Abstract

Background: Brucella melitensis infection is still a major health problem for human and cattle in developing countries and the Middle East. Cell-mediated immunity is the dominant immune response required for protection against brucellosis. So, identifying of new candidates which can induce cellmediated immunity is demanded. Proliferation assay is one of the methods used to evaluate the potential of an antigen to generate memory T-cell. In the present study, the proliferation of speloncyets obtained from immunized mice was evaluated when they were challenged by recombinant Omp31 (rOmp31) in vitro. Methods: Mice were immunized by phosphate buffered saline (PBS), rOmp31 and formalin-killed Brucella melitensis Rev.1 with Freund’s adjuvant. At the day 30, after the last immunization, spleens were removed and homogenized. Purified rOmp31 (2.5 and 5 μg/ml) was added to 2 × 105 spleen cells and then, the speloncytes were incubated at 37°C in 5 CO2 atmosphere. After 48 hours, XTT 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide and MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were used to show the splenocyte proliferation. Findings: The splenocytes of mice immunized with rOmp31or Rev.1 started to proliferate after stimulation with rOmp31. There was a significant difference between SIs obtained from rOmp31 and Rev compared to phosphate buffered saline group (P < 0.01 and P < 0.05, respectively). Conclusion: Based on our findings, rOmp31 could generate a good memory cellular immune response. In addition, the method described here to study the speloncyte proliferation using XTT, is a simple and efficient method. © 2014, Isfahan University of Medical Sciences(IUMS). All rights reserved.