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Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA
Journal of Immunological Methods 336 (2008) 1–8
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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA Larry H. Stanker ⁎, Paul Merrill, Miles C. Scotcher, Luisa W. Cheng United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, United States
a r t i c l e
i n f o
Article history: Received 16 November 2007 Received in revised form 3 March 2008 Accepted 4 March 2008 Available online 9 April 2008 Keywords: Botulinum neurotoxin Monoclonal antibodies Immunoassays Food safety
a b s t r a c t Botulinum neurotoxins (BoNT), produced by the anaerobic bacterium Clostridium botulinum, cause severe neuroparalytic disease and are considered the most toxic biological agents known. While botulism is rare in the U.S. it often is fatal if not treated quickly, and recovery is long, requiring intensive treatment. BoNT is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disulfide bond. The ‘gold standard’ for BoNT detection relies on a mouse bioassay. This is a time consuming (up to 4 days) assay and it lacks specificity, however, it gives a sensitivity (mouse LD50) of approximately 10 pg mL− 1. Most BoNT immunoassays are much less sensitive. In this study we describe the development of four high-affinity (dissociation constants (Kd's) in the low pM range) monoclonal antibodies (mAbs) that specifically bind BoNT serotype A (BoNT/A). These antibodies, designated F1-2, F1-5, F1-40, and F2-43 are IgG1 subclass mAbs with kappa light chains and they specifically bind BoNT serotype A. Western blot analyses following SDSPAGE demonstrate that mAbs F1-2 and F1-5 bind the 100 kDa heavy chain subunit and that mAb F1-40 binds the 50 kDa light chain. The fourth antibody demonstrated strong binding to the 150 kDa holotoxin in the ELISA and on Western blots following electrophoresis on native gels. However binding in Western blot studies was not observed for mAb F2-43 following SDSPAGE. A highly sensitive sandwich ELISA, capable of detecting as little as 2 pg/mL BoNT/A was developed using mAbs F1-2 and F1-40. Such an assay represents a realistic, high sensitivity alternative to the mouse bioassay. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Clostridium botulinum, an anaerobic spore-forming bacterium, produces a family of botulinum neurotoxins (BoNT, EC 3.4.24.69) consisting of seven serotypes, A–G (BoNT/A–BoNT/G) (Gill, 1982). Serotype A is synthesized as a single 1296 amino acid, 150 kDa polypeptide which is cleaved endogenously or exogenously forming a dichain molecule comprised of an ∼100 kDa heavy chain (HC) and an ∼50 kDa light chain (LC)
⁎ Corresponding author. U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, United States. Tel.: +1 510 559 5984; fax +1 510 559 6429. E-mail address: [email protected] (L.H. Stanker). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.03.003
linked by a single disulfide bond, the holotoxin, (Montecucco and Schiavo, 1995). The HC mediates toxin entry into neurons, and the LC functions as a zinc-dependent endoprotease cleaving SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) involved in acetylcholine release resulting in muscular paralysis (Turton et al., 2002). The crystal structure of BoNT/A was determined at a resolution of 3.3 Å (Lacy et al., 1998). An LD50 for BoNT/A of 0.5 ng kg− 1 in mice was reported by Wu et al. (2001). Because of its high toxic potency and ease of production, BoNT is classified by the Centers for Disease Control (CDC) as a Class A bioterrorism agent (Arnon et al., 2001). Currently, BoNT detection in food relies on use of the mouse bioassay, a highly sensitive method with detection limits of 7–20 pg mL− 1. However, the mouse bioassay is time
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consuming (up to 4 days), requires the use of large numbers of animals and often relies on death as an endpoint. Furthermore, a subsequent neutralization bioassay is required in order to confirm toxin presence and identify toxin serotype. An alternative method that is rapid, sensitive, and toxin serotype-specific is highly desirable. In an effort to meet this challenge, a number of immunoassays have been developed, the most common being the enzyme-linked immunosorbent assay (ELISA) (Doellgast et al., 1993; Ferreira et al., 2003; Gessler et al., 2007; Gibson et al., 1988, 1987; Poli et al., 2001; Sharma et al., 2006; Szilagyi et al., 2000; Wictome et al., 1999). For the most part, these assays do not detect toxin at the lowest levels measured in the mouse bioassay. Doellgast et al. (1994a,b) reported an enzyme-linked coagulation assay (ELCA) with sensitivity comparable to the mouse bioassay. However, this assay is complicated, and its high sensitivity is achieved through a novel amplification system that utilizes snake venom coagulation factor. Greater sensitivity was reported by Chao et al. (2004) who used an immunopolymerase chain reaction (IPCR) based assay to detect as little as 50 fg. This IPCR approach also suffers from its complexity and susceptibility to sample matrix effects on assay performance. Simple immunoassay formats, including lateral flow assays have been developed (Gessler et al., 2007) but these again fail to achieve the sensitivity of the mouse bioassay. Recently, high-affinity IgG constructs were generated from single chain Fv (scFv) antibodies using molecular evolution and a yeast display system (Razai et al., 2005). These recombinant antibodies formed the basis of a sensitive ELISA microarray immunoassay and a micro-column sensor capable of detecting BoNT down to 14 fM (1.4 pg mL− 1) (Varnum et al., 2006). These results demonstrated that simple immunoassay formats can be highly sensitive when highaffinity antibodies are incorporated. In this study we pursued the development of high-affinity mouse mAbs to BoNT/A, and demonstrate that they can be used in a simple sandwich ELISA, with luminescence detection, to quantify BoNT/A below the mouse LD50 level.
used in spiking experiments were purchased from a local food store. Luminesence was measured using a Perkin-Elmer Victor-2 microplate reader. Data were exported to Microsoft Excel for further analysis. 2.1.1. Cell culture media Hybridoma medium (HM) consisted of Iscove's modified Dulbecco's Minimal Medium (Sigma #I-7633) containing NaHCO3 (36 mM), and glutamine (2 mM). All hybridoma cells and SP2/0 mouse myeloma cells were maintained in HM supplemented with 10% fetal calf serum (cHM). Hybridomas were selected following cell fusion using HAT selection medium prepared by adding hypoxanthine (5 μM), aminopterin (0.2 μM), and thymidine (0.8 μM) to cHM. Macrophage conditioned medium (MPCM) was prepared as described (Sugasawara et al., 1985). A mixture of 40% cHM and MPCM was used for all cell-cloning procedures. 2.2. Monoclonal antibody production All protocols were approved by the Institutional Animal Care and Use Committee. Female Balb/cJ mice (Simonsen Laboratories, Gilroy, CA) were immunized at 2-week intervals by intraperitoneal injection (IP) of 100 μL of BoNT/A toxoid (approximately 10 μg mL− 1) prepared in RIBI adjuvant as suggested by the manufacturer. Following the third injection, serum was obtained and evaluated for anti-BoNT/A antibodies. After 2 weeks, mice with a strong antibody titer were injected IP with 1 μg of BoNT/A toxoid in PBS. 2.2.1. Fusion procedure Three days following the last IP injection mice were euthanized and their splenocytes were fused with SP2/0 myeloma cells using polyethylene glycol as previously described (Bigbee et al., 1983). Following cell fusion, the cells were suspended in 100 mL of HAT selection medium supplemented with 10% fetal calf serum and 10% MPCM, dispensed into ten, 96-well tissue culture plates, and incubated for 10 to 14 days at 37 °C in 5% CO2 before screening for antibody production.
2. Materials and methods 2.1. Reagents and equipment Solutions at 1 mg mL− 1 of botulinum neurotoxin serotypes A–G and BoNT/A toxoid were purchased from Metabiologics Inc. (Madison, WI). Lyophilized samples of BoNT/A toxin and toxoid (10 μg vial− 1) were purchased from List Biological Laboratories, Campbell, CA. Bovine serum albumin (BSA), ovalbumin (OVA), goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (IgG-peroxidase) #A-4416, polyoxyethylene sorbitan monolaurate (Tween-20), RIBI adjuvant, Protein-G conjugated Sepharose #P-32196, and the following buffers: 0.01 M phosphate buffered saline (PBS) #P-3813, 0.138 M NaCl, 0.0027 M KCl, pH 7.4; and 0.02 M TRIS–buffered saline (TBS) #T-5912, 0.9% NaCl, pH 7.4 were purchased from Sigma Chemical Co. (St. Louis, MO). Black, Maxisorp 96-well Nunc microtiter plates were obtained from PGC Scientific (Gaithersburg, MD), and SuperSignal Femto Max Sensitivity substrate was purchased from Pierce Inc. (Rockford, IL). Non-fat dry milk (NFDM) and milk samples
2.2.2. Screening methods Sera from immunized mice and supernatants from cell fusion plates were screened using a direct-binding ELISA. Microtiter plates were rinsed with distilled water and coated by incubating 100 μL/well of a 0.2 μg mL− 1 or 0.6 μg mL− 1 solution of BoNT/A or 900 kDa BoNT/A complex, respectively, in 0.05 M sodium carbonate buffer, pH 9.6 overnight at 4 °C. The toxin solution was aspirated and non-coated sites blocked by adding 300 μL well− 1 of 3% non-fat dry milk in Tris buffered saline containing 0.05% Tween-20 (NFDM-TBST) and the plates were incubated for 1 h at 37 °C. The plates were then washed 3 times with 0.05% Tween-20. Next, sera or cell culture supernatants were added (100 μL well− 1) and the plates were incubated at 37 °C for 1 h. Plates were again washed 3× and 100 μL well− 1 of a 1/5000 dilution of peroxidase-conjugated goat anti-mouse sera (Sigma, St. Louis, Mo) was added and the plates incubated for 1 h at 37 °C. The plates were then washed 6× with 0.05% Tween-20. Freshly prepared luminescence substrate was added (100 μL/ well) according to the manufacturer's recommendation. The
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plates were incubated for 3 min at room temperature and luminescent counts recorded. Cells from the wells giving positive signals for antibody production were cloned by limiting dilution. Hybridomas were then expanded and small amounts (usually less than 10 mL) of ascites fluids obtained (Covance Research Products, Inc., Denver, PA). Antibody was purified by affinity chromatography on Protein-G Sepharose. Bound antibody was eluted with 0.1 M glycine–HCl, pH 2.7. Protein concentrations were determined with a BCA-kit (Pierce) using the microplate method suggested by the manufacturer. Antibodies were conjugated with biotin using EZ-Link Sulfo-NHS-LC-Biotin (Pierce) as described by the manufacturer using a 50-fold molar excess of biotin reagent. Antibody isotype was determined by ELISA using toxin-coated microtiter plates and horseradish peroxidase-conjugated, isotype-specific antibodies (SouthernBiotech, Birmingham, AL).
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(Pierce, #34075) was added as recommended by the manufacturer. Membranes were placed between vinyl sheets and bands were visualized using BioMax XAR film (Eastman Kodak Co, Rochester, NY) or using a FluoroChem Sp gel imaging system (Alpha Innotech Corp., San Leandro, CA). Native gel electrophoresis was performed as follows. Samples of 200 ng of BoNT/A were incubated for 10 min at 55 °C in native sample buffer (supplemented with Dithiothreitol (DTT) at a final concentration of 100 mM) prior to loading onto a 10% native gel. NativeMark protein standards (Invitrogen) were diluted 1/20 in native sample buffer, and loaded directly onto the gel without heating. Electrophoresis was carried out at 200 V for 80 min. For Western blotting, the gel was subjected to horizontal blotting onto a nitrocellulose membrane in native transfer buffer (0.025 M Tris, 0.192 M glycine, pH 8.6) at 180 mA for 100 min. The blot was briefly washed in TBS-T (20 mM Tris, 0.9% NaCl, 0.05% Tween-20, pH 7.4) and transferred to 20 mL blocking buffer, 3% NFDM-TBS-T and incubated at room temperature for 1 h. The membranes were then incubated in primary antibody using 10 mL blocking buffer containing the appropriate mAb at 5 μg mL− 1 (F1-2, F-5, F1-40) or 10μg − 1 mL (F2-43) at 4 °C overnight. The blot was washed 4× in TBS-T, for 4 min. each at room temperature. The membranes were then processed with goat anti-mouse peroxidase-conjugated IgG (1/5000) for 1 h at room temperature prior to chemiluminescense detection using SuperSignal West Dura Extended Duration Substrate according to the manufacturer's protocol (Pierce Biotechnology Inc.). Blots were visualized using a Fluorchem SP unit as described above.
2.2.3. Capture ELISA Microtiter wells were coated with capture antibody by adding 100 μL well− 1 of a 1 μg mL− 1 solution in PBS pH 7.4 of mAb F1-2 to each well and incubating overnight at 4 °C. Plates were then blocked by addition of 300 μL of 3% NFDM-TBST and incubating the plates for 1 h at 37 °C. Finally, plates were washed 3× with 0.05% Tween-20 and stored for up to 14 days at 4 °C before use. Toxin standards and samples were then added (100 μL well− 1 in 5% NFDM-TBST buffer) and the plates incubated for 1 h at 37 °C. The plates were then washed 6× in 5% Tween-20, 100 μL of a 3 μg mL− 1 solution of biotinconjugated mAb F1-40 was added to each well, and the plates were incubated at 37 °C for 1 h. Next, the plates were washed 9× with 0.05% Tween-20 and 100 μL well− 1 of a 1/20,000 dilution of streptavidin-HRP (Zymed, S. San Francisco, CA) in 3% NFDM-TBST was added and the plate incubated for 1 h at 37 ° C. Finally the plates were washed 12× with 0.05% Tween20 and luminescence substrate was added.
The binding dissociation constant (Kd) was measured by ELISA using the method described by Friguet et al. (1985). Data analysis and calculation of Kd used the following relationships described by Bobrovnik (2003).
2.3. Electrophoresis
ðRLUo RLUi Þ=RLUi ¼ KaLi
ð1Þ
All gel electrophoresis equipment, buffers, gels and PVDF membranes were purchased from PAGEgel (San Diego, CA). Direct visualization of proteins following SDS-PAGE used silver staining (Silver Express, Invitrogen Inc., Grand Island, NY) as described by the manufacturer. Toxins were separated by SDS-PAGE using 4–20% gels as recommended by the manufacturer. Briefly, samples (heated at 70 °C for 10 min) were suspended in electrophoresis sample buffer and 10 μL containing 100 ng of protein were loaded into each well and separated by electrophoresis at 200 V (constant). Following electrophoresis, proteins were electrophoretically transferred to PVDF membranes using a PAGEgel transfer cell, (180 mA constant for 90–120 min). Membranes were blocked with 3% NFDM-PBS–0.05% Tween-20 buffer (PBST) for 1 h at 22 °C on a rocking platform. The membranes were then washed 3×, 5 min each with rocking in PBST, primary anti-toxin antibody was added (1 μg mL− 1), and the membranes were incubated overnight at 4 °C. The membranes were again washed 3× in PBST. Peroxidase conjugated goat anti-mouse IgG (whole molecule) antiserum (Sigma, #A4416) diluted 1/5000 in PBST was added and the blots incubated with rocking at 22 °C for 1 h. The membranes were again washed (3×, 5 min), substrate
Ka ¼ 1=Kd
ð2Þ
2.4. Antibody affinity measurements
where RLUo equals the activity measured when no toxin was present; RLUi, the activity measured when different concentrations of toxin (Li) ranging between 4.2–6600 ×10− 12 M was added to a constant concentration of antibody. The reaction mixtures were incubated up to 48 h and the percentage of unbound
Table 1 ELISA comparison of antibody activity in immunized mouse serum using a luminescent versus a colorimetric substrate Antisera dilution factor 500 1000 10,000 100,000 500,000 1,000,000 2,000,000 Blank
Luminescent (counts)
Signal: noise ratio
Colorimetric (absorbency)
Signal: noise ratio
5,557,000 5,078,000 2,228,934 477,000 98,000 55,352 28,426 1750
3175 2901 1274 273 56 32 16
1.60 1.40 0.21 0.09 0.08 0.07 0.07 0.06
22.6 23.3 35 1.5 1.3 1.2 1.2
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Fig. 1. Determination of BoNT serotype binding specificity of mAb F1-2, (horizontal strip) F1-5 (solid), F1-40, (stippled), and F2-43 (diagonal strip) by ELISA. Antibody binding to microtiter wells coated with a 0.2 μgmL− 1 of the BoNT serotypes A–G was measured. The data shown represent the average of three experiments, error bars represent + one standard deviation (SD).
antibody determined by ELISA. The value of Ka was determined graphically as the slope of the linear relation depicted in Eq. (1).
initial antisera dilutions using the colorimetric ELISA. Since significantly lower antibody concentrations would be expected in the hybridoma supernatants being tested in the initial screening, detection with a high level of confidence required the more sensitive assay. Hybridoma cells 10–14 days following cell fusion were screened for antibodies that bound BoNT/A toxoid. In general, N95% of the culture wells contained between 1 and 6 hybridoma “colonies”, equivalent to N10,000 total hybridoma colonies in 2000 culture wells. Positive signals (signal-tonoise ratios of 5 or greater) were observed for approximately 130 supernatants. The cells from these wells were expanded, tested, and cloned by limiting dilution to produce hybridoma lines. Of these hybridomas, only four antibodies bound toxin and toxoid, the rest bound only the formalin fixed toxoid but not active 150 kDa holotoxin. Future studies will concentrate on using active toxin as the immunogen. The toxin-binding antibodies, referred to as F1-2, F1-5, F1-40, and F2-43, were further characterized. Isotype analysis indicated that these four mAbs were IgG1 antibodies, with kappa light chains. Antibodies were then produced in mouse ascites fluids and purified by affinity chromatography using Protein-G coated Sepharose. 3.1. Binding specificity
3. Results In an effort to select only high-affinity mAbs, the initial ELISA screens utilized a low level of coating antigen (20 ng/well) and a sensitive chemiluminescence-based reporter system. The sensitivity of this approach was first evaluated with antisera obtained from immunized mice in a dilution series, comparing the endpoint dilution values and signal-to-noise ratios using a chromogenic versus a luminescent substrate (Table 1). These data suggest that the luminescent assay was at least 1000-times more sensitive than the chromogenic assay for detection of bound antibody. More importantly, the signal-to-noise ratio for the chromogenic and luminescent substrates at the endpoint values was 1.5 versus 16, respectively. Positive signals, readily distinguished above background, were observed only in the
Antibody binding to different serotypes of BoNT was evaluated by ELISA (Fig. 1). The four mAbs tested bound BoNT serotype A with virtually no binding to the other six serotypes. To determine whether the antibodies specifically bound the HC or LC, each antibody was studied in Western blot experiments probing reduced versus non-reduced 150 kDa holotoxin following separation by SDS-PAGE (Fig. 2). mAbs F1-2, F1-5, and F1-40 labeled the intact 150 kDa holotoxin present in the non-reduced samples. mAbs F1-2, and F1-5 specifically bound HC when the samples were reduced (Fig. 2, lanes 2 and 4), but no labeling of the LC band was observed. In contrast, mAb F1-40 labeled the LC but not the heavy chain under reducing conditions (Fig. 2, lane 6). mAb F2-43 did not
Fig. 2. Antibody binding on Western blots of reduced and non-reduced BoNT/A. BoNT/A was separated on 4–20% SDS-PAGE in the presence and absence of 1 mM DTT, transferred to membranes and probed with mAbs F1-2 (lanes 1–2) F1-5 (lanes 3–4), F1-40 (lanes 5–6) and F2-43 (lanes 7–8). Markers, Bio-Rad prestained Markers (Bio-Rad, Richmond, CA).
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label either the intact BoNT holotoxin or the separated chains under denaturing conditions (Fig. 2, lanes 7 and 8). F2-43 antibody binding was observed by Western blots following native (non-SDS) PAGE (Fig. 3, lanes 5 and 6). In these experiments, F1-2 bound holotoxin and heavy chain but not light chain, in agreement with the results following SDSPAGE. Under these conditions, mAb F2-43 bound the holotoxin and both the heavy and light chains in the reduced sample (Fig. 3, lane 6). The 150 kDa BoNT/A holotoxin is associated with a number of non-toxic proteins and is secreted as a protein complex or progenitor toxin found in three forms; a 12S, 16S, and 19S toxins (Sakaguchi et al., 1984; Fujinaga et al., 2000). The 900 kDa BoNT/A complex consists of neurotoxin, various haemagglutinin (HA) proteins, and a 120 kDa non-toxic protein without HA activity designated non-toxic non-HA (NTNH). Results from direct-binding ELISA experiments comparing antibody binding to the 150 kDa holotoxin and the 900 kDa toxin complex are shown in Fig. 4. These results demonstrate that mAbs F1-2-, F1-5 and F1-40 strongly bind both the 150 kDa holotoxin and the toxin complex. Three times more toxin complex was used as coating antigen in these experiments since toxin is reported to account for approximately 33% of the 900 kDa complex (Maksymowych et al., 1999). SDS-PAGE analysis of the 900 kDa complex in our laboratory suggested that between 28 and 29% of the total protein present was 150 kDa holotoxin in agreement with the earlier published studies (results not shown). In sharp contrast, mAb F2-43 had significantly greater reactivity, higher luminescence, when the 900 kDa toxin complex was used as antigen in the ELISA than when the 150 kDa holotoxin was used to coat plates (Fig. 4). 3.2. Antibody affinity The dissociation constant for each mAb against purified 150 kDa toxins was determined by ELISA and analyzed as described by Bobrovnik (2003). The Kd values were calculated as the inverse of the slope determined by linear regression analysis of a Scatchard plot of the data and ranged from 8 to
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Fig. 4. Binding of antibodies F1-2, F1-5, F1-40, and F2-43 to purified 150 kDa BoNT/A holotoxin (open bar) and BoNT/A 900 kDa complex (solid bar) by ELISA. Microtiter wells were coated with holotoxin at 0.2 μg mL− 1 and with BoNT/A complex at 0.6 μg m− 1.
62 pM (Table 2). While antibodies with pM dissociation constants are not common, anti-BoNT antibodies with pM dissociation constants recently have been described (Razai et al., 2005). 3.3. Sandwich ELISA A sensitive antigen-capture ELISA to detect toxin was established (see Fig. 5). Each of the four mAbs described here were labeled with biotin and then utilized as either the capture or detection antibody in order to determine the optimum combination. The data shown in Fig. 6 indicate that the highest number of luminescent units measured was obtained when mAb F1-2 (8.9 × 105 counts) or mAb F1-5 (7.4 × 105 counts) was used as capture antibody and biotinylated mAb F1-40 was used as detector antibody. Significantly lower activity was observed using any of the other combinations of capture and detector antibody. As expected, low or no counts were observed when the same mAb was used as both the capture and detector antibody. Binding of biotinylated mAb F1-5 was inhibited when mAb F1-2 was used as capture antibody. Likewise, greatly reduced binding of biotinylated mAb F1-2 was observed when mAb F1-5 was used as capture antibody. These results suggest that F1-2 and F1-5 bind the same or adjacent (possibly overlapping) epitopes. A sandwich
Table 2 Capture dissociation constant measured by ELISA Antibody
Fig. 3. Antibody binding by Western blotting following native PAGE. BoNT/A 150 kDa holotoxin was electrophoresed on native gels in the presence (lanes 4 and 6) or absence (lanes 3 and 5) of 2 mM DTT. Membranes there probed with mAb F1-2 (lanes 3–4) and mAb F2-43 (lanes 5–6). Lanes 1–2 represent proteins detected by silver staining.
F1-2 F1-5 F1-40 F2-43
Antigen 150 kDa toxin
900 kDa complex
16.6 ± 0.2 × 10− 12 M 8.0 ± 0.1 × 10− 12 M 61.9 ± 12.9 × 10− 12 M 16.32 ± 3.6 × 10− 12 M
nd 48 ± 17 × 10− 12 M Nd Nd
nd = not done. Data represents the average of three separate experiments ± one standard deviation.
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Fig. 5. Diagramatic representation of a sandwich ELISA used to detect BoNT/A. Biotin, B; Avidin, A; Horseraddish peroxidase, E.
ELISA incorporating biotinylated F1-40 as detector antibody and mAb F1-2 as capture antibody was further studied. A linear standard curve was observed using 150 kDa holotoxin standard with a limit of quantitation (LOQ) of 10 pg mL− 1 for BoNT/A (Fig. 7). 3.4. Detection of BoNT/A and BoNT 900 kDa complex in milk matrices The performance of the above sandwich ELISA was evaluated using milk samples spiked with 150 kDa BoNT/A
or with the 900 kDa BoNT/A complex. Undiluted milk samples could be analyzed following centrifugation at 14,000 ×g for 15 min at 6 °C in order to defat the samples or by simply diluting the samples (10-fold) before analysis. Recovery of BoNT/A from spiked milk samples (skim, 2% milk, and whole milk) is summarized in Tables 3 and 4. Toxin recovery varied from 83–113% using samples spiked with the 150 kDa BoNT/A holotoxin at levels ranging from 10.0–0.3 ng mL− 1, and from 79–106% when samples were spiked with the 900 kDa BoNT/ A toxin complex. 4. Discussion The four high-affinity anti-BoNT/A mAbs reported represent only a small percentage of the mAbs detected at screening. All of the mAbs detected bound toxoid whereas
Fig. 6. Sandwich ELISA for detection of BoNT/A. Each of the four mAbs was used as capture antibody or as the biotinylated antibody. Relative Luminescent Counts were measured for each antibody combination using BoNT/A at 10 ng/mL.
Fig. 7. Detection of BoNT/A 150 kDa holotoxin using mAb F1-2 (HC specific) as capture antibody and MAb F1-40 (LC-specific) as detector antibody. Points represent the average of three determinations, bars equal ± one SD. Horizontal dashed line equals the average of the zero spike plus three SD, approximately 1800 counts.
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Table 3 Percent recovery of BoNT/A from milk using a sandwich immunoassay Spike level (ng mL− 1)
Skim milk 2% Milk Whole milk
10
5
2.5
1.25
0.625
0.312
98.4 ± 5.8 101.8 ± 3.3 113.1 ± 7.0
86.7 ± 7.8 86.8 ± 2.9 85.0 ± 4.5
83.5 ± 4.8 83.8 ± 4.8 94.6 ± 5.5
90.3 ± 5.4 92.3 ± 6.7 96.7 ± 5.3
92.2 ± 13.3 98.8 ± 19.3 91.1 ± 4.5
92.9 ± 19.9 107.2 ± 14.5 83.7 ± 4.4
SD = standard deviation. Spike level represents the amount of BoNT/A in milk before a 10-fold dilution of the sample. Data is the average of three replicates ± one SD.
only a few cross-reacted with biologically active toxin. While this distribution was expected, it demonstrates that use of toxoid as an immunogen is an inefficient route to produce toxin-specific antibodies. The low dissociation constant measured for these mAbs (low pM range) suggests that they bind an epitope on the 150 kDa holotoxin. Furthermore, the low Kd measured is comparable to that reported for antiBoNT/A scFv binding molecules following their affinity maturation and conversion to IgG (Razai et al., 2005). Recently, sensitive detection of BoNT/A in buffer and blood plasma, down to 1.4 pg mL− 1, has been achieved using highaffinity recombinant antibodies (Varnum et al., 2006). The antibodies reported here also appear to have high specificity, in that they only bound the A serotype. Three of the antibodies (F1-2, F1-5, and F1-40) bound the 900 kDa toxin complex as well as the 150 kDa holotoxin suggesting that their epitopes are exposed in the toxin complex. The stronger binding of F2-43 in ELISA to the 900 kDa toxin complex versus the 150 kDa toxin suggests that part of the binding epitope may be contributed by a non-toxic associated protein (NAP) consistent with the hypothesis that F2-43 has a complex conformational epitope. Precise identification of the epitopes for each of the four mAbs described here has yet to be completed. However, the Western blot results following SDS-PAGE demonstrate that the epitopes for mAbs F1-2 and F1-5 are located on the HC. In contrast, the epitope for F1-40 is located on the LC. mAb F2-43 demonstrated stronger binding to the 900 kDa toxin complex than to the150 kDa holotoxin in the ELISA and binding was observed following native gel electrophoresis of the 150 kDa holotoxin to both HC and LC in Western blot experiments. However, no binding of this mAb to holotoxin toxin or isolated chains could be demonstrated in blots of SDS gels. These observations suggest that mAb F2-43 binds a discontinuous (conformational) epitope. The ELISA data suggest that maximal binding may involve both toxin and NAP, possibly the NAP stabilizes the epitope resulting in improved antibody binding. Studies using recombinant peptides and using phage display libraries are in progress to more precisely define the epitopes of these mAbs. The high affinities and distinct epitopes (i.e., HC versus LC) of these mAbs have allowed them to be used in the development of a sensitive sandwich ELISA to detect BoNT/A holotoxin and the 900 kDa toxin complex, using F1-2 as capture antibody and biotin-conjugated F1-40 as detector antibody (Fig. 5). This sandwich ELISA is capable of detecting as little at 2 pg mL− 1 (13.3 fM) in buffer, a detection limit similar to that reported using recombinant antibodies (Varnum et al., 2006). The LOD, defined as three standard deviations above the average blank
is approximately 5 pg mL− 1 (33.3 fM), substantially lower than that observed in the mouse bioassay (20 pg mL− 1). This low level of detection allows for simple dilution of complex food samples in order to eliminate interferences while still maintaining a detection limit relevant for food monitoring. Sample interference were eliminated in milk by centrifugation at 4 °C for 15 min (defatting the sample), but this step is time consuming, not easily automated, and limits field applications. A preferable method to eliminate interferences involves simple dilution of the samples (10-fold) and this still maintains a LOD equivalent to 1/15,000 of the reported human oral LD50 (Brin, 1997) in a 237 mL serving (8 oz glass). Thus development of high-affinity monoclonal antibodies should result in faster, more sensitive assays. Such antibodies can be formatted into a variety of assays including traditional ELISA, bead-based assays, array-based assays, and lateral flow devices. Likewise, they can be used for immunoaffinity applications such as toxin capture and purification from a complex food matrix. A challenge for immunoassay is that it does not report on the activity of the toxin, only its presence and concentration. In an independent study Razoolie et al., used the HC specific mAb, F1-2 conjugated to magnetic beads, to concentrate toxin from milk followed by activity determination using a SNAPtide functional assay (Rasooly, R., Stanker, L.H., Carter, J.M., Do, P.M., Cheng, L.W., He, X., Brandon, D.L. In vitro peptide cleavage assay for detection of Botulinum Neurotoxin-A activity in food. (Submitted)). All of the experiments reported here use BoNT serotype A subtyp A1 obtained from the Hall A strain of C. botulinum. Of the four subtypes known, A1–A4, type A1 is the most commonly available. Antibody binding to one subtype does not necessarily guarantee binding to the other subtypes. Studies are underway to determine which subtype these mAbs bind.
Table 4 Percentage recovery of BoNT/A 900 kDa complex from milk using a sandwich immunoassay Spike level (ng mL− 1)
Skim 2% milk Whole milk
30.0
15.0
7.5
3.8
1.9
106.1 ± 2.0 91.5 ± 1.6 81.9 ± 2.2
90.3 ± 1.0 89.5 ± 3.7 84.4 ± 7.6
84.8 ± 3 83.5 ± 3.2 80.8 ± 5.3
79.12 ± 0 84.3 ± 16.3 87.1 ± 31.5
92.6 ± 20 106.1 ± 13.1 98.8 ± 23.4
SD = standard deviation. Spike level represents the amount of 900 kDa Complex added before a 10fold dilution of the sample. Data is the average of three replicates ± SD.
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References Arnon, S.S., Schechter, R., Inglesby, T.V., Henderson, D.A., Barlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Hauer, J., Layton, M., Lillibridge, S., Osterholm, M.T., O'Toole, T., Parker, G., Perl, T.M., Russell, P.K., Swerdlow, D.L., Tonat, K., 2001. Botulinum toxin as a biological weapon: medical and public health management. JAMA 285, 1059. Bigbee, W.L., Vanderlaan, M., Fong, S.S.N., Jensen, R.H., 1983. Monoclonal antibodies specific for the M- and N-forms of human glycophorin A. Mol. Immunol. 20, 1353. Bobrovnik, S.A., 2003. Determination of antibody affinity by ELISA. Theor. J. Biochem. Biophys. Methods 57, 213. Brin, M.F., 1997. Botulinum toxin: chemistry, pharmacology, toxicity, and immunology. Muscle Nerve 6, S146. Chao, H.Y., Wang, Y.C., Tang, S.S., Liu, H.W., 2004. A highly sensitive immunopolymerase chain reaction assay for Clostridium botulinum neurotoxin type A. Toxicon 43, 27. Doellgast, G.J., Triscott, M.X., Beard, G.A., Bottoms, J.D., Cheng, T., Roh, B.H., Roman, M.G., Hall, P.A., Brown, J.E., 1993. Sensitive enzyme-linked immunosorbent assay for detection of Clorstrium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay. J. Clin. Microbiol. 31, 2402. Doellgast, G.J., Beard, G.A., Bottoms, J.D., Cheng, T., Roh, B.H., Roman, M.G., Hall, P.A., Triscott, M.X., 1994a. Enzyme-linked immunosorbent assay and enzyme-linked coagulation assay for detection of Clostridum botulinum neurotoxins A, B, and E and solution-phase complexes with dual-label antibodies. J. Clin. Microbiol. 32, 105. Doellgast, G.J., Triscott, M.X., Beard, G.A., Bottoms, J.D., 1994b. Enzyme-linked immunosorbent assay-enzyme-linked coagulation assay for detection of antibodies to Clostridum botulinum neurotoxins A, B, and E and solutionphase complexes. J. Clin. Microbiol. 32, 851. Ferreira, J.L., Maslanka, S., Johnson, E., Goodnough, M., 2003. Detection of botulinal; neurotoxins A, B, E, and F by amplified enzyme-linked immunosorbent assay: collaborative study. J. AOAC Int. 86, 314. Friguet, B., Chaffotte, A.F., Djavadi-Ohaniance, L., Goldberg, M.E., 1985. Measurements of the true affinity constant in solution of antigen– antibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Methods 77, 305. Fujinaga, Y., Inoue, K., Nomura, T., Sasaki, J., Marvaud, J.C., Popoff, M.R., Kozoki, S., Oguma, K., 2000. Identification and characterization of functional subunits of Clorstridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes. FEBS Lett. 467, 179. Gessler, F., Pagel-Wieder, S., Avondet, M.A., Böhnel, H., 2007. Evaluation of lateral flow assays for the detection of botulinum neurotoxin type A and their application in laboratory diagnosis of botulism. Diagn. Microbiol. Infect. Dis. 57, 243. Gibson, A.M., Modi, N.K., Roberts, T.A., Shone, C.C., Hambleton, P., Melling, J., 1987. Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system. J. Appl. Bacteriol. 63, 217. Gibson, A.M., Modi, N.K., Roberts, T.A., Hambeton, P., Melling, J., 1988. Evaluation of a monoclonal antibody-based immunoassay for detecting
type B Clostridium botulinum toxin produced in pure culture and an inoculate model cured meat system. J. Appl. Bacteriol. 64, 285. Gill, M., 1982. Bacterial toxins: a table of lethal amounts. Microbiol. Rev. 46, 86. Lacy, D.B., Tepp, W., Cohen, A.C., DasGupta, B.R., Stevens, R.C., 1998. Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nat. Struct. Biol. 5, 898. Maksymowych, A.B., Reinhard, M., Malizio, C.J., Goodnough, M.C., Johnson, E.A., Simpson, L.I., 1999. Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockage. Infect. Immun. 67, 4708. Montecucco, C., Schiavo, G., 1995. Structure and function of tetanus and botulinum neurotoxins. Quart. Rev. Biophys. 28, 423. Poli, M.A., Rivera, V.R., Neal, D., 2001. Developmentr of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F. Toxicon 40, 797. Razai, A., Garcia-Rodriguez, C., Lou, J., Geren, I.N., Forsyth, C.M., Robles, Y., Tsai, R., Smith, T.J., Smith, L.A., Siegel, R.W., Feldhaus, M., Marks, J.D., 2005. Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A. J. Mol. Biol. 351, 158. Sakaguchi, G., Kozaki, S., Ohishi, I., 1984. Structure and function of botulinum toxins. Bacterial Protein Toxins. Structure and function of botulinum toxins. Academic Press, London, pp. 435–443. Sharma, S.K., Ferreira, J.L., Eblen, B.S., Whiting, R.C., 2006. Detection of type A, B, E, and F, Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies. Appl. Environ. Microbiol. 72, 1231. Sugasawara, R.J., Cahoon, B.E., Karu, A.E., 1985. The influence of murine macrophage-conditioned medium on cloning efficiency, antibody synthesis, and growth rate of hybridomas. J. Immunol. Methods 79, 263. Szilagyi, M., Rivera, V.R., Neal, D., Merrill, G.A., Poli, M.A., 2000. Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes A and B. Toxicon 38, 381. Turton, K., Chaddock, J.A., Acharya, K.R., 2002. Botulinum and tetanus neurotoxins: structure, function and therapeutic utility. Trends Biochem. Sci. 27, 552. Varnum, S.M., Warner, M.G., Dockendorff, B., Anheier Jr., N.C., Lour, J., Marks, J.D., Smith, L.A., Feldhaus, M.J., Grate, J.W., Bruckner-Lea, C.J., 2006. Enzymeamplified protein microarray and fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies. Anal. Chim. Acta 570, 137. Wictome, M., Newton, K., Jameson, K., Hallis, B., Dunnigan, P., Mackay, E., Clarke, S., Taylor, R., Gaze, J., Foster, K., Shone, C., 1999. Development of an in vitro bioassay for Clostridium botulinum type B neurotoxin in foods that is more sensitive than the mouse bioassay. Appl. Environ. Microbiol. 65, 3787. Wu, H.C., Yeh, C.T., Huan, Y.L., Tarn, L.J., Lung, C.C., 2001. Characterization of neutralizing antibodies and identification of neutralizing epitope mimics on the Clostridium botulinum neurotoxin type A. Appl. Environ. Microbiol. 67, 3201.
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA Larry H. Stanker ⁎, Paul Merrill, Miles C. Scotcher, Luisa W. Cheng United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, United States
a r t i c l e
i n f o
Article history: Received 16 November 2007 Received in revised form 3 March 2008 Accepted 4 March 2008 Available online 9 April 2008 Keywords: Botulinum neurotoxin Monoclonal antibodies Immunoassays Food safety
a b s t r a c t Botulinum neurotoxins (BoNT), produced by the anaerobic bacterium Clostridium botulinum, cause severe neuroparalytic disease and are considered the most toxic biological agents known. While botulism is rare in the U.S. it often is fatal if not treated quickly, and recovery is long, requiring intensive treatment. BoNT is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disulfide bond. The ‘gold standard’ for BoNT detection relies on a mouse bioassay. This is a time consuming (up to 4 days) assay and it lacks specificity, however, it gives a sensitivity (mouse LD50) of approximately 10 pg mL− 1. Most BoNT immunoassays are much less sensitive. In this study we describe the development of four high-affinity (dissociation constants (Kd's) in the low pM range) monoclonal antibodies (mAbs) that specifically bind BoNT serotype A (BoNT/A). These antibodies, designated F1-2, F1-5, F1-40, and F2-43 are IgG1 subclass mAbs with kappa light chains and they specifically bind BoNT serotype A. Western blot analyses following SDSPAGE demonstrate that mAbs F1-2 and F1-5 bind the 100 kDa heavy chain subunit and that mAb F1-40 binds the 50 kDa light chain. The fourth antibody demonstrated strong binding to the 150 kDa holotoxin in the ELISA and on Western blots following electrophoresis on native gels. However binding in Western blot studies was not observed for mAb F2-43 following SDSPAGE. A highly sensitive sandwich ELISA, capable of detecting as little as 2 pg/mL BoNT/A was developed using mAbs F1-2 and F1-40. Such an assay represents a realistic, high sensitivity alternative to the mouse bioassay. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Clostridium botulinum, an anaerobic spore-forming bacterium, produces a family of botulinum neurotoxins (BoNT, EC 3.4.24.69) consisting of seven serotypes, A–G (BoNT/A–BoNT/G) (Gill, 1982). Serotype A is synthesized as a single 1296 amino acid, 150 kDa polypeptide which is cleaved endogenously or exogenously forming a dichain molecule comprised of an ∼100 kDa heavy chain (HC) and an ∼50 kDa light chain (LC)
⁎ Corresponding author. U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, United States. Tel.: +1 510 559 5984; fax +1 510 559 6429. E-mail address: [email protected] (L.H. Stanker). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.03.003
linked by a single disulfide bond, the holotoxin, (Montecucco and Schiavo, 1995). The HC mediates toxin entry into neurons, and the LC functions as a zinc-dependent endoprotease cleaving SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) involved in acetylcholine release resulting in muscular paralysis (Turton et al., 2002). The crystal structure of BoNT/A was determined at a resolution of 3.3 Å (Lacy et al., 1998). An LD50 for BoNT/A of 0.5 ng kg− 1 in mice was reported by Wu et al. (2001). Because of its high toxic potency and ease of production, BoNT is classified by the Centers for Disease Control (CDC) as a Class A bioterrorism agent (Arnon et al., 2001). Currently, BoNT detection in food relies on use of the mouse bioassay, a highly sensitive method with detection limits of 7–20 pg mL− 1. However, the mouse bioassay is time
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consuming (up to 4 days), requires the use of large numbers of animals and often relies on death as an endpoint. Furthermore, a subsequent neutralization bioassay is required in order to confirm toxin presence and identify toxin serotype. An alternative method that is rapid, sensitive, and toxin serotype-specific is highly desirable. In an effort to meet this challenge, a number of immunoassays have been developed, the most common being the enzyme-linked immunosorbent assay (ELISA) (Doellgast et al., 1993; Ferreira et al., 2003; Gessler et al., 2007; Gibson et al., 1988, 1987; Poli et al., 2001; Sharma et al., 2006; Szilagyi et al., 2000; Wictome et al., 1999). For the most part, these assays do not detect toxin at the lowest levels measured in the mouse bioassay. Doellgast et al. (1994a,b) reported an enzyme-linked coagulation assay (ELCA) with sensitivity comparable to the mouse bioassay. However, this assay is complicated, and its high sensitivity is achieved through a novel amplification system that utilizes snake venom coagulation factor. Greater sensitivity was reported by Chao et al. (2004) who used an immunopolymerase chain reaction (IPCR) based assay to detect as little as 50 fg. This IPCR approach also suffers from its complexity and susceptibility to sample matrix effects on assay performance. Simple immunoassay formats, including lateral flow assays have been developed (Gessler et al., 2007) but these again fail to achieve the sensitivity of the mouse bioassay. Recently, high-affinity IgG constructs were generated from single chain Fv (scFv) antibodies using molecular evolution and a yeast display system (Razai et al., 2005). These recombinant antibodies formed the basis of a sensitive ELISA microarray immunoassay and a micro-column sensor capable of detecting BoNT down to 14 fM (1.4 pg mL− 1) (Varnum et al., 2006). These results demonstrated that simple immunoassay formats can be highly sensitive when highaffinity antibodies are incorporated. In this study we pursued the development of high-affinity mouse mAbs to BoNT/A, and demonstrate that they can be used in a simple sandwich ELISA, with luminescence detection, to quantify BoNT/A below the mouse LD50 level.
used in spiking experiments were purchased from a local food store. Luminesence was measured using a Perkin-Elmer Victor-2 microplate reader. Data were exported to Microsoft Excel for further analysis. 2.1.1. Cell culture media Hybridoma medium (HM) consisted of Iscove's modified Dulbecco's Minimal Medium (Sigma #I-7633) containing NaHCO3 (36 mM), and glutamine (2 mM). All hybridoma cells and SP2/0 mouse myeloma cells were maintained in HM supplemented with 10% fetal calf serum (cHM). Hybridomas were selected following cell fusion using HAT selection medium prepared by adding hypoxanthine (5 μM), aminopterin (0.2 μM), and thymidine (0.8 μM) to cHM. Macrophage conditioned medium (MPCM) was prepared as described (Sugasawara et al., 1985). A mixture of 40% cHM and MPCM was used for all cell-cloning procedures. 2.2. Monoclonal antibody production All protocols were approved by the Institutional Animal Care and Use Committee. Female Balb/cJ mice (Simonsen Laboratories, Gilroy, CA) were immunized at 2-week intervals by intraperitoneal injection (IP) of 100 μL of BoNT/A toxoid (approximately 10 μg mL− 1) prepared in RIBI adjuvant as suggested by the manufacturer. Following the third injection, serum was obtained and evaluated for anti-BoNT/A antibodies. After 2 weeks, mice with a strong antibody titer were injected IP with 1 μg of BoNT/A toxoid in PBS. 2.2.1. Fusion procedure Three days following the last IP injection mice were euthanized and their splenocytes were fused with SP2/0 myeloma cells using polyethylene glycol as previously described (Bigbee et al., 1983). Following cell fusion, the cells were suspended in 100 mL of HAT selection medium supplemented with 10% fetal calf serum and 10% MPCM, dispensed into ten, 96-well tissue culture plates, and incubated for 10 to 14 days at 37 °C in 5% CO2 before screening for antibody production.
2. Materials and methods 2.1. Reagents and equipment Solutions at 1 mg mL− 1 of botulinum neurotoxin serotypes A–G and BoNT/A toxoid were purchased from Metabiologics Inc. (Madison, WI). Lyophilized samples of BoNT/A toxin and toxoid (10 μg vial− 1) were purchased from List Biological Laboratories, Campbell, CA. Bovine serum albumin (BSA), ovalbumin (OVA), goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (IgG-peroxidase) #A-4416, polyoxyethylene sorbitan monolaurate (Tween-20), RIBI adjuvant, Protein-G conjugated Sepharose #P-32196, and the following buffers: 0.01 M phosphate buffered saline (PBS) #P-3813, 0.138 M NaCl, 0.0027 M KCl, pH 7.4; and 0.02 M TRIS–buffered saline (TBS) #T-5912, 0.9% NaCl, pH 7.4 were purchased from Sigma Chemical Co. (St. Louis, MO). Black, Maxisorp 96-well Nunc microtiter plates were obtained from PGC Scientific (Gaithersburg, MD), and SuperSignal Femto Max Sensitivity substrate was purchased from Pierce Inc. (Rockford, IL). Non-fat dry milk (NFDM) and milk samples
2.2.2. Screening methods Sera from immunized mice and supernatants from cell fusion plates were screened using a direct-binding ELISA. Microtiter plates were rinsed with distilled water and coated by incubating 100 μL/well of a 0.2 μg mL− 1 or 0.6 μg mL− 1 solution of BoNT/A or 900 kDa BoNT/A complex, respectively, in 0.05 M sodium carbonate buffer, pH 9.6 overnight at 4 °C. The toxin solution was aspirated and non-coated sites blocked by adding 300 μL well− 1 of 3% non-fat dry milk in Tris buffered saline containing 0.05% Tween-20 (NFDM-TBST) and the plates were incubated for 1 h at 37 °C. The plates were then washed 3 times with 0.05% Tween-20. Next, sera or cell culture supernatants were added (100 μL well− 1) and the plates were incubated at 37 °C for 1 h. Plates were again washed 3× and 100 μL well− 1 of a 1/5000 dilution of peroxidase-conjugated goat anti-mouse sera (Sigma, St. Louis, Mo) was added and the plates incubated for 1 h at 37 °C. The plates were then washed 6× with 0.05% Tween-20. Freshly prepared luminescence substrate was added (100 μL/ well) according to the manufacturer's recommendation. The
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plates were incubated for 3 min at room temperature and luminescent counts recorded. Cells from the wells giving positive signals for antibody production were cloned by limiting dilution. Hybridomas were then expanded and small amounts (usually less than 10 mL) of ascites fluids obtained (Covance Research Products, Inc., Denver, PA). Antibody was purified by affinity chromatography on Protein-G Sepharose. Bound antibody was eluted with 0.1 M glycine–HCl, pH 2.7. Protein concentrations were determined with a BCA-kit (Pierce) using the microplate method suggested by the manufacturer. Antibodies were conjugated with biotin using EZ-Link Sulfo-NHS-LC-Biotin (Pierce) as described by the manufacturer using a 50-fold molar excess of biotin reagent. Antibody isotype was determined by ELISA using toxin-coated microtiter plates and horseradish peroxidase-conjugated, isotype-specific antibodies (SouthernBiotech, Birmingham, AL).
3
(Pierce, #34075) was added as recommended by the manufacturer. Membranes were placed between vinyl sheets and bands were visualized using BioMax XAR film (Eastman Kodak Co, Rochester, NY) or using a FluoroChem Sp gel imaging system (Alpha Innotech Corp., San Leandro, CA). Native gel electrophoresis was performed as follows. Samples of 200 ng of BoNT/A were incubated for 10 min at 55 °C in native sample buffer (supplemented with Dithiothreitol (DTT) at a final concentration of 100 mM) prior to loading onto a 10% native gel. NativeMark protein standards (Invitrogen) were diluted 1/20 in native sample buffer, and loaded directly onto the gel without heating. Electrophoresis was carried out at 200 V for 80 min. For Western blotting, the gel was subjected to horizontal blotting onto a nitrocellulose membrane in native transfer buffer (0.025 M Tris, 0.192 M glycine, pH 8.6) at 180 mA for 100 min. The blot was briefly washed in TBS-T (20 mM Tris, 0.9% NaCl, 0.05% Tween-20, pH 7.4) and transferred to 20 mL blocking buffer, 3% NFDM-TBS-T and incubated at room temperature for 1 h. The membranes were then incubated in primary antibody using 10 mL blocking buffer containing the appropriate mAb at 5 μg mL− 1 (F1-2, F-5, F1-40) or 10μg − 1 mL (F2-43) at 4 °C overnight. The blot was washed 4× in TBS-T, for 4 min. each at room temperature. The membranes were then processed with goat anti-mouse peroxidase-conjugated IgG (1/5000) for 1 h at room temperature prior to chemiluminescense detection using SuperSignal West Dura Extended Duration Substrate according to the manufacturer's protocol (Pierce Biotechnology Inc.). Blots were visualized using a Fluorchem SP unit as described above.
2.2.3. Capture ELISA Microtiter wells were coated with capture antibody by adding 100 μL well− 1 of a 1 μg mL− 1 solution in PBS pH 7.4 of mAb F1-2 to each well and incubating overnight at 4 °C. Plates were then blocked by addition of 300 μL of 3% NFDM-TBST and incubating the plates for 1 h at 37 °C. Finally, plates were washed 3× with 0.05% Tween-20 and stored for up to 14 days at 4 °C before use. Toxin standards and samples were then added (100 μL well− 1 in 5% NFDM-TBST buffer) and the plates incubated for 1 h at 37 °C. The plates were then washed 6× in 5% Tween-20, 100 μL of a 3 μg mL− 1 solution of biotinconjugated mAb F1-40 was added to each well, and the plates were incubated at 37 °C for 1 h. Next, the plates were washed 9× with 0.05% Tween-20 and 100 μL well− 1 of a 1/20,000 dilution of streptavidin-HRP (Zymed, S. San Francisco, CA) in 3% NFDM-TBST was added and the plate incubated for 1 h at 37 ° C. Finally the plates were washed 12× with 0.05% Tween20 and luminescence substrate was added.
The binding dissociation constant (Kd) was measured by ELISA using the method described by Friguet et al. (1985). Data analysis and calculation of Kd used the following relationships described by Bobrovnik (2003).
2.3. Electrophoresis
ðRLUo RLUi Þ=RLUi ¼ KaLi
ð1Þ
All gel electrophoresis equipment, buffers, gels and PVDF membranes were purchased from PAGEgel (San Diego, CA). Direct visualization of proteins following SDS-PAGE used silver staining (Silver Express, Invitrogen Inc., Grand Island, NY) as described by the manufacturer. Toxins were separated by SDS-PAGE using 4–20% gels as recommended by the manufacturer. Briefly, samples (heated at 70 °C for 10 min) were suspended in electrophoresis sample buffer and 10 μL containing 100 ng of protein were loaded into each well and separated by electrophoresis at 200 V (constant). Following electrophoresis, proteins were electrophoretically transferred to PVDF membranes using a PAGEgel transfer cell, (180 mA constant for 90–120 min). Membranes were blocked with 3% NFDM-PBS–0.05% Tween-20 buffer (PBST) for 1 h at 22 °C on a rocking platform. The membranes were then washed 3×, 5 min each with rocking in PBST, primary anti-toxin antibody was added (1 μg mL− 1), and the membranes were incubated overnight at 4 °C. The membranes were again washed 3× in PBST. Peroxidase conjugated goat anti-mouse IgG (whole molecule) antiserum (Sigma, #A4416) diluted 1/5000 in PBST was added and the blots incubated with rocking at 22 °C for 1 h. The membranes were again washed (3×, 5 min), substrate
Ka ¼ 1=Kd
ð2Þ
2.4. Antibody affinity measurements
where RLUo equals the activity measured when no toxin was present; RLUi, the activity measured when different concentrations of toxin (Li) ranging between 4.2–6600 ×10− 12 M was added to a constant concentration of antibody. The reaction mixtures were incubated up to 48 h and the percentage of unbound
Table 1 ELISA comparison of antibody activity in immunized mouse serum using a luminescent versus a colorimetric substrate Antisera dilution factor 500 1000 10,000 100,000 500,000 1,000,000 2,000,000 Blank
Luminescent (counts)
Signal: noise ratio
Colorimetric (absorbency)
Signal: noise ratio
5,557,000 5,078,000 2,228,934 477,000 98,000 55,352 28,426 1750
3175 2901 1274 273 56 32 16
1.60 1.40 0.21 0.09 0.08 0.07 0.07 0.06
22.6 23.3 35 1.5 1.3 1.2 1.2
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Fig. 1. Determination of BoNT serotype binding specificity of mAb F1-2, (horizontal strip) F1-5 (solid), F1-40, (stippled), and F2-43 (diagonal strip) by ELISA. Antibody binding to microtiter wells coated with a 0.2 μgmL− 1 of the BoNT serotypes A–G was measured. The data shown represent the average of three experiments, error bars represent + one standard deviation (SD).
antibody determined by ELISA. The value of Ka was determined graphically as the slope of the linear relation depicted in Eq. (1).
initial antisera dilutions using the colorimetric ELISA. Since significantly lower antibody concentrations would be expected in the hybridoma supernatants being tested in the initial screening, detection with a high level of confidence required the more sensitive assay. Hybridoma cells 10–14 days following cell fusion were screened for antibodies that bound BoNT/A toxoid. In general, N95% of the culture wells contained between 1 and 6 hybridoma “colonies”, equivalent to N10,000 total hybridoma colonies in 2000 culture wells. Positive signals (signal-tonoise ratios of 5 or greater) were observed for approximately 130 supernatants. The cells from these wells were expanded, tested, and cloned by limiting dilution to produce hybridoma lines. Of these hybridomas, only four antibodies bound toxin and toxoid, the rest bound only the formalin fixed toxoid but not active 150 kDa holotoxin. Future studies will concentrate on using active toxin as the immunogen. The toxin-binding antibodies, referred to as F1-2, F1-5, F1-40, and F2-43, were further characterized. Isotype analysis indicated that these four mAbs were IgG1 antibodies, with kappa light chains. Antibodies were then produced in mouse ascites fluids and purified by affinity chromatography using Protein-G coated Sepharose. 3.1. Binding specificity
3. Results In an effort to select only high-affinity mAbs, the initial ELISA screens utilized a low level of coating antigen (20 ng/well) and a sensitive chemiluminescence-based reporter system. The sensitivity of this approach was first evaluated with antisera obtained from immunized mice in a dilution series, comparing the endpoint dilution values and signal-to-noise ratios using a chromogenic versus a luminescent substrate (Table 1). These data suggest that the luminescent assay was at least 1000-times more sensitive than the chromogenic assay for detection of bound antibody. More importantly, the signal-to-noise ratio for the chromogenic and luminescent substrates at the endpoint values was 1.5 versus 16, respectively. Positive signals, readily distinguished above background, were observed only in the
Antibody binding to different serotypes of BoNT was evaluated by ELISA (Fig. 1). The four mAbs tested bound BoNT serotype A with virtually no binding to the other six serotypes. To determine whether the antibodies specifically bound the HC or LC, each antibody was studied in Western blot experiments probing reduced versus non-reduced 150 kDa holotoxin following separation by SDS-PAGE (Fig. 2). mAbs F1-2, F1-5, and F1-40 labeled the intact 150 kDa holotoxin present in the non-reduced samples. mAbs F1-2, and F1-5 specifically bound HC when the samples were reduced (Fig. 2, lanes 2 and 4), but no labeling of the LC band was observed. In contrast, mAb F1-40 labeled the LC but not the heavy chain under reducing conditions (Fig. 2, lane 6). mAb F2-43 did not
Fig. 2. Antibody binding on Western blots of reduced and non-reduced BoNT/A. BoNT/A was separated on 4–20% SDS-PAGE in the presence and absence of 1 mM DTT, transferred to membranes and probed with mAbs F1-2 (lanes 1–2) F1-5 (lanes 3–4), F1-40 (lanes 5–6) and F2-43 (lanes 7–8). Markers, Bio-Rad prestained Markers (Bio-Rad, Richmond, CA).
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label either the intact BoNT holotoxin or the separated chains under denaturing conditions (Fig. 2, lanes 7 and 8). F2-43 antibody binding was observed by Western blots following native (non-SDS) PAGE (Fig. 3, lanes 5 and 6). In these experiments, F1-2 bound holotoxin and heavy chain but not light chain, in agreement with the results following SDSPAGE. Under these conditions, mAb F2-43 bound the holotoxin and both the heavy and light chains in the reduced sample (Fig. 3, lane 6). The 150 kDa BoNT/A holotoxin is associated with a number of non-toxic proteins and is secreted as a protein complex or progenitor toxin found in three forms; a 12S, 16S, and 19S toxins (Sakaguchi et al., 1984; Fujinaga et al., 2000). The 900 kDa BoNT/A complex consists of neurotoxin, various haemagglutinin (HA) proteins, and a 120 kDa non-toxic protein without HA activity designated non-toxic non-HA (NTNH). Results from direct-binding ELISA experiments comparing antibody binding to the 150 kDa holotoxin and the 900 kDa toxin complex are shown in Fig. 4. These results demonstrate that mAbs F1-2-, F1-5 and F1-40 strongly bind both the 150 kDa holotoxin and the toxin complex. Three times more toxin complex was used as coating antigen in these experiments since toxin is reported to account for approximately 33% of the 900 kDa complex (Maksymowych et al., 1999). SDS-PAGE analysis of the 900 kDa complex in our laboratory suggested that between 28 and 29% of the total protein present was 150 kDa holotoxin in agreement with the earlier published studies (results not shown). In sharp contrast, mAb F2-43 had significantly greater reactivity, higher luminescence, when the 900 kDa toxin complex was used as antigen in the ELISA than when the 150 kDa holotoxin was used to coat plates (Fig. 4). 3.2. Antibody affinity The dissociation constant for each mAb against purified 150 kDa toxins was determined by ELISA and analyzed as described by Bobrovnik (2003). The Kd values were calculated as the inverse of the slope determined by linear regression analysis of a Scatchard plot of the data and ranged from 8 to
5
Fig. 4. Binding of antibodies F1-2, F1-5, F1-40, and F2-43 to purified 150 kDa BoNT/A holotoxin (open bar) and BoNT/A 900 kDa complex (solid bar) by ELISA. Microtiter wells were coated with holotoxin at 0.2 μg mL− 1 and with BoNT/A complex at 0.6 μg m− 1.
62 pM (Table 2). While antibodies with pM dissociation constants are not common, anti-BoNT antibodies with pM dissociation constants recently have been described (Razai et al., 2005). 3.3. Sandwich ELISA A sensitive antigen-capture ELISA to detect toxin was established (see Fig. 5). Each of the four mAbs described here were labeled with biotin and then utilized as either the capture or detection antibody in order to determine the optimum combination. The data shown in Fig. 6 indicate that the highest number of luminescent units measured was obtained when mAb F1-2 (8.9 × 105 counts) or mAb F1-5 (7.4 × 105 counts) was used as capture antibody and biotinylated mAb F1-40 was used as detector antibody. Significantly lower activity was observed using any of the other combinations of capture and detector antibody. As expected, low or no counts were observed when the same mAb was used as both the capture and detector antibody. Binding of biotinylated mAb F1-5 was inhibited when mAb F1-2 was used as capture antibody. Likewise, greatly reduced binding of biotinylated mAb F1-2 was observed when mAb F1-5 was used as capture antibody. These results suggest that F1-2 and F1-5 bind the same or adjacent (possibly overlapping) epitopes. A sandwich
Table 2 Capture dissociation constant measured by ELISA Antibody
Fig. 3. Antibody binding by Western blotting following native PAGE. BoNT/A 150 kDa holotoxin was electrophoresed on native gels in the presence (lanes 4 and 6) or absence (lanes 3 and 5) of 2 mM DTT. Membranes there probed with mAb F1-2 (lanes 3–4) and mAb F2-43 (lanes 5–6). Lanes 1–2 represent proteins detected by silver staining.
F1-2 F1-5 F1-40 F2-43
Antigen 150 kDa toxin
900 kDa complex
16.6 ± 0.2 × 10− 12 M 8.0 ± 0.1 × 10− 12 M 61.9 ± 12.9 × 10− 12 M 16.32 ± 3.6 × 10− 12 M
nd 48 ± 17 × 10− 12 M Nd Nd
nd = not done. Data represents the average of three separate experiments ± one standard deviation.
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Fig. 5. Diagramatic representation of a sandwich ELISA used to detect BoNT/A. Biotin, B; Avidin, A; Horseraddish peroxidase, E.
ELISA incorporating biotinylated F1-40 as detector antibody and mAb F1-2 as capture antibody was further studied. A linear standard curve was observed using 150 kDa holotoxin standard with a limit of quantitation (LOQ) of 10 pg mL− 1 for BoNT/A (Fig. 7). 3.4. Detection of BoNT/A and BoNT 900 kDa complex in milk matrices The performance of the above sandwich ELISA was evaluated using milk samples spiked with 150 kDa BoNT/A
or with the 900 kDa BoNT/A complex. Undiluted milk samples could be analyzed following centrifugation at 14,000 ×g for 15 min at 6 °C in order to defat the samples or by simply diluting the samples (10-fold) before analysis. Recovery of BoNT/A from spiked milk samples (skim, 2% milk, and whole milk) is summarized in Tables 3 and 4. Toxin recovery varied from 83–113% using samples spiked with the 150 kDa BoNT/A holotoxin at levels ranging from 10.0–0.3 ng mL− 1, and from 79–106% when samples were spiked with the 900 kDa BoNT/ A toxin complex. 4. Discussion The four high-affinity anti-BoNT/A mAbs reported represent only a small percentage of the mAbs detected at screening. All of the mAbs detected bound toxoid whereas
Fig. 6. Sandwich ELISA for detection of BoNT/A. Each of the four mAbs was used as capture antibody or as the biotinylated antibody. Relative Luminescent Counts were measured for each antibody combination using BoNT/A at 10 ng/mL.
Fig. 7. Detection of BoNT/A 150 kDa holotoxin using mAb F1-2 (HC specific) as capture antibody and MAb F1-40 (LC-specific) as detector antibody. Points represent the average of three determinations, bars equal ± one SD. Horizontal dashed line equals the average of the zero spike plus three SD, approximately 1800 counts.
L.H. Stanker et al. / Journal of Immunological Methods 336 (2008) 1–8
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Table 3 Percent recovery of BoNT/A from milk using a sandwich immunoassay Spike level (ng mL− 1)
Skim milk 2% Milk Whole milk
10
5
2.5
1.25
0.625
0.312
98.4 ± 5.8 101.8 ± 3.3 113.1 ± 7.0
86.7 ± 7.8 86.8 ± 2.9 85.0 ± 4.5
83.5 ± 4.8 83.8 ± 4.8 94.6 ± 5.5
90.3 ± 5.4 92.3 ± 6.7 96.7 ± 5.3
92.2 ± 13.3 98.8 ± 19.3 91.1 ± 4.5
92.9 ± 19.9 107.2 ± 14.5 83.7 ± 4.4
SD = standard deviation. Spike level represents the amount of BoNT/A in milk before a 10-fold dilution of the sample. Data is the average of three replicates ± one SD.
only a few cross-reacted with biologically active toxin. While this distribution was expected, it demonstrates that use of toxoid as an immunogen is an inefficient route to produce toxin-specific antibodies. The low dissociation constant measured for these mAbs (low pM range) suggests that they bind an epitope on the 150 kDa holotoxin. Furthermore, the low Kd measured is comparable to that reported for antiBoNT/A scFv binding molecules following their affinity maturation and conversion to IgG (Razai et al., 2005). Recently, sensitive detection of BoNT/A in buffer and blood plasma, down to 1.4 pg mL− 1, has been achieved using highaffinity recombinant antibodies (Varnum et al., 2006). The antibodies reported here also appear to have high specificity, in that they only bound the A serotype. Three of the antibodies (F1-2, F1-5, and F1-40) bound the 900 kDa toxin complex as well as the 150 kDa holotoxin suggesting that their epitopes are exposed in the toxin complex. The stronger binding of F2-43 in ELISA to the 900 kDa toxin complex versus the 150 kDa toxin suggests that part of the binding epitope may be contributed by a non-toxic associated protein (NAP) consistent with the hypothesis that F2-43 has a complex conformational epitope. Precise identification of the epitopes for each of the four mAbs described here has yet to be completed. However, the Western blot results following SDS-PAGE demonstrate that the epitopes for mAbs F1-2 and F1-5 are located on the HC. In contrast, the epitope for F1-40 is located on the LC. mAb F2-43 demonstrated stronger binding to the 900 kDa toxin complex than to the150 kDa holotoxin in the ELISA and binding was observed following native gel electrophoresis of the 150 kDa holotoxin to both HC and LC in Western blot experiments. However, no binding of this mAb to holotoxin toxin or isolated chains could be demonstrated in blots of SDS gels. These observations suggest that mAb F2-43 binds a discontinuous (conformational) epitope. The ELISA data suggest that maximal binding may involve both toxin and NAP, possibly the NAP stabilizes the epitope resulting in improved antibody binding. Studies using recombinant peptides and using phage display libraries are in progress to more precisely define the epitopes of these mAbs. The high affinities and distinct epitopes (i.e., HC versus LC) of these mAbs have allowed them to be used in the development of a sensitive sandwich ELISA to detect BoNT/A holotoxin and the 900 kDa toxin complex, using F1-2 as capture antibody and biotin-conjugated F1-40 as detector antibody (Fig. 5). This sandwich ELISA is capable of detecting as little at 2 pg mL− 1 (13.3 fM) in buffer, a detection limit similar to that reported using recombinant antibodies (Varnum et al., 2006). The LOD, defined as three standard deviations above the average blank
is approximately 5 pg mL− 1 (33.3 fM), substantially lower than that observed in the mouse bioassay (20 pg mL− 1). This low level of detection allows for simple dilution of complex food samples in order to eliminate interferences while still maintaining a detection limit relevant for food monitoring. Sample interference were eliminated in milk by centrifugation at 4 °C for 15 min (defatting the sample), but this step is time consuming, not easily automated, and limits field applications. A preferable method to eliminate interferences involves simple dilution of the samples (10-fold) and this still maintains a LOD equivalent to 1/15,000 of the reported human oral LD50 (Brin, 1997) in a 237 mL serving (8 oz glass). Thus development of high-affinity monoclonal antibodies should result in faster, more sensitive assays. Such antibodies can be formatted into a variety of assays including traditional ELISA, bead-based assays, array-based assays, and lateral flow devices. Likewise, they can be used for immunoaffinity applications such as toxin capture and purification from a complex food matrix. A challenge for immunoassay is that it does not report on the activity of the toxin, only its presence and concentration. In an independent study Razoolie et al., used the HC specific mAb, F1-2 conjugated to magnetic beads, to concentrate toxin from milk followed by activity determination using a SNAPtide functional assay (Rasooly, R., Stanker, L.H., Carter, J.M., Do, P.M., Cheng, L.W., He, X., Brandon, D.L. In vitro peptide cleavage assay for detection of Botulinum Neurotoxin-A activity in food. (Submitted)). All of the experiments reported here use BoNT serotype A subtyp A1 obtained from the Hall A strain of C. botulinum. Of the four subtypes known, A1–A4, type A1 is the most commonly available. Antibody binding to one subtype does not necessarily guarantee binding to the other subtypes. Studies are underway to determine which subtype these mAbs bind.
Table 4 Percentage recovery of BoNT/A 900 kDa complex from milk using a sandwich immunoassay Spike level (ng mL− 1)
Skim 2% milk Whole milk
30.0
15.0
7.5
3.8
1.9
106.1 ± 2.0 91.5 ± 1.6 81.9 ± 2.2
90.3 ± 1.0 89.5 ± 3.7 84.4 ± 7.6
84.8 ± 3 83.5 ± 3.2 80.8 ± 5.3
79.12 ± 0 84.3 ± 16.3 87.1 ± 31.5
92.6 ± 20 106.1 ± 13.1 98.8 ± 23.4
SD = standard deviation. Spike level represents the amount of 900 kDa Complex added before a 10fold dilution of the sample. Data is the average of three replicates ± SD.
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