Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10–100 CFU) required to establish the infection. In this study, we developed sandwich ELISA with detection range of 102 to 108 cells mL−1 and limit of detection at 103 cells mL−1 by employing polyclonal rabbit IgG (capture antibody, 10 µg mL−1) and mice IgG (detection antibody, 50 µg mL−1) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 102 cells mL−1 along with non co-operative interaction of protein albumin. Further, kinetic evaluation study using detection antibody against cell envelope antigen was performed whereby, Equilibrium Dissociation Constant (KD) and Maximum Binding Capacity (Bmax) were found to be 16.48 pM and 81.67 m° for Brucella abortus S99 and 0.42 pM and 54.50 m° for Brucella melitensis 16 M, respectively. During interference study, sandwich ELISA assay cross-reacted with either of the polyclonal antibody of above Brucella species. Upon validation, no cross-reactivity observed with bacteria-closely related to Brucella. In conclusion, developed semi-quantitative sandwich immunoassay is sensitively rapid in whole cell detection of Brucella and will be useful in development of detection assays from environmental and clinical matrices.

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Brucellosis is the most widespread and serious zoonotic disease worldwide which affects livestock, sylvatic wildlife, marine dwellers, and humans. It is acquired through Alphaproteobacteria which belong to the genus Brucella and is categorized as a potential bio-threat agent. In this study, we developed a rapid and direct differential whole cell (WC) agglutination-based assay for its on-field detection. The recombinant outer membrane (rOmp28) protein-derived specific mice IgG polyclonal antibodies (pAbs) of Brucella were purified using affinity chromatography and conjugated with functionalized gold nanoparticles (AuNPs) for rapid agglutination. A positive blot of 32 kDa protein revealed specific immuno-reactivity of rOmp28-pAbs using immunoblot analysis. For the synthesis of AuNPs, the conventional “Turkevich method” was optimized at a concentration < 1 mM of gold precursor for obtaining 50-nm-sized particles. Also, their physico-chemical characteristics were analyzed using UV-visible spectrophotometry, Fourier transform infra-red spectroscopy (FT-IR), Raman spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential (ζ, ZP), and fluorescence spectroscopy. Furthermore, these AuNPs were functionalized with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to prepare modified carboxylated AuNPs. For bioconjugation with Brucella rOmp28 IgG pAbs, antibody-conjugated functionalized AuNP constructs were prepared and characterized using FT-IR analysis with strong N–H deformations. Subsequently, these bioconjugated AuNPs were used to develop a direct-differential slide agglutination assay with a detection limit of 104 CFU mL−1. The sensitivity of this assay was compared with standard double-antibody sandwich ELISA (S-ELISA) using rOmp28 IgG pAbs with an LOD of 103 CFU mL−1 and a detection range of 102–108 CFU mL−1. No intraspecies cross-reactivity was observed based on evaluation of its specificity with a battery of closely related bacterial species. In conclusion, the increased sensitivity and specificity of the developed agglutination assay obtained using bioconjugated functionalized AuNPs is ≥ 98% for the detection of Brucella. Therefore, it can be used as an alternate rapid method of direct WC detection of bacteria as it is simple, robust, and cost-effective, with minimal time of reaction in the case of early disease diagnosis.

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.