Poster presentado en Med-Vet-Net Association 5th International Scientific Conference (OneHealth: Zoonoses - Emerging Threats)

27 de junio de 2017

Moreno I., Perez-Sancho M., Gonzalez S., Infantes-Lorenzo JA., Ugarte-Ruiz M., Rodriguez-Bertos A., Dominguez L. y Dominguez M.

Almost 10% of all food-borne outbreaks occurred on EU in 2015 were caused by staphylococcal enterotoxins (SEs) (EFSA. 2016). Diarrhea, vomiting and cramps are clinical signs traditionally associated with food poisoning by classical SEs (SEA-SEE) (Kadariya et al., 2014). Nevertheless, is it foreseen that additional SEs are involved in cases of illness, although their clinical importance has to be elucidated. Immunological detection assays for SEs are important to control and survey food-borne diseases associated with S. aureus. To this end we have developed an antibody-based enterotoxin B Staphylococcus aureus sandwich ELISA which improves the current detection level of SEB reported in the literature (Wu et al., 2016).

METHODS AND RESULTS

Development of monoclonal antibodies (mAb) against SEB.
BALB/c mice were immunized with three i.p. injections of SEB done two weeks apart. The first of 50 µg/0.1 ml emulsified in CFA, and the other two of 25 µg/0.1 ml emulsified in IFA. Cell hybridacion was carried out following traditional methods (Goding, J. W. 2004). Culture supernatants were screened for anti-SEB reactivity by indirect ELISA, Western blot and capture ELISA. Positive wells were clone twice by limiting dilution in Clonacell-Hy medium. Twelve mAb were selected whose characteristics are summarized in Table 1

Development of polyclonal antibody (pAb) against SEB.
Anti-SEB pAb were obtained in rabbits immunized three times with SEB at three week intervals first with 200 ug SEB in CFA and the following two with 100 ug of SEB in IFA. Rabbits were bled and their sera tested against SEB by indirect and capture ELISA and by western-blotting.

Capture ELISA
Plates were coated (16h, 4ºC) with 50 µl of a mixture (1.1)of purified mAb, SIM 247-3.1.5. and SIM 255-13.2.3 (5 µg/ml PBS). After coating, plates were blocked (30 min, 37ºC) with 75 µl of 2% BSA and then washed 3 times with PBS-Tween 0.05% (PBST). To develop the standard dose-response curve 50 ul/well of three-fold serially diluted of SEB (from 10 ng/ml to 0.001ng/ml) were incubated for 2 h at 37ºC. Afther incubation biotin-labelled mAb SIM 247-3.2.5 (1/500 PBST-diluted) was added (1h at 20ºC). At the end 50 µl of 1/2000 PBST-diluted streptavidin-HRP added; plates were incubated (30 min, 20 ºC), washed three times and 100 µl/well of OPD/H2O2 reagent (0.4 mg/ml of o-phenylenediamine in 0.2 M disodium phosphate-0.1 M citric acid, pH 4.9, supplemented with 1/1000 volume of hydrogen peroxide) were added and incubated (5 min., 20 ºC, in the dark). The reaction was terminated by addition of 50 µl/well of 3 N H2SO4 and the absorbance recorded at OD 492 nm in a microtiter plate reader (Anthos 2020). The standard curve obtained is shown in Figure 1. To improve the sensitivity of the assay we developed a sandwich ELISA using biotin-labelled rabbit antibody anti-SEB (Figure 2).

ACKNOWLEDGEMENS
Collaboration with the staff of VISAVET and ISCIII is greatly acknowledged.
TAVS-CM R&D Grant Programme of Regional Interest of the Madrid Regional Government 2013. S2013/ABI-2747. 2014-2018.

REFERENCES
European Food Safety Authority, European Centre for Disease Prevention and Control. 2016. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. EFSA Journal. 14(12): e04634.
Goding, J. W. 2004. Monoclonal Antibodies: principles and practice. Academic Press.
Kadariya J, Smith TC, Thapaliya D. 2014. Staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge in public health. Biomed Res Int;2014:827965
Wu, S., Duan, N., Gu, H., Hao, L., Ye, H., Gong, W., & Wang, Z. 2016. A Review of the Methods for Detection of Staphylococcus aureus Enterotoxins. Toxins.(Basel), 8(7).

	Centro Nacional de Microbiología (CNM). Instituto de Salud Carlos III (ISCIII).
	Servicio de Zoonosis Emergentes, de Baja Prevalencia y Agresivos Biológicos (NED). Servicio de Zoonosis de Transmisión Alimentaria y Resistencia a Antimicrobianos (ZTA). Servicio de Patología y Veterinaria Forense (SAP). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).

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