Abstract
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.
Highlights
Brucellosis is re-emerging infectious bacterial zoonosis caused by small gram-negative, cocco-bacilli, facultative intracellular bacteria belongs to genus Brucella[1]
The obtained Ab titer with whole cell based I-ELISA for mice and rabbit is ≥ 64000 with optical density (OD) value comparatively more with anti-sera raised against WC Ag of Brucella abortus S99
Characterization with western blot revealed that group 1 were the prominent proteins against which mice IgG detection polyclonal antibody (pAb) was reacting at 1:100 dilution in both the Brucella spp. respectively
Summary
Brucellosis is re-emerging infectious bacterial zoonosis caused by small gram-negative, cocco-bacilli, facultative intracellular bacteria belongs to genus Brucella[1]. Surface Plasmon Resonance (SPR) validates immobilization of IgG antibody, viable WC and DNA by detecting high molecular analytes whereby ligand binding (association) increases mass on the chip surface and on dissociation decreases the mass Such changes produce direct detectable signal by affecting the refractive index, RI30,31. In our proposed study, we have raised pAbs against intact whole cell of Brucella and developed a semi-quantitative S-ELISA employing SPR based evaluation and validation (with WC spiked serum) of mice IgG detection antibody in direct whole cell detection of Brucella. We characterized these high affinity antibodies in recognizing certain cell-surface components to develop a conventional S-ELISA for fast, direct and convenient detection of Brucella
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