under same condition, equal sensitivity to BoNT/D of 5 pM or 200 pM concentration to the parental neuro-2a cell under same condition, and no sensitivity to BoNT/E of 10-400 pM concentration; (b) obtaining cell lysate of the cell of (a), said cell lysate comprising proteins of the cell of (a) or isolating proteins from the cell of (a); (c) contacting the cell lysate or the isolated proteins with an agent which specifically binds synaptosomal nerve-associated protein 25 (SNAP25.sub.FL) or botulinum toxin-cleaved SNAP25 fragment (SNAP25.sub.197); (d) detecting the presence of a complex between the agent and the SNAP25.sub.FL and/or SNAP25.sub.197, and (e) determining the activity of botulinum toxin in the sample, wherein the higher the amount of the agent-antigen SNAP25.sub.FL and/or SNAP25.sub.197 complex detected the higher the level of botulinum toxin activity in the sample.
2. The method of claim 1, wherein the botulinum toxin is botulinum toxin type A (BoNT/A).
3. The method of claim 1, which further comprises, prior to step (a), culturing the cell in a culture medium supplemented with ganglioside GT1b trisodium salt (GT1b).
4. The method of claim 3, wherein the culture medium further comprises creatine and arginine.
5. The method of claim 3, wherein the concentration of GT1b is 25-75 μg/ml.
6. The method of claim 4, wherein concentration of arginine is about 5 mM.
7. The method of claim 1, wherein the agent is an antibody comprises: a heavy-chain CDR1 region which is any one selected from the group consisting of SEQ ID NOs: 11 to 13, 28 to 33, and 55 to 56; a heavy-chain CDR2 region which is any one selected from the group consisting of SEQ ID NOs: 14 to 16, 34 to 39, and 57 to 58; a heavy-chain CDR3 region which is any one selected from the group consisting of SEQ ID NOs. 17 to 19, 40 to 46, and 59 to 60; a light-chain CDR1 region which is any one selected from the group consisting of SEQ ID NOs: 20 to 22, 47 to 49, and 61 to 62; a light-chain CDR2 region which is any one selected from the group consisting of SEQ ID NOs: 23 to 24, 50 to 51, and 63 to 64; and a light-chain CDR3 region which is any one selected from the group consisting of SEQ ID NOs: 25 to 27, 52 to 54, and 65 to 66.
8. A method for detecting botulinum toxin in a sample, comprising the steps of: (a) contacting a cell with the sample comprising botulinum toxin or suspected of comprising botulinum toxin, wherein the cell is from a cell line clonally derived from parental neuro-2a cell (accession number KCTC AC28106), wherein the clonally derived cell line has homogenous cell population, and wherein the clonal cell line comprises cells susceptible to intoxication by botulinum toxin type A (BoNT/A) by about 25 pM or less of BoNT/A, and the cell shows higher sensitivity to BoNT/A, BoNT/B, BoNT/C, and BoNT/F compared to the parental neuro-2a cell under same condition, equal sensitivity to BoNT/D of 5 pM or 200 pM concentration to the parental neuro-2a cell under same condition, and no sensitivity to BoNT/E of 10-400 pM concentration; (b) obtaining cell lysate of the cell of (a), said cell lysate comprising proteins of the cell of (a), or isolating proteins from the cell of (a); (c) contacting the cell lysate or the isolated proteins with an agent which specifically binds synaptosomal nerve-associated protein 25 (SNAP25.sub.FL) or botulinum toxin-cleaved SNAP25 fragment (SNAP25.sub.197); (d) detecting the presence of a complex between the agent and the SNAP25.sub.FL and/or SNAP25.sub.197; and (e) determining that when agent-antigen SNAP25.sub.FL and/or SNAP25.sub.197 complex is detected, the botulinum toxin is present in the sample.
9. The method of claim 8, wherein the botulinum toxin is botulinum toxin type A (BoNT/A).
10. The method of claim 8, which further comprises, prior to step (a), culturing the cell in a culture medium supplemented with ganglioside GT1b trisodium salt (GT1b).
11. The method of claim 10, wherein the culture medium further comprises creatine and arginine.
12. The method of claim 10, wherein the concentration of GT1b is 25-75 μg/ml.
13. The method of claim 11, wherein concentration of arginine is about 5 mM.
14. The method of claim 8, wherein the agent is an antibody comprises: a heavy-chain CDR1 region which is any one selected from the group consisting of SEQ ID NOs: 11 to 13, 28 to 33, and 55 to 56; a heavy-chain CDR2 region which is any one selected from the group consisting of SEQ ID NOs: 14 to 16, 34 to 39, and 57 to 58; a heavy-chain CDR3 region which is any one selected from the group consisting of SEQ ID NOs. 17 to 19, 40 to 46, and 59 to 60; a light-chain CDR1 region which is any one selected from the group consisting of SEQ ID NOs: 20 to 22, 47 to 49, and 61 to 62; a light-chain CDR2 region which is any one selected from the group consisting of SEQ ID NOs: 23 to 24, 50 to 51, and 63 to 64; and a light-chain CDR3 region which is any one selected from the group consisting of SEQ ID NOs: 25 to 27, 52 to 54, and 65 to 66.
Description
DESCRIPTION OF DRAWINGS
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MODE FOR INVENTION
[0129] Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Materials and Method 1: Reagents for Cell Development
[0130] 0.25% trypsin EDTA (GIBCO™ 25200056), 12-well plate (Corning CLS3513), 24-well plate (Falcon 353047), 6-well plate (Falcon 353046), antibiotic antimycotic solution (AA) (100×) (Sigma A5955), boric acid (Sigma B6768), collagen from human placenta (Sigma C5533), DMSO (Sigma D2650), DPBS (Welgene LB001-02), DTT (Sigma D0632), fetal bovine serum (FBS) (YI Frontier US-FBS-500), gelatin solution (Sigma G1393), GlutaMAX™ (GIBCO™ 35050061), glycerol (Affymatrix USB 16374), MEM (GIBCO™ 11095080), MEM non-essential amino acid (NEAA) (100×) (GIBCO™ 11140050), PCR Mycoplasma Detection Set (Takara Bio 6601), poly-D-lysine hydrobromide (Sigma P6407), polysorbate (Sigma P7949), RIPA buffer (10×) (abcam ab156034), sodium pyruvate (GIBCO™ 11360070), sodium tetraborate (Sigma 221732), T75 flask (Falcon BD353136), TAKARA EX TAQ™ (Takara Bio RR001), TRYPLE™ Express Enzyme (1×) (GIBCO™ 12604021).
Materials and Method 2. Preparation of Botulinum Toxin A Stock Solution, Diluent, and BoNT/A Toxic Medium
[0131] Purified botulinum toxin serotype A(BoNT/A) was provided by Hugel (EXBII1501).
Materials and Method 2-1. Preparation of BoNT/A Working Stock Solution
[0132] Purified BoNT/A was provided by Hugel (EXBII1501). BoNT/A was diluted to make working stock solution (10 nM) using a toxin dilution buffer consisting of 50 mM sodium phosphate, pH 7.0, 1 mM DTT, 0.05% polysorbate, 20% glycerol, and 0.2 mg/ml of acetylated-BSA. BoNT/A working stocks were stored in aliquots at −80° C. prior to use. And BoNT/A stock solution was prepared as follows.
(1) Master Stock (200 U/ml): Re-suspend the lyophilized BoNT/A (200 U) in 1 ml of intoxication medium or saline solution, and leave it at RT for 10 min.
(2) Stock A (50 U/ml): Aliquot 150 μl of the master stock in a sterile microfuge tube with 450 μl of intoxication medium at RT (i.e., 1:4 dilution of master stock).
(3) Stock B (5 U/ml): Aliquot 20 μl of the stock A in a sterile microfuge tube with 180 μl of intoxication medium at RT (i.e., 1:10 dilution of stock A).
(4) Stock C (0.5 U/ml): Aliquot 20 μl of the stock B in a tube containing 180 μl of intoxication medium at RT (1:10 dilution of stock B).
Materials and Method 2-2. Sample Preparation for Standard Curve
[0133] BoNT/A Standard Reference Samples for Standard Curve prepared as follows.
1. Standard Stock A (50 pM, 113.6 U/ml): Re-suspend the lyophilized BoNT/A (100 U) in 880 μl of intoxication medium or saline solution, and leave it at RT for 10 min.
2. Standard Stock B (10 pM, 22.7 U/ml): Aliquot 50 μl of Stock A in a sterile microfuge tube with 200 μl of intoxication medium at RT (i.e., 1:5 dilution of Stock A).
3. Standard Stock C (2 pM, 4.54 U/ml): Aliquot 50 μl of Stock B in a sterile microfuge tube with 200 μl of intoxication medium at RT (i.e., 1:5 dilution of Stock B).
Materials and Method 3. Plate Coating for Neuronal Cell Culture
[0134] Culture plate was coated overnight with either gelatin solution (0.1% in 1×PBS), collagen Type IV (0.1 mg/ml), or poly-D-lysine (PDL) (50 μg/ml). Freeze-dried collagen was reconstituted in deionized H.sub.2O to a final concentration of 0.1 mg/ml. PDL solution was prepared by dissolving 5 mg powder in 0.1 M borate buffer, pH 8.5, to the working concentration of 50 μg/ml. Culture plate was rinsed twice with 1×DPBS and air-dried in the tissue culture hood.
Materials and Method 4. Propagation of Cell Lines and Culture Medium
[0135] Thirteen neuronal cell lines were collected from 5 different institutes (Table 1). Neuronal cells were purchased from the American Tissue Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ), Japanese Collection of Research Bioresources (JCRB), Korean Collection for Type Culture (KCTC), and Korean Cell Line Bank (KCLB). They were maintained and propagated in the recommended medium. Mycoplasm contamination of all cell lines were monitored using PCR Mycoplasma Detection Set (Takara Bio Inc. 6601) at every 4 passages of N2-42F cells or 10 passages of SiMa and hybridoma cells. The PCR test was performed according to the recommended procedure. In brief, culture supernatant was collected and incubated for 3-4 days. PCR (50 μl) was performed with aliquots (3 μl) of culture supernatant, 1×PCR buffer, dNTP mixture, MCGp F1/R2 primers, and TAKARA EX TAQ™ (Takara Bio RR001). Aliquots (10 μl) of PCR products were resolved on a 1% agarose gel and visualized by ethidium staining.
TABLE-US-00001 TABLE 1 Cell line Source Culture Medium SK-N-SH KCLB MEM, 300 mg/L glutamine, 30011.sup.1 25 mM HEPES, 25 mM NaHCO.sub.3, 10% FBS SH-SY5Y KCLB MEM, 20 mM HEPES, 22266.sup.1 25 mM NaHCO.sub.3, 10% FBS IMR-32 KCLB RPMI1640, 300 mg/L glutamine, 10127.sup.1 25 mM HEPES, 25 mM NaHCO.sub.3, 10% FBS Neuro-2a KCTC MEM, 10% FBS AC28106.sup.2 SK-N-MC KCTCHC18501.sup.2 DMEM, 10% FBS N1E-115 ATCCCRL2263.sup.3 DMEM, 10% FBS NG108-15 ATCC DMEM, 0.1 mM hypoxanthin, HB12317.sup.3 0.4 mM aminopterin, 16 mM thymidine, 10% FBS, 1.5 g/L NaHCO.sub.3 BE(2)-M17 ATCC EMEM + F12, 10% FBS CRL2267.sup.3 SiMa DSMZ RPMI1640, ACC164.sup.4 2 mM glutamine, 10% FBS KP-N-RT-BM-1 JCRB RPMI1640, 10% FBS 1FO50432.sup.5 KP-N-YN JCRBIFO50431.sup.5 RPMI1640, 10% FBS NH-6 JCRB 0832.sup.5 Alpha-MEM, 10% FCS NH-12 JCRB 0833.sup.5 Alpha-MEM, 10% FCS TGW JCRB 0618.sup.5 EMEM, 10% FBS .sup.1: KCLB; Korean Cell Line Bank .sup.2: KCTC; Korean Collection for Type Culture .sup.3: ATCC; American Tissue Culture Collection .sup.4: DSMZ; Deutsche Sammlung von Mikroorganismen und Zellkulturen .sup.5: JCRB; Japanese Collection of Research Bioresources FBS; fetal bovine serum, FCS; fetal calf serum
Materials and Method 5: Reagents for Antibodies Development
[0136] 2-mercaptoethanol (Sigma M3148), 4-iodopheylboronic acid (Sigma 471933), 10×TBS (BIO-RAD 170-6435), 10× Tris/Glycine/SDS buffer (Bio-Rad 161-0772), 12% Mini-PROTEAN® TGX™ (Bio-Rad 456-1046), acetic acid (Merck 100063), alkaline phosphatase (Sigma P0114), AMICON Ultra-15, Ultracel 30K (Millipore UFC903024), antibiotic antimycotic solution (AA) (100×) (Sigma A5955), bromophenol blue (Sigma B0126), DMEM (Gibco™ 11995065), DMSO (Sigma D2650), DMSO (Sigma 472301), EZ-link NHS-PEG.sub.4-Biotin (Thermo Fisher Scientific 21329), ethylene glycol (Sigma 324558) fetal bovine serum (FBS) (YI Frontier US-FBS-500), glycerol (Affymetrix USB 16374), glycine (Bioshop GLN001), glutaraldehyde solution (Sigma G7651), horseradish peroxidase (Sigma P6782), hydrogen peroxide solution (Sigma 216763), LUMINOL (Sigma 123072), magnesium chloride (Sigma M8266), MEM non-essential amino acid (NEAA) (100×) (GIBCO™ 11140050), methanol (Merck 106009), PCR® STRIP TUBE (Axygen PCR-0208-CP-C), polyvinylidene fluoride (PVDF) membrane (Millipore ISEQ00010), potassium chloride (Sigma P9333), PRECISION PLUS PROTEIN™ dual color standards (Bio-Rad 1610374) SDS solution 20% (w/v) (Bio-Rad 161-0418), skim milk (BD DIFCO™ 232-100), sodium acetate (Sigma W302406), sodium bicarbonate (Sigma S6014), sodium borohydride (Sigma 452882), sodium chloride (Merck 106404), sodium (meta)periodate (Sigma S1878), sodium phosphate dibasic (Sigma S7907), sodium phosphate monobasic (Sigma S5011), sodium stannate trihydrate (Sigma 336262), tetrabutylammonium borohydride (Sigma, 230170), T175 (SPL 74175), T75 flask (SPL 70375), Tris (Bioshop TRS001), zinc chloride (Sigma 229997).
Materials and Method 6: Setting of Antibody Manufacturing Method
Materials and Method 6-1. Generation of Polyclonal and Monoclonal Antibodies Using Synthetic Peptides
[0137] Synthetic peptides were conjugated with keyhole limpet hemocyanin (KHL) at either C- or N-terminus, as summarized in
Materials and Method 6-2. Preparation of SNAP25-Affinity Column
[0138] Recombinant SNAP25.sub.197 was concentrated to 10 mg/ml using AMICON Ultra-15 by repeated centrifugation at 1,000×g for 10 min at 4° C., while measuring the protein concentration using the Nano spectrophotometer (Drawell Scientific Instrument Co., Ltd, Shanghai). Aliquot (3 ml) of SNAP25.sub.197 in AMICO Ultra-15 was mixed with 12 ml of coupling buffer (0.1 M HEPES, pH 7.5, 0.1 M NaCl) and re-concentrated by centrifugation at 1,000×g. This buffer exchange was repeated 6 times, and SANP25.sub.197 concentrate was added to 1 ml slurry of Affi-Gel 15 (Bio-Rad) in deionized H.sub.2O. After incubation on a shaker incubator for 1 hr at RT, the Affi-Gel 15 was centrifuged at 1,000×g for 10 min at 4° C. This SNAP25.sub.197-conjugated Affi-Gel 15 (SNAP25-AffiGel) was re-suspended in 10 ml of 10 mM ethanolamine hydrochloride, pH 8.0 and incubated for 1 hr at RT. After washing with 1×PBS, SNAP25-AffiGel was stored in 1×PBS containing 0.2% sodium azide before use.
Materials and Method 6-3. Purification of Polyclonal Antibody
[0139] Rabbit serum was diluted 10 times with 10 mM Tris-HCl, pH 7.5, and centrifuged at 10,000×g for 10 min at 4° C. After filtering through 0.45 μm bottle top filter (NALGENE™), the clear serum diluent was passed through SNAP25-AffiGel three times. SNAP25-AffGel was then washed with 20 CV of 10 mM Tris-HCl, pH 7.5, and 0.5 M NaCl, and bound proteins were sequentially eluted with 12 CV of 0.1 M sodium acetate, pH 5.5, 0.1 M glycine, pH 4.0, and 0.1 M glycine, pH 2.5 and 0.1 M triethylamine, pH 11.5. During elution, protein samples were collected in tubes containing 0.1 ml of 1 M Tris-HCl, pH 8.0. Protein-containing peak fractions were pooled and concentrated to 1 ml using AMICON Ultra-15. After dialysis with four changes of 1×PBS and 10% glycerol (0.5 L) at every 90 min, they were analyzed by SDS-PAGE and ELISA.
Materials and Method 6-4. Antibody Conjugation with Horseradish Peroxidase (HRP)
[0140] For conjugation of HRP, purified B4 or C16 IgG was concentrated to 10 mg/ml using AMICON Ultra-15 through repeated centrifugations at 1,000×g for ˜15 min per centrifugation at 4° C., while providing with excessive volume of the conjugation buffer (0.1 M NaHCO.sub.3, pH 9.5, 0.9% NaCl) at the end of each centrifugation. HRP (5 mg) was solubilized in 1.2 ml of deionized H.sub.2O and mixed with 0.3 ml of 0.1 M sodium periodate in 10 mM sodium phosphate, pH 7.0. After incubation for 20 min at RT, the HRP solution was dialyzed with four changes of 1 mM sodium acetate, pH 4.0, for 6 hr at 4° C. Concentrated antibody (5 mg) and activated HRP (5 mg) were mixed together in a microfuge tube and incubated for 2 hr at RT with light protection. The conjugation reaction was stopped by the addition of 0.1 ml of sodium borohydride (4 mg/ml in deionized H.sub.2O). The HRP-antibody conjugate was dialyzed using Pur-A-Lyzer Maxi 50000 with three changes of 1×PBS and once with 1×PBS/50% glycerol at an hourly interval at 4° C. The HRP-antibody conjugate was stored at 4° C. or −80° C. for a long-term storage before use.
Materials and Method 6-5. Antibody Conjugation with Activated Biotin
[0141] A15 IgG (1 mg/ml) was transferred to Pur-A-Lyzer Maxi 20000 and dialyzed with four changes of a reaction buffer consisting of 0.1 M phosphate, pH 7.2, and 0.15 M NaCl at an hourly interval at 4° C. with light protection. Activated biotin (10 mM in the reaction buffer) (EZ-Link NHS-PEG.sub.4-Biotin) was mixed with 50 μg aliquot of A15 IgG to the final concentration of 0.1 mM, 0.25 mM, or 0.5 mM. The mixture was adjusted to 100 μl with the reaction buffer and incubated for 2 hr at 4° C. with light protection. After adding 2 μl of 0.1 M glycine, the reaction mixture was dialyzed with three changes of 1×PBS and once with 1×PBS/50% glycerol at an hourly interval at 4° C. and stored in a microfuge tube at −20° C. before use. The extent of biotinylation of IgG was estimated using the Pierce Biotin Quantitation Kit (Thermo Scientific 28005) and the reactivity and specificity of biotinylated IgG were examined by sandwich ELISA.
Materials and Method 6-6. Crosslinking of AP to Antibodies
[0142] Conjugation of alkaline phosphatase (AP) to antibody was initiated by adding glutaraldehyde to 0.25% in a mixture (20 ) consisting of reaction buffer (0.1 M sodium phosphate, pH 6.8), 50 μg of AP (2.2 mg/ml) (Sigma-Aldrich P0114-10KU), and 100 μg of purified IgG such as monoclonal antibody A15, polyclonal antibody rA15 IgG, and polyclonal anti-SNAP25 IgG (Sigma-Aldrich S9684). After incubation on ice for 1 hr with light protection, the reaction was provided with 1
aliquot of 1 M ethanolamine and incubated for 1 hr at RT with light protection. AP-IgG conjugate was dialyzed with three changes of 1×PBS and once with a storage buffer (25 mM Tris-HCl, pH 7.5, 1 mM MgCl.sub.2, 0.1 mM ZnCl.sub.2, and 50% glycerol) at an hourly interval at 4° C. Antigen binding specificity and AP activity of AP-IgG conjugate was examined by direct ELISA, described below.
Materials and Method 6-7. Measurement of K.SUB.D .by OCTET RED96
[0143] For kinetics analysis of monoclonal antibody, the Bio-Layer Interferometry (BLI) assay was performed at 30° C. using FORTÉBIO® Octet Red96 instrument, following the procedure recommended by the manufacturer. In brief, recombinant GST-SNAP25.sub.FL or SNAP25.sub.197 was diluted to 125 or 250 nM in 1× kinetics buffer (1×KB)/1×PBS, and the analyte, i.e. purified IgG, was serially diluted to 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250 nM in 1× kinetics buffer. After equilibration in 1× kinetics buffer for 1 min, anti-GST probes (FORTÉBIO 18-5096) were loaded with GST-SANP25.sub.FL or GST-SNAP25.sub.197 for 30 min, followed by dipping in 1× kinetics buffer for 10 min. Subsequent to association and dissociation of analyte for 10 min each, kinetics curves were obtained, and K.sub.D (equilibrium dissociation constant) was estimated using FORTÉBIO® Octet analysis software.
[0144] As an alternative kinetics analysis, anti-mouse IgG Fc capture (AMC) biosensors were loaded with antibody and subjected to association/dissociation with serially diluted GST-SNAP25. In brief, purified IgG was diluted to 100 or 200 nM, whereas GST-SNAP25.sub.FL or SNAP25.sub.197 was serially diluted to 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 nM. After equilibration in 1× kinetics buffer for 1 min, AMC biosensors were loaded with IgGs for 10 min, followed by dipping in 1× kinetics buffer for 10 min. Association and dissociation of analyte were carried out for 10 min each, and K.sub.D was estimated as described above.
Materials and Method 6-8. Direct ELISA
[0145] Immunoplate (Thermo Fisher Scientific A71125) were coated for 2 hr at 37° C. with 1 μg of GST-SNAP25.sub.FL or GST-SNAP25.sub.197 in 0.1 M carbonate buffer, pH 9.5. After washing with 1×PBS, immunoplate was incubated with 300 μl of a blocking buffer (5% nonfat dried milk in 1×PBS) for 15 min at RT. After washing three times with 1×PBST (1×PBS/0.05% Tween-20), the microplate was provided with 100 μl of hybridoma culture supernatant (1:20-10,000 dilution) or ascites fluid (1:1,000-312,500 dilution) and incubated for 1 hr at RT. The microplate was washed three times with 1×PBST, to which aliquots (100 μl per well) of goat anti-mouse IgG-HRP conjugate (1:1,000 dilution) (Ab Frontier LF-SA8001) were added. After incubation for 1 hr at RT, the microplate was washed three times with 1×PBST and HRP reaction was carried out with 50 μL of 1-STEP™ Ultra TMB-ELISA (Thermo Fisher Scientific 34028) at RT for 3-25 min. HRP reaction was stopped by the addition of 50 μL of 1 M H.sub.2SO.sub.4 and the ELISA signal was estimated at 450 nm using BIO-TEK SynergyNeo2.
Materials and Method 6-9. Western Blot Analysis
[0146] Recombinant GST-SNAP25 (20 ng-1 μg per well) or total cell lysate of neuro-2a cells (15 μg per well) were resolved together with Precision protein standards (3 μl per well) by 10% or 12% SDS-PAGE. After 5 min-soaking in a transfer buffer consisting of 48 mM Tris, 38.9 mM glycine, 20% methanol, 0.05% SDS, proteins were transferred to PVDF membrane using TRANS-BLOT® Semi-Dry (Bio-Rad 170-3940) for 45 min at 25 V PVDF membrane was briefly rinsed with 1×TBST (1×TBS/0.05% TWEEN 20) and incubated with a blocking buffer (5% nonfat dried milk in 1×TBST) for 15 min at RT. Subsequently, PVDF membrane was incubated with either hybridoma culture supernatant (1:100 dilution in blocking solution) for 45 min at RT. Polyclonal anti-SNAP25 IgG (Sigma 59684) (1:8,000 dilution) and anti-SNAP25.sub.197 IgG (R&D MC6050) (1:100 dilution) were used as positive controls. After washing with 1×TBST three times for 15 min, PVDF membrane was incubated with either goat anti-rabbit IgG-HRP conjugate (1:10,000 dilution) or goat anti-mouse IgG-HRP conjugate (1:10,000 dilution) for 45 min at RT. After washing 3 times with 1×TBST, recombinant GST-SNAP25 or endogenous SNAP25 was detected and quantified using ECL solution (see below) and Bio-Rad CHEMIDOC™ MP Imaging system (Bio-Rad Universal hood III).
EXAMPLES
Example 1: Screening of BoNT/A-Sensitive Neuronal Cell
Example 1-1. Comparative Analysis of Neuronal Cell Lines for their Sensitivity to BoNT/A
[0147] Neuronal cells were seeded at 2×10.sup.5 cells/well in a 24-well plate and 24 hr later, they were treated with 2 nM BoNT/A in medium indicated in Table 1 for 3 days. Total cell lysates (TCLs) were prepared using 1×RIPA, and aliquots (3.5 μg) were subjected to 12% SDS-PAGE.
[0148] SNAP25.sub.FL and SNAP25.sub.197 were analyzed by Western blot using rabbit polyclonal IgG (Sigma S9684), specific for SNAP25, as described in Materials and Methods. ECL solution was formulated and optimized by AbBio Inc. Working ECL solution was prepared by mixing Sol A and Sol B in 1:1 ratio before use. Sol A and Sol B is comprised as Table 2. And sensitivity of cell lines to BoNT/A (2 nM) were represented in Table 3.
TABLE-US-00002 TABLE 2 ECL solution Composition SolA 0.1 M Tris-HCl, pH 8.8, 2.5 mM LUMINOL in DMSO, 4 mM 4-iodopheylboronic acid, 0.2 mM tetrabutylammonium borohydride, 2% ethylene glycol, and 0.02% TRITON X-100. SolB 0.1 M Tris-HCl, pH 8.8, 10.6 mM hydrogen peroxide, and 0.012% sodium stannate
TABLE-US-00003 TABLE 3 Cell line Sensitivity to BoNT/A (2 nM) SK-N-SH No SH-SY5Y No IMR-32 No Neuro-2a Yes SK-N-MC No N1E-115 No NG108-15 No BE(2)-M17 No SiMa Yes KP-N-RT-BM-1 Yes KP-N-YN No NH-6 No NH-12 No TGW No
[0149] As summarized in Table 3, except for SiMa cell, only neuro-2a and KP-N-RT-BM1 (K-BM1) cells yielded detectable levels of SNAP25 cleavage.
Example 1-2. Screening of BoNT/A-Sensitive Neuronal Cell
[0150] Neuro-2a, SiMa and KP-N-RT-BM-1 cells were separately cultured on a 24-well plate under the same conditions as materials and method, and the cleavage phenomenon of SNAP25 protein was analyzed by Western blot analysis The results are shown in
[0151] Neuro-2a was further used for clonal selection over K-BM1 for the following reason. Firstly, the in vivo potency of BoNT/A, which is conventionally measured by mouse lethality (i.e. mouse LD.sub.50), could be better recapitulated with mouse cell line. In fact, neuro-2a is a mouse cell line, but K-BM1 is a human cell line. Secondly, K-BM1 tends to grow at a much slower rate than neuro-2a (data not shown). It should be noted that the population doubling time of SiMa cells, a human neuroblastoma cell line, is reported to be 34 to 100 hrs (DSMZ ACC-164). Thirdly, neuro-2a cells appear to be a mixed population of several cell types under the microscope. Thus, it is likely that BoNT/A-sensitive cells exist in the heterogeneous neuro-2a cell population.
Example 2: Clone Selection for BoNT/A-Sensitive Neuronal Cell
[0152] Among the Neuro-2a cell populations selected in Example 1, clones highly sensitive to BoNT/A were selected.
Example 2-1. Clonal Culture of Neuro-2a Cells
[0153] Neuro-2a cells were propagated in 1MEM supplemented with 10% fetal bovine serum (FBS), 1× non-essential amino acids (1×NEAA), 1× sodium pyruvate, 1×GLUTAMAX™, and 1× antibiotic antimycotic (1×AA). When cells reached ˜80-90% confluence, they were treated with 1× TrypLE (GIBCO™ 12604021) to make a single cell suspension. After determining the viable cell number using the hemocytometer and trypan blue, cells were diluted to the density of 10 cells per ml of medium. Then, 100 μl aliquots (˜one cell equivalent) were added to 7×96-well culture plate. Colony growth was periodically monitored by microscopic examination. At ˜60% confluence, cells were transferred to a 24-well culture plate. Later, they were equally divided, and one half was stored in the liquid nitrogen tank, and the rest was tested for the sensitivity to BoNT/A intoxication.
[0154] Neuro-2a clonal cells were sub-cultured from the 24-well plate to 96-well microplate. On the following day, culture medium was replaced with PRMI1640 supplemented with 2 mM L-alanyl-L-glutamine, 1×B27, 1×N2, 1×NEAA (differentiation medium) for 2 days to induce neuronal differentiation. On the 4.sup.th day, GT1b was added to the final concentration of 25 μg/ml (1× intoxication medium). After incubation for 24 hr, the culture medium was replaced with the intoxication medium containing BoNT/A (0.1 nM), and cells were incubated for additional 2 days. After removing medium by aspiration, cells were treated with 1×SDS sample buffer, and aliquots were examined for the extent of SNAP25 cleavage by 12% SDS-PAGE and Western blot analysis. This clone selection procedure was repeated three times, and the results confirmed in each clone selection procedure are shown in
Example 2-2. Recovery, Proliferation, and Storage of Freezed Cells
[0155] Recovery of freezed cells was performed as follows.
(1) Rapidly thaw (<2 minute) the cell stock vial, retrieved from a liquid nitrogen freezer, by gentle agitation in a 37° C. water bath.
(2) Once thawed, decontaminate the cell stock vial by spraying with 70% ethanol.
(3) Unscrew the top of the vial in a laminar flow tissue culture hood, and transfer the content to a sterile 15-ml conical tube containing 9 ml of pre-warmed complete medium.
(4) After gentle centrifugation (125×g for 10 min), remove the supernatant by aspiration and re-suspend the cells in 2 ml of the complete medium.
(5) Pipet gently to loosen the pellet and break apart clumps, and transfer the cell suspension to T75 flask containing 25 ml of the complete medium.
(6) Incubate the flask at 37° C. in a CO.sub.2 incubator.
Cell culture was performed as follows.
(1) When cells become ˜90% confluent, remove the culture medium by aspiration and rinse the culture flask once with 1×DPBS.
(2) Add 1 ml of 0.25% trypsin-EDTA and incubate the flask for 10 min at 37° C.
(3) Add 9 ml of the complete medium, and break apart cell clumps by repeated pipetting.
(4) Transfer the cell suspension to a 15 ml-conical tube, and centrifuge it at 800×g for 5 min.
(5) Discard the supernatant by aspiration and re-suspend the cell pellet with 10 ml of the complete medium.
(6) Determine the density of viable cells using the hemocytometer.
(7) Transfer 3 ml aliquots of the cell suspension to fresh T75 flasks.
(8) Incubate for 3 days until cells reach ˜90% confluency.
Cell freezing for establish cell bank was performed as follows.
(1) Subculture ˜90% confluent cells in two T75 flasks to eight T75 flasks.
(2) When cells reach a late growth phase (˜2.0×107 cells/flask), remove the culture medium and prepare a single-cell suspension through treatment with 0.25% trypsin-EDTA.
(3) Re-suspend the cell pellet at a density of 5×106 cells/ml with a freezing medium.
(4) Aliquot 1 ml of cell suspension into cryogenic stock vials.
(5) Place tubes in the isopropanol-filled freezing container (be sure to tighten the tube cap).
(6) Transfer the freezing container to −80° C. overnight.
(7) On the following day, transfer frozen cells immediately to the liquid nitrogen vapor phase storage.
Example 3: Identification of N2-42F Characteristics and Culture Environment
Example 3-1. Confirmation of Doubling Time of N2-42F
[0156] The cleavage times of Neuro-2a cells and N2-42F, which were confirmed to be capable of detecting truncated forms of the SNAP25 protein, were determined.
[0157] N2-42F and neuro-2a cells were seeded at 1.5×10.sup.5 cells per well in a 6-well culture plate. On a daily basis for 6 days, total viable cells were counted using the hemocytometer and trypan blue staining. Doubling time was calculated using viable cell numbers obtained during the exponential growth phase as follows Table 4. And the results are shown in Table 5 and
TABLE-US-00004 TABLE 4 Doubling time = T X ln2/ln(Xe/Xb)
TABLE-US-00005 TABLE 5 Cell line Doubling time (hr) Neuro-2a 24 ± 4.7 N2-42F 24 ± 2.9
[0158] As shown in Table 5 and
Example 3-2. Confirmation of Morphology of N2-42F
[0159] Allergan reported the isolation of BoNT/A-sensitive clone (H1) from SiMa cells (PLoS One. 2012; 7(11):e49516). Thus, it is expected that like neuro-2a, SiMa cells also represent a heterogeneous cell population of mixed cell types but their sub-clones such as H1 and N2-42F are homogeneous cell types. This was confirmed by microscopic examination of neuro-2a, N2-42F, and SiMa cells. As shown in
[0160] From the above results, it can be seen that N2-42F corresponds to a homogeneous cell type as a single clone among various clones constituting Neuro-2a.
Example 3-3. Confirmation of Culture Conditions of N2-42F
[0161] In efforts to optimize the BoNT/A intoxication condition, N2-42F cells were grown in culture plates coated with different matrices. Unlike parental neuro-2a cells that are routinely propagated in non-coated culture plates, N2-42F cells exhibit a clear preference for culture plates coated with poly-D-lysine (PDL) (
Example 4: Confirmation of the Susceptibility of N2-42F to Botulinum Neurotoxins
[0162] Using SiMa cells as control, the BoNT/A sensitivity of N2-42F cells was thoroughly examined in triplicate sets of 96-well plate culture provided with 1× intoxication medium containing varying concentrations of BoNT/A. After incubation for 4 days, cells were treated with 50 μl of 1×SDS sample buffer, and aliquots (12 μl) were subjected to Western blot analysis using polyclonal anti-SNAP25 IgGs (Sigma S9684), as described in Materials and Methods. Endogenous SNAP25 cleavage was insignificantly detected in both SiMa and N2-42F cells treated with 0.93 pM or lower concentrations of BoNT/A. Treatment with 2.78-25 pM BoNT/A led to 25-72% cleavage of SNAP25 in N2-42F cells and 33-75% with SiMa cells (
[0163] Including BoNT/A, there are seven serologically different botulinum neurotoxins from BoNT/A to BoNT/G. Similar to BoNT/A, BoNT/B has also been licensed for the pharmaceutical application such as MYOBLOC® or NEUROBLOC®. Thus, it was explored if N2-42F cells can be used in any cell-based potency assays for different serotypes of botulinum neurotoxins. For this purpose, differentiating N2-42F and Neuro-2a cells were compared for their susceptibility to different neurotoxin complexes (Metabiologics C08RA188-RA194).
[0164] As shown in
Example 5: Confirmation of Stability of N2-42F
[0165] Passage stability could a key feature of neuronal cells to be used as host in any cell-based assay platforms. N2-42F cells were continuously propagated for multiple passages. The cells in early passages were used to make the master cell bank as described in Materials and Methods. And at every 5 passages, cells were also stored in the liquid nitrogen tanks for the passage stability. In brief, N2-42F cells stored at passage 5 (P5) and 15 (P15) were restored from the liquid nitrogen tank and at ˜90% confluence, they were compared in the BoNT/A sensitivity using the 1× intoxication medium containing 0.1 nM BoNT/A following the procedure described in
[0166] This result indicates that similar to H1 clone of SiMa cells identified by Allergan, the BoNT/A sensitivity is a stably inheriting property of N2-42F cells. which makes them a suitable host, the second of its kind next to the H1 clone of SiMa cells, in a cell-based assay platform.
Example 6: Determination of BoNT/a Potency Based on N2-42F
Example 6-1. Experimental Method
[0167] 96-well plates were coated in the manner described in Materials and methods. While air-drying the 96-well culture plate, prepare a total of 7 ml of N2-42F cell suspension in the density of 5.5×10.sup.5 cells/ml. About 90% confluent N2-42F cells in one T75 flask would be sufficient for three 96-well culture plates. Transfer the cell suspension to a sterile buffer reservoir, and dispense aliquots (100 μl) of the cell suspension into each well using a multichannel pipette, and incubate the 96-well culture plate in a CO2 incubator. Outer wells of a 96-well plate should be filled with aliquots (100 μl) of 1×AA solution to avoid the dreaded edge effects.
[0168] On the day after the cells were dispensed, all cell culture medium was removed from the 96-well plate using a multi-channel pipette, and 100 μl of RPMI 1640 was added to rinse. The BoNT/A intoxication medium was then treated to each 96-well plate and incubated for 4 days at 37° C., 5% CO.sub.2. In addition, the capture antibody, B4 IgG, was prepared in an amount of 7 ml, which was then divided into 50 in ELISA plates and stored at 4° C. overnight.
[0169] For measurement, the BoNT/A intoxication medium was removed from the 96-well plate using a multi-channel pipette and each well was treated with 60 μl of a lysis buffer (pH 7.5, 20 mM HEPES, 1% TRITON-200 mM NaCl, 1 mM EGTA, and 5 mM EDTA, added with proteolysis inhibitor immediately before use), and incubated 4° C. for 20 minutes with shaking at a speed of 500 rpm. Thereafter, the dissolution buffer contained in each well was obtained, and centrifuged 4,000 rpm for 20 minutes at 4° C.
[0170] The ELISA plate was washed 3 times with the washing buffer, added with 300 μl aliquots of the blocking buffer to each well, incubated for 15 min at RT, and washed twice with the washing buffer after remove the blocking buffer.
[0171] 50 μl aliquots of TCL was transferred from the 96-well culture plate to the ELISA plate coated with the capture antibody (B4), and the ELISA plate was incubated for 4 hr at 4° C. on a microplate shaker at 200 rpm. Finally, the plate washed 3 times with the wash buffer.
[0172] For detection of SNAP25 and SNAP25.sub.197, detection antibodies were added to ELISA plate (50 μl per well), and the plate was incubated for 1 hr at RT on a Thermo shaker incubator (200 rpm). And the plate was rinsed three times with the wash buffer, 50 μl aliquots of 1-Step™ Ultra TMB-ELISA was added to the plate, the HRP reaction was terminated by adding 2 M sulfuric acid (50 μl/well) after 5 min. The HRP reaction was measured at 450 nm. The value at A.sub.450 may represent the relative amount of SNAP25.sub.197.
Example 6-2. Preparation of Standard Curve and Determination of the BoNT/a Potency
[0173] Standard curve and BoNT/A Potency were tested in the following manner.
Calculate the average A450 value of control wells where sandwich ELISA was carried out with no BoNT/A treatment (i.e. 0 pM). Subtract the average control A450 value from test A450 values, and calculate the normalized average test A450 value. And then, plot the normalized average A450 values on Y axis against BoNT/A potency on X axis using Prism 5.0 (GraphPad Software, La Jolla, Calif.). Analyze the plot by successively selecting “analyze”, “nonlinear regression (curve fit)”, and “sigmoidal dose-response”, which will yield a EC50 value. Prepare the standard curve using the normalized A450 values of test wells treated with 0.1-0.93 pM BoNT/A Standard Reference (see Appendix 2, Section D). And use the standard curve equation with R2 value of 0.95 or higher to determine the BoNT/A potency of test samples.
Example 7: Generation of Monoclonal Antibodies Specific for SNAP25
[0174] Allergan used a 13-amino acid (AA) residue peptide N-CDSNKTRIDEANQ-C(SEQ ID NO: 91) to raise antibody. The peptide was designed to be identical to the C-terminal end of SNAP25.sub.197 generated upon BoNT/A digestion. Since SNAP25.sub.FL also has the identical amino acid sequence in it, it would be reasonable to posit that the specificity of monoclonal antibodies recognizing SNAP25.sub.197 is attributable to as yet unidentified feature of SNAP25.sub.197 rather than its primary amino acid sequence. SNAP25 consists of 206 AA residues (
[0175] A total of 10 peptide antigens were designed to meet the following criteria (Table 6). First, alpha helical regions exhibiting relatively lower antigenicity were excluded. Second, peptide sequences are non-redundant and unique, the properties of which are essential to reduce cross reactivity of antibodies.
TABLE-US-00006 TABLE 6 Immunogens/AA SEQ Position in SNAP25 ID NOs AA Sequence N peptide 1-13 SEQ ID NO: 1 KLH-C-MAEDADMRNELEE 1-13 SEQ ID NO: 2 MAEDADMRNELEE-C-KLH 19-38 SEQ ID NO: 3 KLH-C- DQLADESLESTRRMLQLVEE 51-70 SEQ ID NO: 4 KLH-C- DEQGEQLERIEEGMDQINKD M peptide 122-136 SEQ ID NO: 5 KLH-C-DEREQMAISGGFIRR C peptide 170-184 SEQ ID NO: 6 KLH-C-EIDTQNRQIDRIMEK 180-194 SEQ ID NO: 7 KLH-C-RIMEKADSNKTRIDE 180-197 SEQ ID NO: 8 KLH-C-RIMEKADSNKTRIDEANQ 186-197 SEQ ID NO: 9 KLH-C-DSNKTRIDEANQ 189-201 SEQ ID NO: 10 KLH-C-KTRIDEANQPATK
[0176] Of a total of 10 peptides described in Table 6, M and N peptides were used to generate monoclonal antibodies capable of detecting both SNAP25.sub.FL and SNAP25.sub.197. With C peptides, it was anticipated to obtain three different monoclonal antibodies with the binding specificity toward (1) SNAP25.sub.FL, (2) SNAP25.sub.197, and (3) both SNAP25.sub.FL and SNAP25.sub.197. Throughout our research, two commercially available antibodies were used as control in ELISA and Western blot analysis. They include rabbit polyclonal anti-SNAP25 IgGs (Sigma-Aldrich) and MC6050 (R&D). The former recognizes both SNAP25.sub.FL and SNAP25.sub.197, but the latter is a monoclonal antibody specific for SNAP25.sub.197.
[0177] Individual synthetic peptide was injected to two rabbits to raise polyclonal serum and 4 mice to raise monoclonal antibody, as described in Materials and Methods. In particular, the procedure to establish hybridoma cells producing monoclonal antibody was summarized in
[0178] A representative result of initial hybridoma screening is shown in
[0179] For the single-cell clonal selection, hybridoma clones were seeded at a single-cell density in one 96-well plate. Four days later, culture supernatants were tested for the reactivity to recombinant SNAP25 by direct ELISA, and five clones yielding relatively strong ELISA signals were selected for further analysis. For example, in case of clone 4, over 80% sub-clones, grown in a 96-well plate, were tested positive in direct ELISA (
[0180] Single-cell clonal selection repeated two more rounds, as summarized in
Example 8: Production and Purification of Monoclonal Antibodies
[0181] Hybridoma cells were expanded in T175 flasks and used to make master cell bank. For production of monoclonal antibody, hybridoma cells were recovered from the stock vial stored at the vapor phase of liquid nitrogen. When cells reached about 90% confluency, they were sub-cultured at 1:4-10 in multiple T175 flasks. After incubation for 3-4 days, cells were collected by centrifugation and re-suspended in serum-free medium at 1.0×10.sup.6 cells/ml. After incubation for 4 days, culture supernatants were collected and used to purify IgG using protein G column (HITRAP® Protein G HP column), as described in Materials and Methods. Yield of monoclonal antibody purified using culture supernatants varied batch to batch of hybridoma culture and also for individual hybridoma clone. Over a dozen times of purification with different batches of culture supernatant (165-542 ml) yielded about 2.45-5.52 mg of C16 IgG (Table 7).
TABLE-US-00007 TABLE 7 Monoclonal Volume of cell culture Yield antibody supernatant ( ) (mg) C16 IgG.sub.1 185 2.78 235 4.32 542 2.90 331 2.45 291 2.60 233 5.52 225 3.87 253 3.55 190 3.16 165 3.35 269 3.76 283 3.92 277 4.41 299 4.13 237 3.43
[0182] Purification of other monoclonal antibodies is summarized in Table 8.
TABLE-US-00008 TABLE 8 Polyclonal Volume of cell culture Yield antibody supernatant ( ) (mg) A15 IgG.sub.1 403 6.37 210 2.22 202 4.20 217 3.90 171 3.45 226 3.16 233 5.52 205 4.04 200 3.63 177 5.11 B23 IgG.sub.1 44.5 0.17 186 1.14 B20 IgG.sub.1 45 0.58 B16 IgG.sub.1 44.5 0.39 B4 IgG.sub.1 42 1.34 292 4.32 282 6.42 285 5.45 275 3.40 C7 IgG.sub.2a 152 5.18 161 3.30 F14 IgG.sub.1 263 2.56
[0183] On average, about 1-2 mg of IgG was obtained from 100 ml of culture supernatant. Though data not shown, the purity of IgGs and their antigen binding specificity were validated by SDS-PAGE and ELISA.
Example 9: Purification of Polyclonal Antibody
[0184] Polyclonal antibody was purified from rabbit serum using SNAP25-AffiGel, as described in Materials and Methods. In brief, rabbit serum was diluted 10 times with 10 mM Tris-HCl, pH 7.5, and cleared by centrifugation at 10,000×g for 10 min and filtering through 0.45 μm filter. The clear serum diluent was repeatedly loaded onto a SNAP25-AffiGel three times, and bound proteins were sequentially eluted with 0.1 M sodium acetate, pH 5.5, 0.1 M glycine, pH 4.0, 0.1 M glycine, pH 2.5, and 0.1 M triethylamine, pH 11.5. Protein fractions were collected in tubes containing 0.1 ml of 1 M Tris-HCl, pH 8.0, pooled and concentrated to 1 ml using AMICON Ultra-15. After dialysis with four changes of 1×PBS and 10% glycerol at every 90 min, they were analyzed by SDS-PAGE and ELISA.
[0185] Table 9 summarizes the relative distribution of serum proteins in the SNAP25-AffiGel fractions obtained with 4 different batches of rA15 sera.
TABLE-US-00009 TABLE 9 Rabbit Yield (μg) serum ( ) pH 5.5 pH 4.0 pH 2.5 30 10 85 350 30 1102 525 1160 30 936 504 990 100 64 337 945
[0186] Despite that each serum sample exhibited different protein distribution patterns in SNAP25-AffiGel fractions (Table 9), IgG was detected as a major constituent of all fractions examined by 10% SDS-PAGE (
Example 10: Sequence Analysis of Monoclonal Antibodies
[0187] Total RNA, extracted from hybridoma cells, were reversed transcribed to cDNA using either an oligo-dT anti-sense primer or a gene-specific (murine IgG1 CH and kappa CL) anti-sense primer. Specific murine constant domain primers were used to amplify the cDNA by PCR to determine the isotype of the antibody. Degenerate V.sub.H and V.sub.L, primers were used to amplify the variable domains from the cDNA. For 5′ RACE, a homopolymeric [dC] tail was added to the 3′ end of the cDNA. The heavy and light chains were then amplified with an oligo [dG] sense primer and a gene specific (CH/KC) anti-sense primer. The PCR products were cloned into a blunt or TA vector for sequencing. The sequencing results were aligned to V.sub.H and V.sub.L chains to determine consensus sequences.
[0188] CDR sequences are summarized in three different groups of IgGs according to their antigenic specificity: (1) SNAP25.sub.FL-specific IgGs (Table 10), (2) SNAP25.sub.197-specific IgGs (Table 11), and (3) Bi-specific IgGs, reacting with both SNAP25.sub.FL and SNAP25.sub.197, (Table 12).
TABLE-US-00010 TABLE 10 Identified CDR SEQ ID NOs Sequence In V.sub.H CDR1 SEQ ID NO: 11 GYSITSGYY D2 SEQ ID NO: 12 GYTFTDYN D6 SEQ ID NO: 13 GYTFTNYG E6 V.sub.H CDR2 SEQ ID NO: 14 IRYDGSN D2 SEQ ID NO: 15 IYPYNGDT D6 SEQ ID NO: 16 INTYTGEP E6 V.sub.H CDR3 SEQ ID NO: 17 ARDRDSSYYFDY D2 SEQ ID NO: 18 VRSGDY D6 SEQ ID NO: 19 ARGYYDY E6 V.sub.L CDR1 SEQ ID NO: 20 DHINNW D2 SEQ ID NO: 21 QSLLDSNGKTY D6 SEQ ID NO: 22 QSLLDSDGKTY E6 V.sub.L CDR2 SEQ ID NO: 23 DTT D2 SEQ ID NO: 24 LVS D6, E6 V.sub.L CDR3 SEQ ID NO: 25 QQYWSAPPT D2 SEQ ID NO: 26 WQGTLFPYT D6 SEQ ID NO: 27 WQGTHFPRT E6
TABLE-US-00011 TABLE 11 CDR SEQ ID NOs Sequence Identified In V.sub.H CDR1 SEQ ID NO: 28 GYSITSDYA C4 SEQ ID NO: 29 GFTFNTNA C7, C14 SEQ ID NO: 30 GYTFTNYT C16 SEQ ID NO: 31 GYTFNTYA C24 SEQ ID NO: 32 GFTFSNYG D3 SEQ ID NO: 33 GFTFNTYA C15 V.sub.H CDR2 SEQ ID NO: 34 ISYSVGT C4 SEQ ID NO: 35 IRSKSNNYAT C7, C15 SEQ ID NO: 36 IRSKSDNYAT C14 SEQ ID NO: 37 INPSSDYT C16 SEQ ID NO: 38 IRSKSNNYTT C24 SEQ ID NO: 39 INSNGGTT D3 V.sub.H CDR3 SEQ ID NO: 40 ARKGEYGFAY C4 SEQ ID NO: 41 VYGRSYGGLSY C7 SEQ ID NO: 42 VYGRSYGGLGY C14 SEQ ID NO: 43 VRQVTTAVGGFAY C15 SEQ ID NO: 44 ARRIFYNGRTYAAMDY C16 SEQ ID NO: 45 VGQILYYYVGSPAWFAY C24 SEQ ID NO: 46 ARDRDAMDY D3 V.sub.L CDR1 SEQ ID NO: 47 KSVSTSGYSY C4, C14, C24 SEQ ID NO: 48 KSVSSSGYSY C7, C15, C16 SEQ ID NO: 49 QSIVNSHGNTY D3 V.sub.L CDR2 SEQ ID NO: 50 LAS C4, C7, C14, C15, C16, C24 SEQ ID NO: 51 KVS D3 V.sub.L CDR3 SEQ ID NO: 52 QHSRELPLT C4, C7, C14, C15, C16 SEQ ID NO: 53 QHSRELPWT C24 SEQ ID NO: 54 FQGSHVPWT D3
TABLE-US-00012 TABLE 12 Identified CDR SEQ ID NOs Sequence In V.sub.H CDR1 SEQ ID NO: 55 GFTFSNYG B4 SEQ ID NO: 56 GINIKDYY B23 V.sub.H CDR2 SEQ ID NO: 57 ISSGGSYT B4 SEQ ID NO: 58 IDPGNGDA B23 V.sub.H CDR3 SEQ ID NO: 59 ARREGGGNPYFDY B4 SEQ ID NO: 60 NEIAY B23 V.sub.L CDR1 SEQ ID NOs: 61 QSLVHSNGNTY B4 SEQ ID NO 62 QSLLDSDGKTY B23 V.sub.L CDR2 SEQ ID NO: 63 KVS B4 SEQ ID NO: 64 LVS B23 V.sub.L CDR3 SEQ ID NO: 65 SQNTLVPWT B4 SEQ ID NO: 66 WQGTRFPFT B23
[0189] The V.sub.H and V.sub.L domain sequences of the antibodies produced in the present invention are summarized in Table 13.
TABLE-US-00013 TABLE 13 Antibody SEQ ID NOs Sequence B4 V.sub.H SEQ ID NO: 67 EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYGMSW VRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDN AKNTLYLQMSSLRSEDTAMYYCARREGGGNPYFDY WGQGTTLTVSS V.sub.L SEQ ID NO: 68 DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYL HWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD FTLKISRVEAEDLGVYFCSQNTLVPWTFGGGTKLEIK B23 V.sub.H SEQ ID NO: 69 EVQLQQSGAELVRPGASVKLSCTASGINIKDYYMHW MKQRPEQDLEWIGWIDPGNGDAEYAPKFQGKATMT ADTSSNTAYLQLSSLTSEDTAVYYCNEIAYWGQGTLV TVSA VL SEQ ID NO: 70 DIVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLN WLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK C4 V.sub.H SEQ ID NO: 71 DVKLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNW IRQFPGNKLEWMGYISYSVGTRYNPSLKSRISITRDTS KNQFFLLLKSVTNEDTATYFCARKGEYGFAYWGQGT LVTVSA V.sub.L SEQ ID NO: 72 DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYM HWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTD FTLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELK C7 V.sub.H SEQ ID NO: 73 QVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNW VRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTIS RDDSQSLLYLQMNNLKTEDTAMYYCVYGRSYGGLS YWGQGTLVTVSA V.sub.L SEQ ID NO: 74 DIVMTQSPASLAVSLGQRATISCRASKSVSSSGYSYMH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDF TLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELK C14 V.sub.H SEQ ID NO: 75 EVKLVESGGGLVQPKGSLKLSCAASGFTFNTNAMNW VRQAPGKGLEWVARIRSKSDNYATYYADSVKDRFTIS RDDSPSMLYLQMNNLKTEDTAMYYCVYGRSYGGLG YWGQGTLVTVSA V.sub.L SEQ ID NO: 76 DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYVH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDF TLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELK C15 V.sub.H SEQ ID NO: 77 EVKLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNW VRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTIS RDDSQSMLYLQMNNLKTEDTAMYYCVRQVTTAVGG FAYWGQGTLVTVSE V.sub.L SEQ ID NO: 78 DIVMTQSPASLAVSLGQRTTISCRASKSVSSSGYSYMH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDF TLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELR C16 V.sub.H SEQ ID NO: 79 EVQLQQSGAELARPGASVQMSCKAFGYTFTNYTMH WVRQRPGQGLEWIGFINPSSDYTNYNQKFKDKATLSA DKSSSTAYMQLSSLTSEDSAVYYCARRIFYNGRTYAA MDYWGQGTSVTVSS .sub.VL SEQ ID NO: 80 DIVMTQSPASLAVSLGQRATISCRASKSVSSSGYSYMH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDF TLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELK C24 V.sub.H SEQ ID NO: 81 EVKLVESGGGLVQPKGSLKLSCAASGYTFNTYAMNW VRQAPGKGLEWVARIRSKSNNYTTYYADSVKDRFTIS RDDSQSMLYLQINNLKTEDTAMYYCVGQILYYYVGS PAWFAYWGQGTLVTVSA V.sub.L SEQ ID NO: 82 DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYM HWYQQKPGQPPKLLIFLASNLESGVPARFSGSGSGTDF TLNIFIPVEEEDAATYYCQHSRELPWTFGGGTKLEIK D2 V.sub.H SEQ ID NO: 83 DVKLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNW IRQFPGNKLEWMGYIRYDGSNNYNPSLKNRISITRDTS KNQFFLKLNSVTTEDTASYYCARDRDSSYYFDYWGQ GTALTVSS V.sub.L SEQ ID NO: 84 DIVMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQ QKPGNAPRLLISDTTSLETGVPSRFSGSGSGKDYTLSIT SLQTEDVATYYCQQYWSAPPTFGGGTKLEIK D3 V.sub.H SEQ ID NO: 85 EVQLEESGGGLVQPGGSLKLSCAASGFTFSNYGMSW VRQTPDKRLELVATINSNGGTTYYPDSVKGRFTISRDN AKNTLYLQMSSLKSEDSAMYYCARDRDAMDYWGQ GTSVTVSS V.sub.L SEQ ID NO: 86 DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSHGNTYL EWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD FTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK D6 V.sub.H SEQ ID NO: 87 EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHW VKQSHGKSLEWIGYIYPYNGDTGYNQKFKSKATLTV DNSSSTAYMELRSLTSEDSAVYYCVRSGDYWGQGTT LTVSS V.sub.L SEQ ID NO: 88 DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSNGKTYL NWLLQRPGQSPSRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDLGVYYCWQGTLFPYTFGGGTKLEIK E6 V.sub.H SEQ ID NO: 89 QIQLAQSGPELKKPGETVKISCKASGYTFTNYGMSWV KQAPGKGLKWMGWINTYTGEPTYAADFKGRFAFSLE TSASTAFLQINNLKNEDTATYFCARGYYDYWGQGTT LTVSS V.sub.L SEQ ID NO: 90 DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYL NWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTD FTLKISRVEAEDLGVYYCWQGMFPRTFGGGTKLEIK
[0190] It is interesting to note that V.sub.L CDR3 sequence is shared by SNP25.sub.197-specific IgGs such as C4, C7, C14, C15, and C16 IgG, whereas their V.sub.H sequences tend to be IgG-specific (Table 11). Sequence alignment analysis revealed that not a single CDR sequence of IgGs, listed in Tables 10-12, overlaps with V.sub.L and V.sub.H CDR sequences of previously reported IgGs (US patent U.S. Pat. No. 8,198,034B2). This may be ascribed to employment of a more stringent screening strategy for positive hybridoma cells in the present research. That is, throughout multiple rounds of screening, only triple positive hybridoma clones were selected by direct ELISA using peptide antigen, sandwich ELISA with TCL, and Western blot analysis with TCL. This stringent screening strategy must have contributed to significantly lower K.sub.D values of IgGs obtained in the present research (see below).
Example 11: Kinetics Analysis of Monoclonal Antibodies
[0191] As detailed in Materials and Methods, the kinetics analysis of monoclonal antibody was exclusively carried out by the BLI assay using FORTÉBIO® Octet Red96 instrument, following the procedure recommended by the manufacturer. Firstly, kinetics analysis was performed with SNAP25.sub.197-specific IgGs using anti-GST biosensors loaded with recombinant GST-SNAP25.sub.197 (125 nM or 250 nM).
TABLE-US-00014 TABLE 14 SPR Kinetic Parameters (SNAP25.sub.197) Monoclonal Abs K.sub.on (M.sup.−1s.sup.−1) K.sub.dis (s.sup.−1) K.sub.D (nM) SNAP25.sub.197 C4 2.30 × 10.sup.5 4.10 × 10.sup.-3 17.8 ± 4.45 specfic C7 2.57 × 10.sup.5 4.94 × 10.sup.-3 19.3 ± 3.15 (dipped in C16 2.35 × 10.sup.5 2.15 × 10.sup.-3 9.17 ± 2.71 125 nM) C24 5.29 × 10.sup.5 5.09 × 10.sup.-3 9.62 ± 1.30 SNAP25.sub.197 C4 2.27 × 10.sup.5 1.48 × 10.sup.-3 6.53 ± 1.09 specific C7 2.77 × 10.sup.5 1.22 × 10.sup.-3 4.41 ± 0.68 (dipped in C16 2.36 × 10.sup.5 1.52 × 10.sup.-3 6.44 ± 1.25 250 nM) C24 4.58 × 10.sup.5 2.41 × 10.sup.-3 5.25 ± 1.09
[0192] Considering the nM range of K.sub.D values, kinetics analysis seemed to normally proceed. However, when kinetics analysis was performed with a biosensor dipped in a lower concentration of GST-SNAP25.sub.197 (125 nM), K.sub.D values proportionally decreased, mainly due to changes in dissociation rate constant Kdis (Table 14). It was also at odd that Kdis values (1.22-5.09×10.sup.−3) were 1 or 2 orders of magnitude lower than previously reported ones (3.11×10.sup.−4-6.74×10.sup.−5) for monoclonal antibodies with a similar antigen specificity (US patent U.S. Pat. No. 8,198,034B2).
[0193] Two plausible causes can be considered for relatively high K.sub.D and Kdis values. The first is the use of inappropriate assay buffer, and the second is the inherent limitation of the entire kinetics assay performed with anti-GST biosensor (FortéBio Application Note 14: Biomolecular Binding Kinetics Assays on the Octet Platform). Comparative use of diverse assay buffers in the kinetics assay showed that 1× kinetics buffer was the most appropriate among all buffers tested (data not shown). Therefore, as an alternative kinetics analysis, anti-mouse IgG Fc capture (AMC) biosensors were directly loaded with antibody and subjected to association/dissociation with serially diluted recombinant GST-SNAP25. In brief, purified IgG was diluted to 100 or 200 nM, whereas GST-SNAP25.sub.FL or SNAP25.sub.197 was serially diluted to 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 nM. After equilibration in 1× kinetics buffer for 1 min, AMC biosensors were loaded with IgGs for 10 min, followed by dipping in 1× kinetics buffer for 10 min. Association and dissociation of analyte were carried out for 10 min each, and K.sub.D was estimated as described above.
[0194] Estimated K.sub.D values using FORTEBIO® Octet analysis software are listed in Table 15.
TABLE-US-00015 TABLE 15 Kinetics Parameters with SNAP25.sub.197 Kinetics Parameters with SNAP25.sub.FL IgGs K.sub.on (M.sup.−1 s.sup.−1) K.sub.dis (s.sup.−1) K.sub.D (pM) K.sub.on (M.sup.−1 s.sup.−1) K.sub.dis (s.sup.−1) K.sub.D (pM) Bi-specific .sup.aA15 7.78 × 10.sup.4 1.22 × 10.sup.−7 1.56 ± 0.01 8.39 × 10.sup.4 1.0 × 10.sup.−7 1.19 ± 0.62 .sup.aB4 1.0 × 10.sup.5 5.56 × 10.sup.−7 0.48 ± 0.02 9.51 × 10.sup.4 1.0 × 10.sup.−7 0.10 ± 0.38 .sup.aB23 2.54 × 10.sup.4 1.0 × 10.sup.−7 3.27 ± 0.03 9.58 × 10.sup.4 1.0 × 10.sup.−7 0.10 ± 0.42 SNAP25.sub.197- .sup.bC4 9.28 × 10.sup.4 1.0 × 10.sup.−7 1.07 ± 0.60 (not determined) specific .sup.bC7 7.78 × 10.sup.4 1.0 × 10.sup.−7 1.28 ± 0.75 .sup.aC14 7.67 × 10.sup.4 1.0 × 10.sup.−7 1.30 ± 0.75 .sup.bC16 1.60 × 10.sup.6 2.57 × 10.sup.−6 1.62 ± 0.46 .sup.aC24 8.68 × 10.sup.4 1.0 × 10.sup.−7 1.15 ± 0.87 .sup.bD3 1.11 × 10.sup.5 1.0 × 10.sup.−7 0.90 ± 0.59 SNAP25.sub.FL- .sup.bD2 (not determined) 1.64 × 10.sup.5 1.0 × 10.sup.−7 0.61 ± 0.28 specific .sup.bD6 3.52 × 10.sup.4 1.0 × 10.sup.−7 2.84 ± 0.02 .sup.bE6 5.38 × 10.sup.4 1.0 × 10.sup.−7 1.86 ± 0.59
[0195] The most noticeable would be Kdis values (i.e. 2.57×10.sup.−6-1.0×10.sup.−7) (Table 15), which is two to three orders of magnitude lower than those obtained with anti-GST biosensor (Table 14). In fact, dissociation rates were too low to be accurately measured using FORTÉBIO® Octet, so the lowest limit of Kdis value, 1.0×10.sup.−7, was tentatively given for six IgGs, as described in Table 15.
[0196] Estimation of K.sub.D values with these tentative dissociation rate constants yielded 0.48-3.27 pM. It should be noted that AMC biosensors loaded with SNAP25.sub.197-specific IgGs did not show statistically significant extent of association of GST-SNAP25.sub.FL up to 1 μM. Similarly, GST-SNAP25.sub.197 did not associate with AMC biosensors loaded with SNAP25.sub.FL-specific IgGs. These binding specificities of IgGs are in a good agreement with their reactivity in ELISA.
Example 12: Antigen Binding Specificity of Monoclonal Antibodies
[0197] Monoclonal antibodies were comparatively examined for their reactivity toward SNAP25.sub.FL and SNAP25.sub.197 employing direct ELISA and Western blot analysis. First, direct ELISA was performed with a purified IgG (50 ng per well) in a microplate coated with GST-SNAP25.sub.FL or GST-SNAP25.sub.197, as described in Materials and Methods. HRP reaction was carried out for 5 min and the extent of HRP activity was determined by measuring A.sub.450 using Bio-Tek SynergyNeo2. A.sub.450 values were obtained after subtracting background A.sub.450, measured without IgG, and the ratio of A.sub.450 with SNAP25.sub.197 to A.sub.450 with SNAP25.sub.FL was calculated and presented as Ratio.sub.197/206 in Table 16. Consistent with the result obtained by the BLI assay, Ratio.sub.197/206 values for bi-specific IgGs such as A15, B4, and B23, were close to 1.0. Ratio.sub.197/206 values for SNAP.sub.197-specific IgGs such as C7, C14, C16, and C24, were over 950, but SNAP25.sub.FL-specific IgGs yielded 0.02˜0.03 of Ratio.sub.197/206.
TABLE-US-00016 TABLE 16 A.sub.450 with A.sub.450 with IgGs SNAP25.sub.FL SNAP25.sub.197 Ratio.sub.197/206 A15 1.383 1.445 1.04 B4 2.163 1.797 0.83 B23 1.901 1.754 0.92 C4 0.001 0.919 919 C7 0.001 1.199 1.037 C14 0.001 0.953 953 C16 0.001 0.996 996 C24 0.001 0.973 973 D3 0.125 1.616 12.93 D2 1.448 0.048 0.03 D6 1.878 0.031 0.02 E6 1.808 0.039 0.02
[0198] Western blot analysis was performed with hybridoma culture supernatants (1:100 dilution) as the source of primary antibody. Aliquots (0.5 μg) of GST-SNAP25.sub.FL and GST-SNAP25.sub.197, resolved on a denaturing gel and subsequently transferred to PVDF membrane, were tested as antigen. Monoclonal antibodies reacted with denatured SNAP25 antigen with the same specificity as in the ELISA assay (
Example 13: Conjugation of Horseradish Peroxidase (HRP) to Antibodies
[0199] Among monoclonal antibodies tested, C16 IgG exhibited the most reproducible retention of antigenic affinity and specificity after being conjugated with HRP (data not shown). In a typical HRP conjugation reaction, C16 IgG (5 mg) was incubated with activated HRP (5 mg) for 2 hr at RT with light protection, as described in Materials and Methods. After the addition of 0.1 ml of sodium borohydride (4 mg/ml), the HRP-antibody conjugate was dialyzed against 1×PBS and 1×PBS/50% glycerol at 4° C. As shown in
[0200] Two different C16 IgG-HRP conjugates were examined for the reactivity toward SNAP25.sub.197 in direct ELISA. A 96-well microplate was coated with 0-200 pg of GST-SNAP25.sub.197, mimicking approximately up to ˜0.8% cleavage of endogenous SNAP25 in cells grown in a microplate well. Aliquots (200 ng) of C16 IgG-HRP conjugates were capable of detecting as low as 50 pg of GST-SNAP25.sub.197 when measured for 30 min with 50 μl of 1-Step™ Ultra TMB-ELISA (
Example 14: Conjugation of Biotin to Antibodies
[0201] Sandwich ELISA developed by Allergan utilizes two antibodies: a SNAP25.sub.197-specific IgG as capture antibody and polyclonal SNAP25-specific IgG as detection antibody. By contrast, sandwich ELISA invented by the present research utilizes two monoclonal antibodies as capture either detection antibody, which makes its quality control more feasible and easier. This novel sandwich ELISA exhibits high levels of repeatability, reproducibility and accuracy. Yet the addition of a second detection antibody such as IgG conjugated with alkaline phosphatase or biotin could further improve the accuracy of sandwich ELISA through normalization of SNAP25 captured in each well.
[0202] As the first attempt, a bi-specific monoclonal antibody A15 was conjugated with biotin, as described in Materials and Methods. As shown in
[0203] The reactivity of A15 IgG conjugated with 0.1 mM biotin was tested in sandwich ELISA. In brief, TCLs (60 μl) were prepared from N2-42F cells treated with the indicated concentration of BoNT/A. A microplate coated with B4 IgG was incubated with 50 μl aliquots of TCL, and. endogenous SNAP25.sub.197 captured by B4 IgG was detected by incubation with A15-biotin conjugate and streptavidin-AP conjugate (1:500 dilution in 1×TBS). As a bi-specific monoclonal antibody, A15 IgG has been characterized to bind both SNAP25.sub.FL and SNAP25.sub.197 with comparable affinity and specificity (see
Example 15: Direct Cross-Linking of Alkaline Phosphatase (AP) to Antibodies
[0204] As an alternative approach to the second detection antibody, purified IgGs were directly cross-linked to AP using glutaraldehyde as the extent of cross-linking can be regulated by glutaraldehyde concentration. Two polyclonal and three monoclonal antibodies were cross-linked to AP using glutaraldehyde. Except C16, they were all bi-specific antibodies reacting with both SNAP25.sub.FL and SNAP25.sub.197, a key property as the second detection antibody for normalization. C16 IgG was used as control since HPR conjugation was efficient, yet not affecting the antigen specificity of C16 (
TABLE-US-00017 TABLE 17 Ratio of Incubation Antibody IgG to AP time/temperature Analysis Cl 1:3 2 hr/4° C. + 2 hr/RT SDS-PAGE C16 1:1~2:1 3-24 hr/RT or 37° C. A15 1:1~2:1 5 hr/37° C. SDS-PAGE rA15 2:1 2 hr/4° C. hr/RT & direct ELISA Sigma IgG 2:1 1-4 hr/4° C. or RT
[0205] Upon completion of glutaraldehyde cross-linking, the resulting AP-IgG conjugates were analyzed by SDS-PAGE. A representative result showing the electrophoretic resolution of AP-IgG conjugates and the subsequent visualization by Coomassie blue staining is shown in
[0206] Following the confirmation of the cross-linking by SDS-PAGE analysis, AP-rA15 IgG and AP-Sigma IgG conjugates (100 ng per well) were comparatively examined for the antigen reactivity and specificity in direct ELISA using a microplate coated with 2 ng of GST-SNAP25 mixtures containing varying extents of GST-SNAP25.sub.197. AP-Sigma IgG conjugates did not yield any ELISA signal, whereas AP-rA15 IgG conjugates led to a result similar to that with biotinylated A15 IgG (compare
[0207] The results in
Example 16: Optimization of Culture of N2-42F, and Treatment with BoNT/A
[0208] Having acquired key reagents such as neuronal cells highly sensitive to BoNT/A and monoclonal antibodies specific for SNAP25, a series of experiments were carried out to optimize all steps in the cell-based potency assay, including the intoxication medium, sensitizer, BoNT/A treatment time, and capture/detection antibody pairing.
Example 16-1. Optimization of BoNT/A Intoxication Time
[0209] First, the BoNT/A processing time (toxinization time) was optimized.
[0210] Protocol A (
[0211] As described above, towards establishment of the optimal intoxication time for N2-42F cells, Protocol A was modified by prolonging the cell culture time in either 1× intoxication medium devoid of GT1b or in 1× intoxication medium containing BoNT/A. These changes did not improve the extent of SNAP25 cleavage and moreover, N2-42F cells grown in the 1× intoxication medium for more than 4 days looked very unhealthy under the microscope (data not shown). Based on this observation, a novel Protocol B was established, where the culture time in 1× intoxication medium supplemented with both GT1b and BoNT/A was shortened (
Example 16-2. Optimization of Culture Medium
[0212] The osmolarity and temperature influenced the BoNT/A sensitivity of BOCELL™ (US patent U.S. Pat. No. 9,526,345B2). Also, the BoNT/A sensitivity of NG108-15 cells was significantly improved by optimizing neural differentiation medium (J Biomol Screen. 2016 January; 21(1):65-73). Since the BoNT/A sensitivity reflects the extent of BoNT/A uptake through two independent receptors on cell surface, a polysialoganglioside (PSG) receptor and a protein receptor (SV2) (J Neurochem. 2009 June; 109(6):1584-95), the enhanced BoNT/A sensitivity by optimization of culture medium or higher temperature has to do with the facilitated cellular intake of BoNT/A. To this end, N2-42F cells was tested for the BoNT/A sensitivity in three different culture media: RPMI1640, NEUROBASAL™, and MEM, all supplemented with 1×N2, 1×B27, and 1×GT1b. While culturing in the indicated medium, N2-42F cells were treated with varying concentrations (0.93˜25 pM) of BoNT/A cells according to Protocol B. When measured by Western analysis, the BoNT/A sensitivity of N2-42F cells was measured the highest with RPMI1640 (
[0213] Under the condition described in
Example 16-3. Identification and Optimization of Sensitizers
[0214] Taking advantage of having fully characterized monoclonal antibodies specific for SNAP25 (see below), the BoNT/A sensitivity of N2-42F cells in the 1× intoxication medium containing varying concentrations of BoNT/A (0.03-5.5 pM) were examined by sandwich ELISA assay following Protocol B, while testing if the BoNT/A sensitivity is affected by arginine (see above) or any compounds with demonstrated effects on neural survival or differentiation, including ATP (Trends Neurosci. 2000 December; 23(12):625-33), creatine (J Neurochem. 2005 October; 95(1):33-45), and lipoic acid (J Neurosci Res. 2014 January; 92(1):86-94). As shown in
[0215] In an optimization study for the toxinized medium, it was believed that the BoNT/A sensitivity of N2-24F cells was affected by factors other than osmotic pressure. Because the sensitivity was higher in RPMI1640 medium than in Neurobasal™ or MEM medium. For arginine in the medium composition, it contained 1.15 mM in RPMI1640, 0.6 mM in MEM, and 0.4 mM in Neurobasal™. Since arginine is a precursor amino acid of creatine, arginine alone was further examined for its effects on the BoNT/A sensitivity using 2× intoxication medium containing 2-10 mM arginine. As shown in
[0216] In 2002, Schengrund and his coworkers provided experimental evidence for the first time that in neuro-2a cells, an efficient SNAP25 cleavage by BoNT/A requires higher than 25 μg/ml GT1b in DMEM (J Biol Chem. 2002 Sep. 6; 277(36):32815-9). Since N2-42F cells are derived from neuro-2a, the requirement of GT1b for BoNT/A activity was examined using the 1× intoxication medium containing 25-75 μg/ml GT1b (1˜3×GT1b) by Western blot analysis. Without GT1b supplementation, 8.3 pM BoNT/A led to about 18% of SNAP25 cleavage (
[0217] Towards optimization of GT1b concentration, the BoNT/A sensitivity of N2-42F cells were tested in 2× intoxication medium containing 25 pM BoNT/A and increasing concentrations of GT1b from 1× to 5×. When measured by Western blot analysis, the SNAP25 cleavage was significantly enhanced by the addition of 1× or 2×GT1b, but with more than 2×GT1b, the SNAP25 cleavage only marginally increased from 63%, 65%, 68%, to 70% (
[0218] The effect of N2(N2 supplement, Thermo Fisher Scientific 17502048)/B27(B27™ Serum free supplement, Thermo Fisher Scientific 17504-044) in neuron cultures on BoNT/A sensitivity was tested. B27, containing all trans-retinol (0.1 mg/L), is known to support motor neuron differentiation of neural progenitor cell (J Cell Biochem. 2008 Oct. 15; 105(3):633-40). N2 contains a subset of B27 components, including insulin and is known to promote (1) differentiation of human embryonic stem cells and (2) proliferation/survival of neural progenitor cell (J Cell Biochem. 2008 Oct. 15; 105(3):633-40). The BoNT/A sensitivity of N2-42F cells in RPMI1640 supplemented with 1×GT1b, 8.3 pM BoNT/A and the indicated concentration of N2/B27 following Protocol B. The SNAP25 cleavage was quantitatively analyzed by Western blot. As shown in
[0219] Using RPMI1640 supplemented with 3×GT1b and 5 mM arginine, the effects of N2/B27 on the BoNT/A activity were further examined. The extent of SNAP25 cleavage in N2-42F cells treated with 8.3 pM BoNT/A was enhanced from 13% to 56%, 69% by 1× and 2×N2/B27 in spite of noticeable increase in overall intracellular level of SNAP25 (
Example 17: Optimization of Sandwich ELISA
Example 17-1. Optimization of Buffers
[0220] Having optimized the culture medium and media supplements for N2-42F cell intoxication, next, sandwich ELISA was systematically evaluated for its performance under diverse conditions summarized in Table 18. And selected optimized conditions summarized in Table 19.
TABLE-US-00018 TABLE 18 Parameters Conditions tested Optimal condition Plate coating 3 matrices Poly-D-lysine matrix Cell density 2 densities 5.5 × 10.sup.4 cells per well Intoxication 3 culture media/ RPMI1640, 2 mM medium/ 6 sensitizers L-alanyl-L-glutamine, sensitizer 0.1 mM NEAA, 2x N2, 2x B27, GT1b (50 μg/ ,) 5 mM arginine Intoxication time 3 time points 96 hr BoNT/A dose 0.03 pM-1 nM 0.03-8.33 pM Capture/ 11 combinations B4 (300 ng/50
) + C16-HRP detection antibodies (200 ng/50
) Lysis buffers 11 buffers 20 mM Hepes-NaOH, pH 7.5, 0.2 M NaCl, 1% TRITON X-100, 1 mM EGTA, 5 mM EDTA Lysate incubation 3 temperatures/ 4 hr at 4° C. 3 time points Blocking buffers 46 buffers 1% polyvinyl alcohol (Mw 145,000), 3% skim milk in 1x PBS Incubation of 4 temperatures/ 1 hr at RT detection antibody 4 time points
TABLE-US-00019 TABLE 19 Parameters Condition tested Optimal Lysis buffers 50 mM Tris-HCl, pH 8.0,150 mM NaC1, 1% TRITON 20 mM Hepes- X-100, 2 mM EGTA, 0.01-0.1% SDS, ± 5 mM EDTA NaOH, pH 7.5, 20 or 50 mM Hepes-NaOH, pH 7.5, 0-0.4 M NaCl, 1 or 150 mM NaCl, 2% TRITON X-100, 0-0.1% SDS, 1% TRITON X- 0-1.5 mM MgCl.sub.2, 0-5 mM EDTA, 1-2 mM EGTA 100, 5 mM EDTA, 50 mM Tris-HCl, pH 8.0, 150 mMNaCl, 0.1% SDS, 2 mM EGTA 0.5% sodium deoxycholate, 1% NP-40 Coating buffers 0.5% APTES 0.1 M carbonate 0.1 M sodium acetate buffer, pH 6.0 buffer, pH 9.6 0.1 M sodium phosphate buffer, pH 7.2 0.1 M sodium citrate buffer, pH 2.8 0.1 M carbonate buffer, pH 9.6 Washing buffers 1x PBS/0.05% TWEEN-20 (1x PBST), ± 0.2 M NaCl 1x PBST Blocking 0.2-3% Ac-BSA, 3-5% skim in 1x PBS 3% skim, buffers 1% goat serum in 1x PBS 1% PVA 2-3% skim, 0.05-1% PVA (Mw 47,000 or 145,000) (Mw 145,000) in 1x PBS in 1x PBS 5% skim or 0.1-0.5% Ac-BSA, with 10 or 100% SUPERBLOCK ™ (PBS).sup.a 2-5% ECL.sup.b or ECL PRIME ™ Blocking agent.sup.c ± 0-5% skim or 0.2% Ac-BSA Western BLoT blocking buffer (protein-free).sup.d .sup.a SUPERBLOCK ™ (PBS) (Thermo Fisher Scientific 37518) .sup.b ECL PRIME ™ blocking agent (GE Healthcare UK limited RPN418V) .sup.c ECL (GE Healthcare UK limited RPN2125V) .sup.d Western BLoT blocking buffer (protein free) (Takara T7132A)
Example 17-2. Optimization of Capture Antibody
[0221] Secondly, three bi-specific antibodies, A15, B4, and B23 were compared for their function as capture antibody in sandwich ELISA. TCLs were prepared from N2-42F cells treated with varying concentrations (0-25 pM) of BoNT/A in 1× intoxication medium. TCLs were added to a microplate coated with the indicated antibody (400 ng per well). After incubation for 4 hr at 4° C., the amount of SNAP25.sub.197 among total SNAP25 captured by the indicated antibody was detected and quantified using C16 IgG-HRP conjugates following the procedure described in Materials and Methods. One exception was that the HRP reaction was carried out for 30 min until all test groups yielded positive ELISA signal. As shown
[0222] Optimal B4 IgG quantity was explored with TCLs prepared from N2-42F cells incubated in 2× intoxication medium containing the indicated concentration of BoNT/A following Protocol B. Sandwich ELISA was performed with a microplate coated with increasing amounts (100˜400 ng per well) of B4 IgG. After incubation for 4 hr at 4° C., SNAP25.sub.197 captured was detected and quantified using C16 IgG-HRP conjugates as described above. It should be noted that the HRP reaction was carried out for 15 min. As shown
Example 17-3. Optimization of Incubation Conditions for TCL and Detection Antibody
[0223] TCLs, prepared from SiMa cells treated with 8.33 or 25 pM BoNT/A in 1× intoxication medium, were incubated in a microplate under the indicated condition. Subsequently, SNAP25.sub.197 captured was detected and quantified using C16 IgG-HRP conjugates. As shown in
[0224] The optimal incubation condition for detection antibody was explored by direct ELISA. In brief, the microplates were coated with varying amounts (from 10 pg to 1 ng) of recombinant GST-SNAP25.sub.197 that represent the endogenous SNAP25 cleavage from 0.05% to 50% in the standard CBPA. After adding C16 IgG-HRP conjugates (200 ng per well), the microplates were incubated under the indicated condition. Of conditions test, incubation of Cg16 IgG-HRP conjugates at RT for 1 hr yielded the highest ELISA signal for all ranges of GST-SNAP25.sub.197 examined but other conditions also generated acceptable levels of ELISA signal. When considering the subsequent HRP reaction that is carried out at RT, however, in the optimized sandwich ELISA, C16-HRP conjugates are incubated for 1 hr at RT.
Example 17-4. Optimization of Detection Method for C16 IgG-HRP Conjugates
[0225] HRP activity was generally measured using a conventional TMB substrate (1-STEP™ Ultra TMB-ELISA). One drawback in using TMB substrate is EC50 value tends to be influenced by HRP reaction time (
Example 18: Validation and Practical Application of CBPA
Example 18-1. Optimized Timeline of CBPA
[0226] Taken all optimized conditions and reagents together, the finally established optimal CBPA can be performed with only three working days, as depicted in
Example 18-2. Accuracy and Linearity of CBPA
[0227] Following the standard Protocol B, a total of 18 CBPA were performed by three operators.
[0228] A total of 18 CBPA were performed by three operators following Protocol B. N2-42F cells were treated with varying concentrations (0, 0.03, 0.1, 0.2, 0.31, 0.62, 0.93, 2.78, 5.55, 8.33 pM) of BoNT/A in 2× intoxication medium (
[0229] The first series of CBPA yielded 1.39 pM of EC50 with 1.5 fM (3.4 mU relative potency/ml) of detection limit (DL) and 4.6 fM of quantitation limit (QL) (Clin Biochem Rev. 2008 August; 29 Suppl 1:S49-52; Anal Sci. 2007 February; 23(2):215-8). Since the 1.39 pM BoNT/A is equivalent to ˜0.3 U relative potency per assay, the first CBPA is sensitive enough to measure the bio-potency of BoNT/A in place of mouse LD50 assay. A slightly lower EC50 value, 1.24 pM, was obtained by a prolonged HRP reaction employed in the second series of CBPA (
[0230] This result indicates that the quantitation power and detection limit of CBPA, developed in the present research, can be adjusted by modulating HRP reaction time or changing GT1b concentration in the intoxication medium. Linear regression analysis of ELISA signals for N2-42F cells treated with 0.03˜0.93 pM BoNT/A revealed an excellent linear correlation between the relative potency of BoNT/A and ELISA signal in all three series of CBPA (
Example 18-3. Measurement of the Bio-Potency in BOTULAX® Samples
[0231] BoNT/A potency in BOTULAX® was determined by mouse LD50. To measure this bio-potency using CBPA, two different lots (HUB 18009 and HUB 18011) of BOTULAX® (200 U per vial) were dissolved in either intoxication medium (matrix a), deionized H.sub.2O (matrix b), or 5 mM arginine, pH 6.0 (matrix c). After 10 min incubation at RT, BOTULAX® dissolved in medium was serially diluted to 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, and 50 U/ml, but the others were to 0.23, 0.48, 0.70, 0.98, 1.41, 2.11, 4.20, 6.32, and 12.6 U/ml in the optimized intoxication medium. Three different sets of CBPA were carried out using these samples following the standard Protocol B. The EC50 values was determined. As shown in
[0232] Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Accession Number
[0233] Depository authority: Korea Research Institute of Bioscience and Biotechnology; [0234] Accession number: KCTC13712BP; [0235] Deposit date: Nov. 13, 2018.
[0236] N2-42F cell line (accession number: KCTC 13712BP) was deposited with Korea Research Institute of Bioscience and Biotechnology, on Nov. 13, 2018. The subject cell line has been deposited under conditions that assure that access to the cell line will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposit will be 10 available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
[0237] Further, the subject deposits will be stored and made available to the public in accord with 15 the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit 20 should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.