DEVELOPMENT OF SEROLOGICAL TESTS TO DETECT ANTI-GFP ANTIBODIES FOR DIFFERENTIATING BRUCELLA INFECTED AND GFP-VACCINATED SHEEP A. Zabalza1, B. San Román1, P.M. Muñoz2, C. Chacón-Díaz3, E. Moreno3, J.M. Blasco2, M.J. Grilló1 1Animal Health, Instituto de Agrobiotecnología (CSIC-UPNA-Gobierno de Navarra), 31006, Pamplona, Spain; 2Animal Health, CITA of Aragón, 50059, Zaragoza, Spain; 3Programa Internacional de Enfermedades Tropicales (Univ. Nacional – Univ. De Costa Rica), 3000, Heredia, Costa Rica. *E-mail: [email protected] INTRODUCTION AND OBJECTIVES Serological interference in diagnostic tests is a major problem associated to the use of veterinary vaccines, which hamper the differentiation of infected and vaccinated animals (DIVA). To solve this problem in the context of brucellosis, our group is developing new Brucella vaccines tagged with green fluorescent protein (GFP) as xenogenic marker to be used in combination with associated DIVA diagnostic tests. The aim of this work was to develop Gel Diffusion (GD-GFP) and indirect-ELISA (ELISA-GFP) assays able to detect specifically anti-GFP antibodies in sheep. METHODS AND RESULTS 1) Production and characterization of GFP-GST and GFP Affinity chromatography LB-Amp Thrombin pGEX-4T-1gfpmut3 soluble fraction E.coli XL1-Blue GFP expression system Both proteins were obtained in amount (500 µg/mL), purity and antigenicity expected 2) Obtaining hyperimmune sheep sera against GFP-GST and GFP 3) Gel Diffusion (GD-GFP) with GFP-GST and GFP 3 immunization 4 WEEKS 3 2 WEEKS 3 2 WEEKS 3 serum vs. homologous antigen Titration of hyperimmune sheep sera vs. heterologous antigen Serum anti-GFP GFP Total dose: 200 g GFP/sheep in IFA (1:1): GFP + IFA; GFP-GST + IFA; IFA control SERUM Optimal concentration of antigen: 31.25 µg/mL of GFP for GFP-GST and GFP SERUM DILUTION Serum antiGFP-GST All hyperimmunized sera showed antibodies against both proteins Antigen dilution 4) Development of an indirect ELISA-GFP assay Parameters analysed: Serum anti-GFP Serum anti-GFP-GST ANTIGEN FOR COATING (PBST; 37°C, 2 h + 4°C, 15 h) BLOCKING REACTION (PBST 2% milk, 37°C, 1h) (10 µg/mL) NO vs. (5 or 10 µg/mL) vs. YES SERUM DILUTION (37°C, 1h) From 1:60 to 1:61,440 CONJUGATE DILUTION Protein G-peroxidase (25°C, 1h) 1:4000 vs Absorbance (405 nm) GFP-GST Negative serum GFP 3 2 1 0 Serum dilution 1:2000 Antigen selection: GFP-GST performed better than GFP in standard conditions (PBST fixation) 0´ vs 15 or 30 min CONCLUSIONS 1. E.coli XL1-Blue with pGEX-4T-1-gfpmut3 expression system allows to obtain GFP-GST from soluble fraction in purity, antigenicity and amount appropriate to be used in serological diagnostic test. ABTS incubation 15 min ABTS incubation 30 min 3 Absorbance (405 nm) SUBSTRATE INCUBATION ABTS 2 1 1,3 0,9 1,0 0 2. Both GFP-GST and GFP as antigens allow to detect especific anti-GFP antibodies in sheep sera, in GD-GFP test. ACKNOWLEDGMENTS This work financed support by MINECO-CICYT (AGL2010-20247 and AGL2011-30453-C04-01) and CSIC-CRUSA (2010CR0005). A.Z. and B.S.R. contracts were supported by UPNA and CSIC (JAE-Doc Program) respectively. REFERENCES Bradford, M.M. 1976. Analytical Biochemistry 72: 248-254. Chacón-Díaz, C., Muñoz-Rodríguez, M., Barquero-Calvo, E., Guzmán-Verri, C., Chaves-Olarte, E., Grilló, M. J. & Moreno, E. 2011. Vaccine 29: 577-582. Crowther, J. 2001. Methods in molecular biology. The ELISA guidebook. New Yersey: Human Press Inc. Fairbanks, G., Steck, T.L. & Wallach, D.F. 1971. Biochemistry 10 (13): 2606-2617. Kingston, B. & Brent, R. 1990. Current Protocols in Molecular Biology. Terpe, K. 2006. Applied microbiology and biotechnology 72: 211-222. San Román, B., Garrido, V., Muñoz, P.M., Arribillaga, L., García, B., De Andrés, X., Zabaleta, V., Mansilla, C., Farrán, I., Lasa, I., De Andrés, D., Amorena, B., Lasarte, J.J., & Grilló M.J. 2012. Vet. Res. 43(1):31. Tsai, C. M. & Frasch C.E., 1982. Anal. Biochem. 119(1):115-119. ELISA-GFP with sheep sera (n=92) 2 Absorbance (405 nm) 3. The selected ELISA-GFP conditions allowed a 99% of diagnostic specificity with sheep sera. Further analysis with sera from Brucella-GFP vaccinated sheep are necessary to assess the final value of this serological test. Serum dilution GFP-Positive sera GFP-Negative sera 1 0 1:100/15´ 1:100/30´ 1:200/30´ Serum dilution/ ABTS incubation Effect of ABTS incubation and milk blocking: Conditions that allowed the best discrimination (doted red lines) between positive and negative sera were: - Milk blocking - 1:4000 conjugate dilution - 1:100 serum dilution and 15’ or 30’ ABTS incubation; or 1:200 serum dilution and 30’ ABTS incubation Diagnostic Specificity: The selected ELISA-GFP conditions resulted in 99 % specificity