Document Type : Original Article


1 Department of Pathobiology, School of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

2 Department of Basic Sciences, School of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran


Brucellosis is considered as one of the important global zoonotic diseases that causes medical as well as economic problems especially in tropical countries. The illness has no specific pathognomonic signs; therefore, the rapid and accurate diagnosis of the disease has a very important role in preventing the Brucella spillover and treatment. The purpose of this study was to design a new indirect ELISA test for detection of human brucellosis based on using recombinant Brucella abortus outer membrane protein 16.00 kDa (rOMP16) as an antigen. OMP16 gene of B. abortus was initially synthesized and cloned in pET-21d vector and then expressed in Escherichia coli cells. The expression was confirmed by the SDS-PAGE, western blotting and dot blotting. The purified protein was coated in ELISA plates and an indirect ELISA was performed on 70 human serum samples. The results were evaluated with a commercial IgG ELISA kit and Rose Bengal plate agglutination tests as reference tests. Diagnostic performance of designed OMP16 ELISA test in comparison with Rose Bengal plate test revealed 100% of sensitivity, 95.00% of specificity and good Fleiss kappa agreement, whereas, where it was compared to commercial ELISA kit, it revealed very good kappa agreement with 100% of sensitivity and 100% of specificity in cut-off value of 0.13. It was concluded that OMP 16.00 kDa could be acceptable alternative antigen for detecting Brucella IgG antibody with high accuracy.


  1. World animal health information database (WAHIS Interface). Available at: home. Accessed Aug 04, 2022.
  2. Bagheri Nejad R, Krecek RC, Khalaf OH, et al. Brucellosis in the Middle East: Current situation and a pathway forward. PLoS Negl Trop Dis 2020; 14(5): e0008071. doi: 10.1371/journal.pntd.0008071.
  3. Gupte S, Kaur T. Diagnosis of human brucellosis. J Trop Dis 2015; 4(1): doi: 10.4172/2329-891X.1000185.
  4. World organization for animal health. OIE manual of diagnostic test and vaccines for terrestrial animals. Available at: Accessed Aug 04, 2022.
  5. Yagupsky P, Morata P, Colmenero JD. Laboratory diagnosis of human brucellosis. Clin Microbiol Rev 2019; 33(1): e00073-19. doi: 10.1128/CMR.00073-19.
  6. Padilla Poester F, Nielsen K, Ernesto Samartino L, et al. Diagnosis of brucellosis. Open Vet Sci J 2010; 4: 46-60.
  7. Blasco JM, Molina-Flores B. Control and eradication of Brucella melitensis infection in sheep and goats. Vet Clin North Food Anim Pract 2011; 27(1): 95-104.
  8. Smirnova EA, Vasin AV, Sandybaev NT, et al. Current methods of human and animal brucellosis diagnostics. Adv Infect Dis 2013; 3(3): 177-184.
  9. Cloeckaert A, de Wergifosse P, Dubray G, et al. Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay. Infect Immun 1990; 58(12): 3980-3987.
  10. Tabynov K, Kydyrbayev Z, Ryskeldinova S, et al. Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induced a strong T-cell immune response, as well as high protectiveness against abortus infection. Vaccine 2014; 32(18): 2034-2041.
  11. Yang X, He Z, Zhang G, et al. Evaluation of reactivity of monoclonal antibodies against Omp25 of Brucella Front Cell Infect Microbiol 2020; 10: 145. doi: 10.3389/ fcimb.2020.00145.
  12. Tian M, Song M, Yin Y, et al. Characterization of the main immunogenic proteins in Brucella infection for their application in diagnosis of brucellosis. Comp Immunol Microbiol Infect Dis 2020; 70: 101462. doi: 10.1016/j.cimid.2020.101462.
  13. Luo D, Ni B, Li P, et al. Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice. Infect Immun 2006; 74(5): 2734-2741.
  14. Pasquevich KA, Estein SM, García Samartino C, et al. Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus Infect Immun 2009; 77(1): 436-445.
  15. Pasquevich KA, García Samartino C, Coria LM, et al. The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis. J Immunol 2010; 184(9): 5200-5212.
  16. Yin D, Li L, Song X, Li H, et al. A novel multi-epitope recombined protein for diagnosis of human brucellosis. BMC Infect Dis 2016; 16: 219. doi: 10.1186/s12879-016-1552-9.
  17. Sambrook, J, Russell DW. Molecular cloning: a laboratory manual. 3rd New York, USA: Coldspring-Harbour Laboratory Press 2001; 110-111.
  18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-685.
  19. McHugh ML. Interrater reliability: the kappa statistic. Biochem Med (Zagreb) 2012; 22(3): 276-282.
  20. O'Grady D, Byrne W, Kelleher P, et al. A comparative assessment of culture and serology in the diagnosis of brucellosis in dairy cattle. Vet J 2014; 199(3): 370-375.
  21. Ducrotoy MJ, Muñoz PM, Conde-Álvarez R, et al. A systematic review of current immunological tests for the diagnosis of cattle brucellosis. Prev Vet Med 2018; 151: 57-72.
  22. Franco MP, Mulder M, Gilman RH, et al. Human brucellosis. Lancet Infect Dis 2007; 7(12): 775-786.
  23. Vitry MA, Hanot Mambres D, De Trez C, et al. Humoral immunity and CD4+ Th1 cells are both necessary for a fully protective immune response upon secondary infection with Brucella melitensis. J Immunol 2014; 192(8): 3740-3752.
  24. Yingst SL, Izadjoo M, Hoover DL. CD8 knockout mice are protected from challenge by vaccination with WR201, a live attenuated mutant of Brucella melitensis. Clin Dev Immunol 2013; 2013: 686919. doi: 10.1155/2013/686919.
  25. Mohammadi E, Golchin M. Detection of Brucella abortus by immunofluorescence assay using anti-16-kDa outer membrane protein (OMP16) antibody. Comp Clin Path 2017; 26: 1299-1304.
  26. Salih SM, Khorsheed HO, Ya`qob JS. Incidence of brucellosis in Kirkuk Province using simple dilution microagglutination rose Bengal test method. Tikrit Med J 2007; 13(1): 70-74.
  27. Yin D, Bai Q, Zhang J, et al. A novel recombinant multiepitope protein candidate for the diagnosis of brucellosis: A pilot study. J Microbiol Methods 2020; 174: 105964. doi: 10.1016/j.mimet.2020.105964.
  28. Koyuncu I, Kocyigit A, Ozer A, et al. Diagnostic potential of Brucella melitensis Rev1 native Omp28 precursor in human brucellosis. Cent Eur J Immunol 2018; 43(1): 81-89.
  29. Ahmed IM, Khairani-Bejo S, Hassan L, et al. Serological diagnostic potential of recombinant outer membrane proteins (rOMPs) from Brucella melitensis in mouse model using indirect enzyme-linked immunosorbent assay. BMC Vet Res 2015; 11: 275. doi: 10.1186/ s12917-015-0587-2.