Staphylococcal food poisoning (SFP), a form of enteritis, is intoxication rather than a disease resulting from ingestion of food contaminated with preformed staphylococcal enterotoxins (SEs). Symptoms of SFP usually occur within 1-6 hrs after the food intake and are characterized by nausea, vomiting, abdominal cramps and diarrhoea. These symptoms usually subside in 1-3 days but the patient remains sick for 7-10 days due to the result of toxic shock (Do Carmo et al. 2004). Twenty different types of SEs, viz., SEA through SEE, SEG through SER and SEU have already been discovered, however, only a few of the toxin serotypes are frequently associated with food poisoning outbreaks (Fernández et al. 2006). Toxin serotypes frequently associated with food poisoning outbreaks are SEA, SEB, SEC and SED (Smyth et al. 2005).

Staphylococcal enterotoxins are ‘Superantigens’ that bind non-specifically to the major histocompatability complex (MHC) class II antigen, causing massive T cell stimulation coupled with the release of cytokines. The release of cytokines results in stimulation of neuro-receptors in the intestinal tract, and triggers vomiting centre in the brain (Hemalatha et al. 2004). This effect is more severe in case of intoxication involving SEB, which is also a listed biological warfare (BW) agent (Boles et al. 2003). Therefore, it is of utmost importance to develop immunological detection system for SEB, the success of which is dependent on the purity of SEB used for generating anti SEB antibodies.

Conventionally, molecular sieving and ion exchange chromatography have been used either independently or in tandem for purification of Staphylococcal enterotoxins. Isoelectric focusing has been proved useful for purification of small amounts of staphylococcal enterotoxins but on a preparative scale the procedure has generally been used in combination with ion exchange chromatography and gel filtration. Use of these techniques in tandem is tedious, time consuming, expensive and sometimes does not result in purification of staphylococcal enterotoxins to the level of homogeneity. A slight contamination of toxin with other bacterial proteins results in antibodies that are less specific for the enterotoxin and hence have poor detection potential leading to false positive results. Therefore, in the present investigation, the staphylococcal enterotoxin B (seb) gene was cloned and co-expressed with biotin as fusion partner facilitating single step affinity purification. The suitability of the purified recombinant SEB was also studied for raising anti SEB antibodies.

 In this study, SEB producing S. aureus ATCC14458 was used to amplify the toxin gene for cloning and expression. Amplification of seb gene of S. aureus ATCC14458 using primers designed at out laboratory resulted in 721 bp amplicon. The seb amplicon was purified and ligated at 16ºC for 4 hrs with fusion vector, PinPoint Xa-1 T, containing biotin tag. The ligated product was transformed into the expression host E. coli JM109 following the protocol reported earlier (Devvrat et al. 1994). Transformants were plated on LB agar containing ampicillin (100 µg/ml) for the selection of recombinant clones. PCR was performed to screen the clones for the presence of toxin gene. The toxin gene in the vector was sequenced to check the possible point mutation. Clones, found positive by PCR for the presence of seb gene, were checked for the expression after IPTG (isopropyl-β-D-thiogalactopyranoside) induction, lysis and SDS-PAGE. A thick band of r-SEB toxin was observed at approximately 41.4 kDa position upon induction of recombinant clones by IPTG. The position of the r-SEB toxin was also confirmed by western blots developed with streptavidin-alkaline phosphatase conjugate and rabbit anti SEB antibodies raised earlier in the laboratory against natural SEB toxin (Nema et al. 2004).

One of the SEB positive recombinant clones, 1SEBR4, was used to standardize the growth and induction conditions for expression of recombinant SEB (r-SEB). Optimum optical density (OD) for adding the inducer, i.e., IPTG was 0.3-0.4 which was achieved within 2-3 hrs of culture inoculation. The maximum yield of r-SEB was obtained when 1SEBR4 was induced by IPTG at a concentration of 75 µM for four hours. Using these optimized conditions r-SEB was produced and purified from cell lysate using SoftLink^TM Soft Release Avidin Resin (Promega Corp., USA). Batch mode affinity purification was done, wherein the purity was confirmed by the presence of single band of r-SEB on SDS-PAGE and western blot. The advantage of using biotin as purification tag was reflected in the purity of the r-SEB. The yield of purified r-SEB was 6.6% (w/w) of total protein and 13.1 mg/L of culture broth. The r-SEB was subjected to Factor Xa protease digestion to remove the biotin affinity tag so that its possible interference with specific and high titre antibody generation against r-SEB can be avoided. Factor Xa protease concentration of 8% (w/w) was found optimum for complete removal of biotin tag from r-SEB when incubated for 16-18 hrs. The N-terminal sequencing of the protease-digested r-SEB confirmed its proper cleavage by Factor Xa protease. The LC-MS profile of the protease digested r-SEB showed that the amino acid sequence of r-SEB was matching with the natural SEB toxin. Size of the protease-digested r-SEB was estimated to be 29.5 kDa by LC-MS.

The protease digested r-SEB (29.5 kDa) and undigested r-SEB (41.4 kDa) were used for immunizing mice. Serum titres obtained in both the cases were 1:32000 as revealed by western blot. These sera were further evaluated for their cross reactivity with SEA, SEC and SED enterotoxins, non-enterotoxigenic S. aurues strain ATCC6538P, E. coli host JM109, Bacillus subitils, Enterococcus faecalis, Bacillus anthracis, Shigella dysenteriae, Salmonella typhi, Clostridium perfringens type A, Clostridium botulinum types A and E, and Staphylococcus epidermidis using indirect ELISA (Agarwal et al. 2002). No cross reactivity of the sera was observed either with other non-SEB enterotoxins of S. aureus or with the tested strains by the test formats used, i.e., western blot and indirect plate ELISA. Staphylococcal enterotoxins and bacterial species used in this investigation for evaluation of the cross reactivity of the anti r-SEB serum are the ones which are either commonly present in the food or are important biological warfare agents.  Further, the antiserum raised in this study was found more specific for SEB detection than the other commercial antiserum available from M/s Toxin Technology, USA. During comparative evaluation of sera using western blot technique, commercial antiserum showed multiple non-specific bands even at 1:10000 dilution with culture supernatant of SEB producing S. aureus strain ATCC14458 and non-enterotoxigenic S. aureus. Both the antibodies were used at 1:2000, 1:5000 and 1:10000 dilutions which are commonly employed for ELISA and western blot analysis. Antiserum raised in the present investigation did not show cross reactivity even at 1:2000 dilution. This demonstrates the superiority of the antiserum raised against purified r-SEB in this study and the importance of toxin purity for polyclonal antibody production.

Expression of r-SEB with biotin as fusion partner facilitates the single step affinity purification of r-SEB for generation of specific antibodies against it. Removal of biotin tag from the expressed protein is a costly and time-consuming process. In this study, r-SEB without biotin fusion did not show any advantages over r-SEB fused with biotin for raising antiserum in terms of titre and corss-reactivity. Therefore, r-SEB alongwith biotin tag can be used for polyclonal antibody generation saving a lot of downstream processing time and cost. The anti r-SEB serum developed during the present study is specific and highlights the need of pure SEB toxin for generation of specific polyclonal antibodies. This antiserum can be used in a suitable format for development of an immunological system for detection of SEB. This can also be used for quality control of food products as well as for retrospective detection of SEB in cases of sabotage and/ or covert bioterrorism activities.

Authors are thankful to Er. Shri K. Sekhar, Director DRDE Gwalior and Dr. M.C. Varadaraj, Scientist ‘E’, CFTRI, Mysore for their help and support during the course of this investigation.

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