Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk

Journal of Immunological Methods 389 (2013) 18–28

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Research paper

Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk Xiaohua He ⁎, Stephanie McMahon, Craig Skinner, Paul Merrill, Miles C. Scotcher 1, Larry H. Stanker ⁎⁎ Western Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, CA 94710, United States

a r t i c l e

i n f o

Article history: Received 14 November 2012 Accepted 18 December 2012 Available online 30 December 2012 Keywords: Cytotoxicity Immunoassays Milk Monoclonal antibodies Neutralization Shiga toxin 2

a b s t r a c t Human infection by Shiga toxin producing Escherichia coli (STEC) is one of the most prevalent foodborne diseases. Shiga toxin type 2 (Stx2) is the major contributor to hemolytic–uremic syndrome (HUS) and other systemic complications caused by STEC. Although outbreaks of HUS due to the consumption of dairy products occur frequently, very few reports are available on assays for the detection of Stx2 in milk. In this study, we describe the development of five high-affinity monoclonal antibodies (dissociation constants below nM range) against Stx2 using a recombinant toxoid as an immunogen. These antibodies, designated Stx2-1, Stx2-2, Stx2-3, Stx2-4, and Stx2-5 are IgG1 or IgG2a heavy-chain subclass with kappa light-chains, did not cross-react with Stx1 and showed different preferences to variants of Stx2. Western blot analyses demonstrate that mAbs Stx2-2 and Stx2-5 bind both the A- and B-subunits, whereas the other 3 mAbs bind the A-subunit of Stx2a only. All antibodies bound stronger to the native than to the denatured Stx2a except the mAb Stx2-3, which bound equally well to both forms of the toxin. Of the five mAbs, Stx2-5 was capable of neutralizing Stx2a mediated cytotoxicity in Vero cells. Highly sensitive ELISA and immuno-PCR assays, capable of detecting 1 and 0.01 pg/mL of Stx2a in milk, were developed using mAb pair Stx2-1 and Stx2-2. Such assays are useful for routine diagnosis of Stx2 contamination in milk production process, thus reducing the risk of STEC outbreaks. Published by Elsevier B.V.

1. Introduction Shiga toxin-producing Escherichia coli (STEC) are a group of prevalent foodborne pathogens responsible for outbreaks of human gastrointestinal disease. The morbidity and mortality associated with these outbreaks have highlighted the threat

Abbreviations: CD50 , 50% cytotoxic dose; ELISA, Enzyme-linked immunosobent assay; HUS, Hemolytic uremic syndrome; IPCR, Immuno-PCR; LB, Luria–Bertani; LD50, Mean lethal dose; mAb, Monoclonal antibody; PBS, Phosphate-buffered saline; PAGE, Polyacrylamide gel electrophoresis; Stx, Shiga toxin; STEC, Shiga toxin-producing Escherichia coli; SD, Standard deviation. ⁎ Corresponding author. Tel.: +1 510 559 5823; fax: +1 510 559 5768. ⁎⁎ Corresponding author. Tel.: +1 510 559 5984; fax: +1 510 559 6429. E-mail addresses: [email protected] (X. He), [email protected] (L.H. Stanker). 1 Current address: Dupont Industrial Biosciences, 925 Page Mill Road, Palo Alto, CA 94304, United States. 0022-1759/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jim.2012.12.005

these organisms pose to public health (Karch et al., 2005; Gyles, 2007; Manning et al., 2007). STEC possess a number of virulence factors, but Shiga toxins (Stxs) were considered the most critical in disease pathogenesis and are responsible for hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) (Russmann et al., 1994; Friedrich et al., 2002). Stxs are AB5 holotoxins and are comprised of one A subunit (32 kDa) and five B subunits (7.7 kDa) (Fraser et al., 1994, 2004). The Stx A subunit is an enzymatically active N-glycosidase that inhibits the activity of rRNA by cleavage of an adenine base from the 28S rRNA component of the eukaryotic ribosomal 60S subunit, causing protein synthesis to cease resulting in cell death (Endo and Tsurugi, 1988). The Stx B subunit is responsible for binding to host cells through interaction with globotriaosylceramide (Gb3) or globotetraosylceramide (Gb4) receptors present on the surfaces of cells (Lingwood, 1993), leading to subsequent internalization of the toxin. There are

X. He et al. / Journal of Immunological Methods 389 (2013) 18–28

two serologically distinct groups of Stxs, Stx1 and Stx2. Recent epidemiological and molecular typing studies suggested that STEC strains expressing Stx2 were more virulent than strains expressing either Stx1 or both Stx1 and Stx2 (Ostroff et al., 1989; Boerlin et al., 1999). A mean lethal dose (LD50) for Stx2 of 50 ng/kg in mice was reported by Tesh et al. (1993) and Lindgren et al. (1994). In contrast to Stx1, many variants of Stx2 have been identified (Weinstein et al., 1988; Piérard et al., 1998; Bertin et al., 2001; Leung et al., 2003; Strauch et al., 2004). These variants differ from each other in terms of their affinity for host receptors, cytotoxicity, and pathogenicity. Most STEC outbreaks have been traced worldwide to the consumption of bacterial-contaminated food. Ruminants are the main reservoir for STEC strains (Hussein, 2007). Recently, the contamination of STEC in raw milk has drawn attentions. A survey on bulk tank milk from dairies across the United States showed that 4.2% samples contained one or both Stx genes (stx1 and stx2) (Karns et al., 2007). An investigation on 1422 samples from hard cheese and 80 samples from soft cheese through a 3-year monitoring program in Swiss demonstrated that 5.7% of the raw milk cheese samples were STEC-positive (Zweifel et al., 2010). Report from France indicated that the prevalence of STEC in raw milk (n= 205) reached 21% (Perelle et al., 2007). According to the Food and Agriculture Organization of the United Nations, world cheese production is around 18 million tons annually (http://faostat.fao.org) and many cheese varieties are made from unpasteurized milk. Consumption of unpasteurized milk and dairy products has resulted in illnesses due to STEC infection (Honish et al., 2005; Espie et al., 2006). Outbreak associated with drinking pasteurized milk has also occurred (Goh et al., 2002). To limit the scale of such outbreaks, prompt diagnosis and identification of the source of infection is critical. Although substantial research has been done to elucidate the role of Stx2 in these outbreaks, little attention has been paid to the production of Stxs by STEC in milk. One of the reasons is that detection of Stxs in milk is often difficult due to the combination of low toxin concentration and the complex matrix effect present in milk. Historically, the Vero cell cytotoxicity assay has played an important role in establishing a diagnosis of STEC infection and it still remains the “gold standard” for Stx activity. However, like most activity-based assays, such as the mouse bioassays, radioactivity assays, and cell-free translation assays, the Vero cell assay is timeconsuming, requires cell culture facilities, and expensive equipment that is usually not available in many laboratories. Furthermore, a subsequent antibody-based neutralization

bioassay is required in order to confirm the presence of the toxin. Other assays, such as receptor-based assays are less time-consuming and enable the discrimination of different toxins, but detailed evaluation and optimization are needed to establish these methods as analytical tools (Uzawa et al., 2007). Over the past decades, a number of immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), immunoPCR (IPCR) assay have been developed. These assays provide multiple benefits. Notably, they are simple, rapid, robotic, and could be used for high-throughput studies. However, the sensitivity and specificity of these assays are largely dependent on the quality of the antibodies used. Our recent studies on detecting botulinum neurotoxin type A in milk demonstrated that simple immunoassay formats can be highly sensitive when high-affinity antibodies are incorporated (Stanker et al., 2008). While antibodies against Stx2 have been described in the scientific literature, few are commercially available. Their expense and lack of sufficient binding affinity to the native toxins make studies focused on constructing a sensitive immunoassay difficult. In the present study, we describe the development of five high-affinity mouse monoclonal antibodies (mAbs) against Stx2 and demonstrate their performance in different applications. We also established two highly sensitive immunoassays for detection of Stx2 in milk using a pair of mAbs developed in this study. The ELISA was simple and rapid, allowed us to detect 1 pg/mL of Stx2 in milk; while the IPCR assay developed here is so far the most sensitive assay for Stx2, the Limit of Detection (LOD) in milk reached 0.01 pg/mL. 2. Materials and methods 2.1. Construction of Stx2a recombinant toxoid The glutamic acid at position 167 of the A subunit is a critical residue in the active site for enzymatic activity of Stx1 and Stx2 (Hovde et al., 1988; Gordon et al., 1992), therefore, a mutation was introduced at this position. The change of glutamate (Q) to glutamine (E) was directed by PCR using bacterial strain EDL933 (O'Brien et al., 1983) genomic DNA and primer pairs for PCR fragments 1 and 2 (Table 1). The full-length recombinant mutant stx2a was generated by connecting 2 fragments through a second round of PCR using the primer pair, Stx2A-F2 and Stx2B-R1. The DNA fragment from the second round PCR was digested with Nde I and Xho I and cloned into the pQE-T7-2 vector (Qiagen, Valencia, CA). The introduced mutation was confirmed by DNA sequencing

Table 1 Oligonucleotide sets for mutagenesis in stx2A. Primers Primers for PCR fragment 1 Stx2A-F2 (Nde I) Stx2 E167Q-R Primers for PCR fragment 2 Stx2 E167Q-F Stx2B-R1 (Xho I) Second round PCR primers Stx2A-F2 (Nde I) Stx2B-R1 (Xho I) a

Sites of mutagenesis are highlighted in gray.

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Sequencea 5′-GGAATTCCATATGAAGTGTATATTATTTAAATG-3′ 5 -CGTAAGGCTTGTGCTGTGAC-3 5 -GTCACAGCACAAGCCTTACG-3

5′-CCGCTCGAGTCTTACTAGTCATTATTAAACTGCACTTC-3′ As above As above

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X. He et al. / Journal of Immunological Methods 389 (2013) 18–28

using the ABI Prism Big Dye Terminator Sequencing Kit (Applied Biosystems, Foster City, CA). 2.2. Purification of Stx2a recombinant toxoid The plasmid containing mutant stx2a was transformed into BL21(DE3) pLysS competent cells (Promega, Madison, WI) and the cells were grown overnight at 30 °C in Luria–Bertani (LB) medium with 50 μg/mL kanamycin. The overnight culture was diluted at 1:50 in LB with kanamycin and continuously grown at 30 °C to OD600 0.6, then induced with IPTG (1 mM) overnight at 20 °C. The bacteria were sedimented by centrifugation, then lysed in 1:10 volume of phosphate-buffered saline (PBS) by sonication. The lysate was clarified by centrifugation and concentrated by precipitation at room temperature with saturated ammonium sulfate added to a final concentration of 60%. The precipitate was pelleted by centrifugation at 10,000 g for 10 min and resuspended in 0.01 M PBS with 0.138 M NaCl and 0.0027 M KCl, pH7.4 (Sigma, St. Louis, MO). After desalting using a Zeba Spin Desalting Column (7 K MWCO, Pierce Biotechnology, Rockford, IL), samples containing the Stx2a toxoid were affinity purified using a column containing an immobilized monoclonal antibody (mAb) against the Stx2 A-subunit (VT135/6-B9, Sifin Institute, Berlin, Germany). The immunoaffinity column was generated using an AminoLink Plus Immobilization Kit (Pierce, Biotechnology) and toxoid was purified following the manufacturer's instruction. Concentration of the toxoid was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL) and purity of the preparation was examined by sodium dodecyl sulfate-polyacrylmide gel electrophoresis (SDS-PAGE). Loss of toxicity of the toxoid was assessed using the Vero cell cytotoxicity assay (Neal et al., 2010). 2.3. Source of Stx1 and Stx2 variants Pure Stx1 was purchased from List Biological Laboratories, Inc. (Campbell, CA). The Stx2 variants, Stx2a, Stx2c, Stx2d, and Stx2g were purified from culture supernatants of bacterial strains RM10638, RM10058, RM8013, and RM10468 (kindly provided by Dr. Robert E. Mandrell at USDA, ARS, WRRC) and prepared as described previously (He et al., 2012). 2.4. Monoclonal antibody production 2.4.1. Cell culture media Hybridoma medium (HM) consisted of Iscove's modified Dulbecco's Minimal Medium (Sigma #1-7633) 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 cHM and 40% MPCM was used for all cell-cloning procedures. 2.4.2. Immunization and sample collection Female Balb/cJ mice (Simonsen Laboratories, Gilroy, CA) were immunized at 2-week intervals by intraperitoneal

injection (IP) of 100 μL of Stx2a toxoid (50 μg/mL) in Sigma Adjuvant System (Sigma, St. Louis, MO). Following the third injection, serum was obtained (50 μL/mouse) and evaluated for anti-Stx2 antibodies. After 2 weeks, mice with a strong antibody titer were boosted by IP injection with a single dose of Stx2a toxoid (100 μL at 10 μg/mL in PBS without adjuvant). 2.4.3. 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. 2.4.4. Screening methods Sera from immunized mice and supernatants from cell fusion plates were screened using an ELISA. Black Maxisorp 96-well Nunc microtiter plates (Thermo Fisher Scientific Inc., Waltham, MA) were coated with 100 μL/well of a 1 μg/mL of Stx2a in PBS by overnight incubation at 4 °C. The toxin solution was aspirated and non-coated sites were blocked by adding 300 μL/well of 5% non-fat dry milk in 0.02 M Tris buffered saline with 0.9% NaCl, pH 7.4 and 0.05% Tween-20 (NFDM-TBST). The plates were incubated for 1 h at 37 °C and then washed two times with TBST. Next, sera or cell culture supernatants were added (100 μL/well) and the plates were incubated at 37 °C for 1 h. Plates were washed 6 times and 100 μL/well of a 1:5000 dilution of HRP conjugated goat anti-mouse IgG (H +L) (GAM-IgG-HRP) (Promega, Madison, WI) was added and the plates were incubated for 1 h at 37 °C. The plates were then washed six times with TBST. Freshly prepared SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) was added (100 μL/well) according to the manufacturer's recommendation. The plates were incubated for 3 min at room temperature and luminescent counts were measured using the Victor-3 microplate reader (Perkin-Elmer, Shelton, CT). 2.4.5. Antibody production and purification Cells from the wells producing antibodies that bound to Stx2a were cloned by limiting dilution. Hybridomas were then expanded and ascites fluids (10 to 30 mL) were obtained (Covance Research Products, Inc., Denver, PA). Antibodies were purified by affinity chromatography on Protein-G conjugated Sepharose (Sigma, #P-32196) and bound antibodies were eluted with 0.1 M glycine-HCl, pH 2.7. Protein concentrations were determined with the BCA Protein Assay Kit (Pierce). The attachment of biotin to antibodies was performed using a Lightning-Link Biotin Conjugation Kit (Innova Biosciences, Cambridge, UK). Antibody isotype was determined by ELISA using toxin-coated microtiter plates and horseradish peroxidase-conjugated, isotype-specific antibodies (SouthernBiotech, Birmingham, AL). 2.5. Characterization of mAbs 2.5.1. Capture ELISA In order to identify the best antibody pair for a capture ELISA all possible pairs of mAbs were evaluated. Black NUNC

X. He et al. / Journal of Immunological Methods 389 (2013) 18–28

plates were individually coated with each mAb (100 μL/well of a 1 μg/mL solution in PBS) and incubated overnight at 4 °C. Plates were then blocked by adding 300 μL of 3% bovine serum albumin (BSA) in TBST and incubating for 1 h at 37 °C. Next, plates were washed two times with TBST and stored for up to 10 days at 4 °C before use. After toxin standards and samples (100 μL/well in PBS) were added, the plates were incubated for 1 h at 37 °C and then washed six times with TBST. Next, each mAb was biotinylated and used as the detection antibody (100 μL/well of a 1 μg/mL solution in 3% BSA-TBST). The plates were incubated for 1 h at 37 °C. The plates were washed six times with TBST and then 100 μL/well of 1:20,000 dilution of streptavidin-HRP (Invitrogen, Carlsbad, CA) in 3% BSA-TBST was added. The plates were incubated for 1 h at 37 °C. Finally, the plates were washed six times with TBST and SuperSignal West Pico Chemiluminescent Substrate (Pierce) was added. The LOD was defined as the lowest toxin concentration at which the average ELISA reading was three standard deviations above the negative control. 2.5.2. Polyacrylamide gel electrophoresis (PAGE) and Western blot All gel electrophoresis equipment, buffers, gels and PVDF membranes were purchased from Invitrogen. Toxin-specificity of each mAb was analyzed by Western blot. Purified wild type Stx2a was separated by Native- or SDS-PAGE using 4–12% Native or NuPAGE (denatured) Novex Bis–Tris mini gels following the manufacturer's protocol. To visualize proteins directly after gel electrophoresis, 2 μg of toxin was loaded in each lane and gels were stained with Coomassie Blue G-250 (Bio-Rad, Hercules, CA). For Western blot analysis, 0.5 μg of toxin was loaded and separated by PAGE. Proteins were electrotransferred to a PVDF membranes (0.45 μm). The membranes were blocked with 5% NFDM, then probed with mouse serum (1:10,000) or anti-toxin mAbs (20 μg/mL), followed by GAM-IgG-HRP (1:500,000). Bound antibody was detected using the Amersham ECL-Plus Western Blotting Detection System (GE Healthcare, UK) according to the manufacturer's protocol. 2.5.3. Antibody–antigen binding affinity measurements Real time binding assays between purified antibodies and purified Stx2a protein were performed using biolayer interferometry with an Octet QK system (Forte-bio, Menlo Park, CA). The system measures light interference on the surface of a fiber optic sensor, which is directly proportional to the thickness of molecules bound to the surface. Targets of interest are chemically tethered to the surface of the sensor using biotin–streptavidin interactions. Binding of a partner molecule to the tethered target results in thickening of the surface, which is monitored in real time. In this study, the biotinylated mAbs were coupled to kinetics grade streptavidin biosensors (Forte-bio) at 10 μg/mL in PBS. Unbound antibodies were removed from the surface of the sensors by incubation in PBS. Probes coupled to antibody were allowed to bind to Stx2a at seven different concentrations ranging from 2 to 142 nM. Binding kinetics were calculated using the Octet QK software package (Data Acquisition 7.0), which fit the observed binding curves to a 1:1 binding model to calculate the association rate constants. The Stx2a protein was allowed to dissociate by incubation of the sensors in PBS. Dissociation kinetics were calculated using the Octet QK software package, which fit the

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observed dissociation curves to a 1:1 model to calculate the dissociation rate constants. Equilibrium dissociation constants were calculated as the kinetic dissociation rate constant divided by the kinetic association rate constant. Statistical differences between dissociation constants were analyzed by One Way Anova, Tukey's Multiple Comparison Test using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA). Differences between numbers were considered significant at P b 0.05. 2.5.4. IPCR IPCR was performed as described previously (He et al., 2010, 2011). Briefly, 30 μL of 8 μg/mL purified mAb against Stx2 B-subunit (VT136/8-H4, Sifin Institute, Berlin, Germany) in borate buffer was used to coat microtiter wells overnight at 4 ºC. After blocking non-adsorbed sites with 150 μL of blocking buffer for 1 h at RT, 30 μL of Stx2 standards or samples were added to each well and the plates were incubated at RT for 1 h. The plates were washed with TBS-EDTA–Tween (Tris-buffered saline containing 5 mM EDTA and 0.05% Tween 20), 30 μL of streptavidin-conjugated MAb against Stx2 A-subunit, VT135/ 6-B9 (500 ng/mL), was added to each well and incubated at 37 ºC for 30 min. After washing, 30 μL of biotinylated DNA marker (0.5 ng/μL) was added to each well and incubated at RT for 30 min. After thorough washing to remove unbound DNA, the bound DNA was detached from the immunocomplex by restriction enzyme, BamHI, and a 6-μL portion was used as a template in a real-time PCR (20 μL). The LOD was defined as the lowest toxin concentration tested at which the average Ct value was three standard deviations below the Ct value of the negative control (Niemeyer et al., 2007). 2.5.5. Neutralization of Stx2a mediated cytotoxicity in Vero cells An in vitro cytotoxicity assay was used to evaluate the neutralization ability of the mAbs. Fresh Vero cells were seeded on 96-well plates at 1 × 105 cells/mL (100 μL/well) overnight in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and incubated in a humidified incubator (37 °C, 5% CO2). Cells were first treated with Stx2a (10 ng/mL), mAb (20 μg/mL), or Stx2a (10 ng/mL) plus mAb (20 μg/mL) at 4 °C for 1 h, then shifted to 37 °C overnight. The cytotoxicity was assessed using CellTiter-Glo reagent (Promega) according to the manufacturer's instruction, except that the reagent was diluted 1:5 in PBS prior to use. Luminescence was measured with a Victor 3 plate reader (Perkin Elmer). All treatments were performed in triplicate. Cells grown in medium without toxin were used as a negative control (0% toxicity). The cytotoxicity for cells was calculated as follows: [(cps from negative control − cps from samples treated) / cps from negative control] × 100. The relative cytotoxicity after neutralization was calculated by normalizing the toxicity of Stx2 without neutralization by mAb as 100%. 3. Results 3.1. Assessment of the Stx2a toxoid It was reported that the glutamic acid at position 167 of the A-subunit was the active site for enzyme activity for both Stx1 and Stx2 (Hovde et al., 1988; Jackson et al., 1990; Wen et al.,

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2006). Therefore, the glutamate at this position was changed to glutamine. The purity of the toxoid, Stx2 E167Q, prepared in this study was assessed following SDS-PAGE by Coomassie staining and Western blot with a mixture of commercial mAbs, VT135/6-B9 and VT136/8-H4 against the Stx2 A- and B-subunit (Sifin Institute, Germany). Two protein bands were observed with molecular weights of approximately 32 kDa and 7 kDa,

corresponding to the sizes of the A and B subunits of Stx2 and no contaminating proteins were visible in the toxoid preparation (Fig. 1A). Next, the cytotoxicity was assessed in Vero cells to confirm that the toxoid was non-toxic. Fig. 1B shows the observed cell viability (92% and 89%) when cells were treated with this toxoid at concentrations of 5 and 10 ng/mL [500

A

B 100

Cell viability (%)

80

60 Stx2a 40 Stx2 E167Q 20

0 0

5

10

Toxin concentration (ng/mL) Fig. 1. Analysis of genetic toxoid of Stx2a. A. Coomassie staining and Western blot of purified Stx2a toxoid following SDS-PAGE. Lane 1, Coomassie stained SDS-PAGE with 1 μg of purified Stx2a toxoid. Lane 2, Western blot of 0.5 μg of Stx2a toxoid analyzed with mixture of mAbs against Stx2 A- and B-subunits. The A and B subunit positions are indicated by arrows at the right side and their molecular weights are labeled as kilodaltons (kDa) at the left side of the panel. B. Effect of genetic toxoid, Stx2E167Q, on growth of vero cells. The relative cell viability was calculated by normalizing their values to the viability of cells without adding toxin as 100%. The results represent the mean± SD of three replicates from one representative experiment. Three individual experiments were performed.

X. He et al. / Journal of Immunological Methods 389 (2013) 18–28 Table 2 Some characteristics of the Stx2 monoclonal antibodies. Antibody

Isotype

Specificity

KD (×10−9 M)⁎

Stx2-5 Stx2-2 Stx2-1 Stx2-4 Stx2-3

IgG1, kappa IgG2a, kappa IgG1, kappa IgG1, kappa IgG1, kappa

A & B-subunit A & B-subunit A-subunit A-subunit A-subunit

0.28 ± 0.08 0.71 ± 0.06 1.28 ± 0.04 1.46 ± 0.01 1.55 ± 0.03

a b c c c

⁎ Differences between numbers with the same letter were not statistically significant and between numbers with different letters were statistically significant (P b 0.05).

and 1000 times the cytotoxic dose (CD50) of the native toxin, respectively]. We concluded that the toxicity of the toxoid was not completely abrogated, but significantly reduced. 3.2. Isolation and characterization of monoclonal antibodies against Stx2 To identify mAbs against Stx2, we screened 2000 culture wells following two splenocyte–myeloma cell fusions. Positive signals (signal-to-noise of 5 or greater) were observed for 127 of the supernatants. The cells from these wells were expanded, tested, and cloned by limiting dilution to produce hybridoma lines. Of these hybridomas, we chose 5 for further investigation based on mAb affinity, subunit specificity, and neutralization activity. These antibodies designated Stx2-1, Stx2-2, Stx2-3, Stx2-4, and Stx2-5 were further characterized. Table 2 summarized the results of the antibody characterization studies. Isotype analysis demonstrated that mAbs Stx2-1, Stx2-3, Stx2-4, and Stx2-5 have IgG1, and that mAb Stx2-2 has an IgG2a type heavy chain. All of the mAbs possess kappa light-chains. In order to determine the subunit-specificity for each antibody, pure Stx2a was probed by Western blot following SDS-PAGE (Fig. 2A). These results demonstrate that mAbs Stx2-1, Stx2-3, and Stx2-4 bound to the A-subunit. In contrast, mAbs Stx2-2 and Stx2-5 bound to both A- and B-subunits. In addition, an unknown protein band above the A-subunit was bound by these two antibodies. Western blots

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following native gel electrophoresis of Stx2a indicate that all five mAbs were able to bind the native holotoxin (Fig. 2B). Four of the mAbs had weaker binding on the Western blots following SDS-PAGE (denatured Stx2a) compared to binding following native gel electrophoresis. Monoclonal antibody Stx2-3 exhibited strong binding to both denatured and native Stx2a protein (compare Fig. 2A and B). All five mAbs failed to bind to the Stx2a denatured by heat at 100 °C for 5 min when tested by direct binding ELISA (data not shown). To confirm the specificity of the mAbs and to quantitate the affinity of each antibody for the Stx2a protein, we used biolayer interferometry to examine mAb binding to the purified Stx2a protein. As expected from Western blot results, all five mAbs bound to the Stx2a protein. Stx2-5 showed the strongest binding, with a dissociation constant of 0.28 × 10 −9 M. Next was mAb, Stx2-2, with a dissociation constant of 0.71 × 10 −9 M. The dissociation constant of mAbs Stx2-1, Stx2-3, and Stx2-4 for Stx2 were similar and lower, with dissociation constants of 0.13 × 10 −8 M, 0.14 × 10 −8 M, and 0.15 × 10 −8 M, respectively.

3.3. Specificity of mAbs in direct binding ELISA The ability of five mAbs to bind Stx1 and different variants of Stx2 was evaluated by ELISA. In these experiments Stx1 and four Stx2 variants available in the laboratory were absorbed onto microplates in PBS buffer. It appears that the five mAbs do not bind Stx1 and have different binding preferences to the four variants of Stx2 (Fig. 3). The mAb Stx2-2 bound to Stx2a and Stx2g with virtually no binding to the Stx2c and Stx2d. The mAbs Stx2-3 and Stx2-5 bound very well to Stx2c, poorly to Stx2g, and intermediately to Stx2a and Stx2d. The mAbs Stx2-1 bound equally well to Stx2a and Stx2d but poorly to the other toxin variants. The mAb Stx2-4 preferentially bound Stx2d and had lower reactivity to the toxins overall compared with other mAbs. These results indicate that the five mAbs are Stx2-specific and distinct from one another, suggesting that they recognize different epitopes.

Fig. 2. Analysis of mAb binding to Stx2a protein. A. Western blot of Stx2a following SDS-PAGE. Stx2a holotoxin (0.5 μg) was separated by SDS-PAGE. Membranes were probed with mAbs: 1. Stx2-1; 2. Stx2-2; 3. Stx2-3; 4. Stx2-4; and 5. Stx2-5, respectively. The sizes of the Stx2a A- and B-subunits are indicated as kilodalton (kDa) at the left side of the panel. B. Western blot of Stx2a following native PAGE. Stx2a holotoxin (0.5 μg) was separated by native polyacrylamide gel electrophoresis. Membranes were probed with mAbs: 1. Stx2-1; 2. Stx2-2; 3. Stx2-3; 4. Stx2-4; and 5. Stx2-5, respectively. The size of the Stx2a holotoxin is indicated as kilodalton (kDa) at the left side of the panel.

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Relative luminescent counts

450000 400000 350000 300000

Toxin

250000

Stx2a Stx2d

200000

Stx2c

150000

Stx2g

100000

Stx1

50000 0 Stx2-1

Stx2-2

Stx2-3

Stx2-4

Stx2-5

Monoclonal antibody Fig. 3. Determination of binding specificity of mAbs to different variants of Stx2 by direct ELISA. Microtiter wells were coated with 1 μg/mL of the Stx2 variants, Stx2a, Stx2c, Stx2d, Stx2g, and Stx1, respectively. The binding of mAbs, Stx2-1, Stx2-2, Stx2-3, Stx2-4, and Stx2-5 to these variants were measured. The data shown represent the mean ± SD of three replicates from one representative experiment. Three individual experiments were performed.

3.4. Sandwich ELISA and immuno-PCR In order to develop sensitive immunoassays for Stx2 detection, all possible combinations of mAb pairs were evaluated using each of the five mAbs as either the capture or detector antibody (pre-labeled with biotin for ELISA, and streptavidin for IPCR). The data shown in Fig. 4 indicates that the best result was obtained when using mAb Stx2-1 as a capture antibody and biotinylated mAb Stx2-2 as a detector antibody in the ELISA. Significantly lower signals were observed using any of the other combinations of capture and detector antibody. A sandwich ELISA incorporating biotinylated mAb Stx2-2 as a detector antibody and mAb Stx2-1 as a capture antibody was further studied. A linear standard curve with R 2 = 1 was observed using Stx2a at the range of 10 to 1000 pg/mL (Fig. 5). The LOD was between 1 pg/mL for Stx2a in PBS buffer.

To increase detection sensitivity, a sandwich IPCR developed previously (He et al., 2011) was tested using the same pair of antibodies. The LOD for Stx2a reached 0.01 pg/mL in PBS buffer (Table 3). It is worth to note that the mAb (Stx2-5) with the highest affinity (Table 2) did not do so well as a capture antibody. It seems that mAb affinity does not correlate directly with utility for toxin detection in this study.

3.5. Detection of Stx2a in milk The sandwich ELISA and IPCR established above were validated for detection of Stx2a in milk matrices. Undiluted milk (1 mL) was spiked with 10 μL of PBS containing varying amounts of Stx2a and analyzed directly. Results from the assays indicated that the LOD for Stx2a by ELISA was 1 pg/mL

Relative Luminescent Counts

3000000

2500000

Detector mAb Stx2-1

2000000

Stx2-2 Stx2-3 Stx2-4

1500000

Stx2-5

1000000

500000

0 Stx2-1

Stx2-2

Stx2-3

Stx2-4

Stx2-5

Capture mAb Fig. 4. Sandwich ELISA for detection of Stx2a. Each of the five mAbs was used as capture antibody or as the biotinylated detector antibody. Relative luminescent counts were measured for each antibody combination using Stx2a at 10 ng/mL.

X. He et al. / Journal of Immunological Methods 389 (2013) 18–28

25

450000

Relative Luminescent Counts

400000 y = 525x + 3649 R² = 1

350000 300000 250000 200000 150000 100000 50000

Bkg+ 3 SD = 7230 0 0

100

200

300

400

500

600

700

800

Stx2 (pg/mL) Fig. 5. Detection of Stx2a in PBS using mAb Stx2-1 as capture antibody and Stx2-2 as detector antibody. Diamonds represent the average of three determinations ± one SD. Horizontal dashed line equals the average counts from samples without spiking Stx2a plus three SD.

in 2% milk and whole milk. The LOD for Stx2a by IPCR was 0.01 pg/mL in 2% milk and whole milk (Table 3).

4. Discussion

3.6. In vitro toxin neutralization To test the ability of the five mAbs in neutralization against the cytotoxicity of Stx2a, Vero cells (100 μL of 0.5 × 10 5 cells/mL) were seeded in wells of a clear 96-well tissue culture plate and incubated overnight. Cells were then treated with DMEM medium (as a negative control), Stx2a (10 ng/mL), mAb (20 μg/mL), and Stx2a (10 ng/mL) + each mAb (20 μg/mL), respectively. In the absence of mAbs, 73% of the toxin treated cells died within 24 h at a dose of 10 ng/mL. In the presence of individual mAbs Stx2-1, Stx2-2, Stx2-3, and Stx2-4 the cytotoxicity measured was similar to the cells without adding mAb. However, cells treated with Stx2 in the presence of mAb Stx2-5 were totally protected from death and the cell survival rate was similar to the DMEM medium (no-toxin control). No toxicity was observed for cells treated with any individual mAb without the presence of the toxin

Table 3 Detection of Stx2a by ELISA and IPCR.a. Assay

Stx2a

PBS

ELISA

Spike level (pg/mL)

Avg counts

SD

Avg counts

SD

Avg counts

SD

0 1 10

7080 7570 13,980

50 120 480

6730 7155 14,035

10 25 175

6090 7020 8365

50 100 645

Spike level (pg/mL)

Avg Ct

SD

Avg Ct

SD

Avg Ct

SD

0 0.01 0.1

31.79 30.64 29.89

0.32 0.24 0.2

31.46 30.29 29.26

0.35 0.31 0.28

33.53 31.64 30.45

0.53 0.49 0.33

IPCR

(only the mAb Stx2-5 treatment was shown in Fig. 6; data from other mAb treatments were not shown).

2% milk

Whole milk

a The LOD for Stx2a in PBS, 2% milk, and whole milk by ELISA was 1 pg/mL, by IPCR was 0.01 pg/mL.

The aim of the present study was to produce high affinity mAbs that can be used for development of rapid and sensitive assays for Stx2 in milk. For this purpose, a genetic toxoid of Stx2a was generated by changing a single amino acid previously shown to be critical to the enzymatic activity of the A subunit (Hovde et al., 1988). Using genetic toxoid as an immunogen is advantageous since conventional inactivation of toxin by hazardous chemicals like formaldehyde or gluteraldehyde, may result in residual toxicity (Metz et al., 2003). Additionally, genetic toxoid preserves the holotoxin structure lost following toxoid production by formaldehyde or gluteraldehyde treatment. Thus resulting antibodies should have better binding to the native biologically active toxin; while toxoid generated by chemical or physical means is often distorted in structure and therefore, antibodies produced often react with toxoid but not the biologically active toxin (Stanker et al., 2008). In this study, all antibodies screened with the genetic toxoid also bound the wild type, active toxin as shown by results from the ELISA (Figs. 3 and 4) and the bindings of these antibodies (except the Stx2–3) were stronger to the native Stx2a than to the denatured toxin (Fig. 2) based on the density of the protein bands on the Western blots. We suspect that the weak signals on the Western blot following the SDS-PAGE may have resulted from the binding of these mAbs to the residual native fraction of Stx2a or re-natured protein. This hypothesis is supported by our observations of no reactivity between antibodies and the heat-denatured toxins in direct binding ELISA. Among dozens of mAbs characterized, most of them were specific to the Stx2a A-subunit, only two of the mAbs bound to the B-subunit even though the amount of Stx2a B-subunit present in the toxoid preparations was similar to the A-subunit (Fig. 1). This observation is in agreement with

26

X. He et al. / Journal of Immunological Methods 389 (2013) 18–28

80

Cytotoxicity (%)

70 60 50 40 30 20 10 0

Treatment Fig. 6. Neutralization of Stx2a cytotoxicity with mAbs. Vero cells were incubated in DMEM medium containing Stx2a (10 ng/mL) with or without the presence of mAbs Stx2-1, Stx2-2, Stx2-3, Stx2-4, and Stx2-5. The cytotoxicity for cells was calculated as: [(cps from negative control − cps from samples treated) / cps from negative control] × 100. Cells grown in DMEM medium were used as a negative control. The data shown represent the mean ± SD of three replicates from one representative experiment. Three individual experiments were performed.

previously reported studies (Padhye et al., 1989; Wen et al., 2006) suggesting that the Stx2 B-subunit is less immunogenic than the A-subunit in mice. The two mAbs, Stx2-2 and Stx2-5 bound to both the A- and B-subunits (Fig. 2A). These antibodies may recognize an epitope that spans both subunits, or they recognize a common epitope present in both subunits. Alignment of the amino acid sequences of Stx2a A and B subunits using PRSS3 (http://www.ch.embnet.org/software/ PRSS_form.html) did reveal a number of consensus sequences (Table 4) present in both the A and B subunits. In spite of their similarities shown on the Western blot, it is clear from the toxin subtype binding experiments (Fig. 3) that the five mAbs bind different epitopes. Each mAb exhibited a unique reaction profile to four toxin variants. Exclusively binding to a single variant of Stx2 was not observed for any of the mAbs and Table 4 Alignment of amino acid consensus sequences between A- and B-subunits of Stx2a. Subunit

Amino acids

Position

Consensusa

A B A B A B A B A B A B A B A B A B

TIDFSTQQS TIKSSTCES IDFS IEFS GSYFA GSGFA DVTTV DTFTV VTTVSMTTDS VTIKSSTCES MEFS IEFS AVLRFVTVT AQLTGMTVT EDGVRVGRISFNN ESGSGFAEVQFNN QITGDRPVIK QLTGMTVTIK

4–12 50–58 5–8 8–11 47–51 59–63 102–106 17–21 103–112 49–58 143–146 8–11 157–165 42–50 215–227 57–69 261–270 43–52

TIxxSTxxS

a

x is any amino acid.

IxFS GSxFA DxxTV VTxxSxTxxS xEFS AxLxxxTVT ExGxxxxxxFNN QxTGxxxxIK

most, except Stx2-2 bound all tested variants to some extent. Antibody cross-reactivity is not surprising because of the high similarity (>95%) in amino acid sequence among these variants (He et al., 2012). It has been reported that Stx1 and Stx2 share about 60% deduced amino acid sequence homology (Jackson et al., 1987). Reports in the literature about whether these two toxins are antigenically distinct have been contradictory (Wen et al., 2006; Jeong et al., 2010). Our results indicated that the five mAbs developed from the Stx2a toxoid-immunized mice reacted only to Stx2, but not to Stx1 (Fig. 3), supporting that Stx1 and Stx2 are distinct antigens for mice. Among the five mAbs, only Stx2-5 neutralized Stx2a in Vero cells (Fig. 5). Although the prevalence of STEC in raw milk and dairy products has been aware (Karns et al., 2007; Martin and Beutin, 2011), very little is known about the quantity of Stxs produced and the conditions required for Stx production by STEC in food (Weeratna and Doyle, 1991; Abdul-Raouf et al., 1994), in part because of the lack of sensitive methods to detect the toxin in food. Immunoassays for Stxs such as Premier EHEC, rBiopharm Ridascreen Verotoxin Enzyme Immunoassay and etc. are available commercially, but they are usually not sensitive enough for quantitative measurement (Willford et al., 2009). Monoclonal antibodies against Stxs have also been amply described in the literature (Smith et al., 2006, 2009; Willford et al., 2009), but most of them were produced for neutralization or other purposes, and therefore, not necessarily ideal for toxin detection. One of the goals of this study was to develop sensitive assays to detect Stx2 in milk. The sandwich ELISA established here using the mAb, Stx2-1, as a capture antibody and the biotinylated mAb, Stx2-2, as a detector antibody allowed us to detect 1 pg/mL of Stx2a in PBS, which is 1000- to 112,000-fold more sensitive than published results (Weeratna and Doyle, 1991; He et al., 2011; Parma et al., 2012) and no matrix effect was found from milk for the assay. We also compared the performance of IPCR using the mAb pair, Stx2-1/Stx2-2 in this study, with our

X. He et al. / Journal of Immunological Methods 389 (2013) 18–28

previous IPCR results using the popular commercial mAb pair, VT135/6-B9 and VT136/8-H4, and found that the LOD of the current IPCR for Stx2 was 0.01 pg/mL in PBS, 10-fold lower than the LOD of the previous IPCR using the commercial primers (He et al., 2011), although the commercial antibody pair was known to be sensitive and broadly used for detection of Stx2 (Zhang et al., 2008; Rasooly et al., 2010; Clotilde et al., 2011; He et al., 2011). The same detection limit (0.01 pg/mL) was obtained for Stx2 in 2% milk and whole milk using the current IPCR protocol. We believe that the ELISA and IPCR developed in this study could be potentially adapted for early recognition of Stx2 contamination in other agricultural products, environmental and human biological samples, thus prompting implementation of control measures to prevent outbreaks caused by STEC. Acknowledgments This research was supported by USDA-ARS National Program NP108 CRIS project 5325-42000-048-00D. The U.S. Department of Agriculture is an equal opportunity provider and employer. We thank Dr. Robert E. Mandrell and Beatriz Quinones for providing all STEC strains; Dr. Robert Hnasko and Luisa Cheng for reviewing the manuscript.

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