Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

Abstract

bBrucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and promising results for further use in clinical screening and serodiagnosis of human brucellosis. Brucellosis is one of the world's major zoonoses, caused by bacteria of the genus Brucella. It is a serious public health problem in India and in other developing countries. The transmission of Brucella infection and its prevalence in a region depend upon several factors, like food habits, methods of processing milk and milk products, social customs, husbandry practices, climatic conditions, socioeconomic status, and environment hygiene (1). The disease has been recognized as one of the most common laboratory-acquired infections; it has been reported to occur in clinical research and production laboratories. Human brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations, and its diagnosis requires microbiological confirmations by means of isolation from blood cultures or demonstration of the presence of specific antibodies by serological tests (2). Blood cultures provide definite proof of brucellosis but may not provi...

Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies

“…Although Omp31 elicited specific antibody production and bactericidal activity [6, 11, 15], it was considered a poor diagnostic antigen [14]. In this study, we prepared 22 murine mAbs against Omp31 of B. melitensis , of which 50% (11/22) were IgG2a, 23% (5/22) IgG1 and 27% (6/22) IgM isotypes, respectively.…”

“…Omp31 plays an important role in cellular and humoral immune protective responses against Brucella infection [4–11]. Previously we fully evaluated Omp31 epitopes in specific T-cell response in sheep vaccinated with attenuated B. melitensis vaccine [12].…”

Evaluation of Reactivity of Monoclonal Antibodies Against Omp25 of Brucella spp.

“…Purified mAbs were used as the primary antibody at optimum working concentration. The microwell plate was incubated with a 1:10,000 dilution of goat antimouse IgG and IgM HRP-conjugate antibodies as reported previously (Tiwari et al, 2013;Ahmed et al, 2015). In peptide-ELISA, peptides P1-P10 were used to identify antigenic epitopes recognized by each mAb (Qiu et al, 2012;Li et al, 2017).…”

Comparative Analysis of Immune Responses to Outer Membrane Antigens OMP10, OMP19, and OMP28 of <i>Brucella abortus</i>

“…Recent studies have focused on various OMPs, such as OMP28, OMP2b, OMP10, OMP16, and OMP19 (14)(15)(16). Of these, OMP28 displays significant potential as a diagnostic candidate that may trigger protective effects in vivo and show high efficiency in serological tests against brucellosis (17)(18)(19)(20). Attempts have been made to clone these OMPs and other proteins to investigate if their immunogenicities may allow LPS-free diagnosis following infection with Brucella spp.…”