Development and validation of immunoassay for whole cell detection of Brucella abortus and Brucella melitensis

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.