1. Development and validation of immunoassay for whole cell detection of Brucella abortus and Brucella melitensis.
- Author
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Hans R, Yadav PK, Sharma PK, Boopathi M, and Thavaselvam D
- Subjects
- Animals, Antibodies, Bacterial blood, Brucella abortus immunology, Brucella melitensis immunology, Brucellosis immunology, Brucellosis microbiology, Female, Immunologic Tests, Mice, Mice, Inbred BALB C, Rabbits, Antibodies, Bacterial immunology, Antigens, Bacterial immunology, Brucella abortus isolation & purification, Brucella melitensis isolation & purification, Brucellosis diagnosis, Immunoassay standards
- 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 10
2 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.- Published
- 2020
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