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 (K_D) and Maximum Binding Capacity (B_max) 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.