Introduction. Brucellosis is an important bacterial zoonosis, re-emerging as a serious public health concern in developing countries. Two major species, Brucella melitensis and Brucella abortus, 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.Hypothesis. 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 Brucella.Aim. Immunoassay-based WC detection of Brucella species in important sub-clinical matrices at lower limits of detection.Methodology. 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 Brucella. 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 Brucella.Results. Double-antibody S-ELISA was developed using WC Ag-derived rabbit IgG (capture antibody at 10 µg ml-1) and rOmp28-derived mice IgG (detection antibody at 100 µg ml-1) with a detection range of 102 to 108 cells ml-1 and a limit of detection at 102 cells ml-1. 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 B. melitensis 16M and B. abortus 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 Brucella with high affinity for rOmp28 Ag analysed on immunoblots. The rOmp28-derived mice IgG revealed two Brucella species at P/N ratios of 11.8 and 6.3, respectively. Upon validation, S-ELISA detected Brucella WCs in human whole blood and sera samples with no cross-reactivity to other related bacteria.Conclusion. The developed S-ELISA is specific and sensitive in early detection of Brucella from different matrices of clinical and non-clinical disease presentation.
Keywords: Brucella; polyclonal antibody; rOmp28; sandwich ELISA; whole-cell detection.