Immunoassay-based evaluation of rOmp28 protein as a candidate for the identification of species.

Brucellosis is an important bacterial zoonosis, re-emerging as a serious public health concern in developing countries. Two major species, and , cause recurrent facile infection in human. Therefore, rapid and accurate diagnosis for early disease control and prevention is needed in areas with low disease burden. This study evaluated the sandwich enzyme-linked immunosorbent assay (ELISA) (S-ELISA) immunoassay for potential use of whole-cell (WC) and recombinant outer-membrane protein (rOmp28)-derived IgG polyclonals in sensitive detection of . Immunoassay-based WC detection of species in important sub-clinical matrices at lower limits of detection. We purified recombinant rOmp28 with Ni-NTA gel affinity chromatography and produced IgG polyclonal antibodies (pAbs) using BALB/c mice and New Zealand white female rabbits against different antigens (Ags) of . Checkerboard sandwich ELISA and P/N ratio (optical density of 'P' positive test sample to 'N' negative control) were used for evaluation and optimization of the study. The pAbs were characterized using Western blot analysis and different matrices were spiked with WC Ag of . Double-antibody S-ELISA was developed using WC Ag-derived rabbit IgG (capture antibody at 10 µg ml) and rOmp28-derived mice IgG (detection antibody at 100 µg ml) with a detection range of 10 to 10 cells ml and a limit of detection at 10 cells ml. A P/N ratio of 1.1 was obtained with WC pAbs as compared to 0.6 and 0.9 ratios with rOmp28-derived pAbs for detecting 16M and S99, respectively. An increased P/N ratio of 4.4 was obtained with WC Ag-derived rabbit IgG as compared to 4.2>4.1>2.4 ratios obtained with rabbit IgGs derived against cell envelope (CE), rOmp28 and sonicated antigen (SA) of with high affinity for rOmp28 Ag analysed on immunoblots. The rOmp28-derived mice IgG revealed two species at P/N ratios of 11.8 and 6.3, respectively. Upon validation, S-ELISA detected WCs in human whole blood and sera samples with no cross-reactivity to other related bacteria. The developed S-ELISA is specific and sensitive in early detection of from different matrices of clinical and non-clinical disease presentation.