Related Experiment Video
Updated: Aug 23, 2025
Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Development of a diagnostic indirect ELISA test for detection of Brucella antibody using recombinant outer membrane
Mehdi Golchin1, Somayye Mollayi1, Elham Mohammadi1
1Department of Pathobiology, School of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran.
Abstract:
Brucellosis is considered as one of the important global zoonotic diseases that causes medical as well as economic problems especially in tropical countries. The illness has no specific pathognomonic signs; therefore, the rapid and accurate diagnosis of the disease has a very important role in preventing the Brucella spillover and treatment. The purpose of this study was to design a new indirect ELISA test for detection of human brucellosis based on using recombinant Brucella abortus outer membrane protein 16 kDa (rOMP16) as an antigen. OMP16 gene of B. abortus was initially synthesized and cloned in pET-21d vector and then expressed in Escherichia coli cells. The expression was confirmed by the SDS-PAGE, western blotting and dot blotting. The purified protein was coated in ELISA plates and an indirect ELISA was performed on 70 human serum samples. The results were evaluated with a commercial IgG ELISA kit and Rose Bengal plate agglutination tests as reference tests. Diagnostic performance of designed OMP16 ELISA test in comparison with Rose Bengal plate test revealed 100% of sensitivity, 95.00% of specificity and good Fleiss kappa agreement, whereas, where it was compared to commercial ELISA kit, it revealed very good kappa agreement with 100% of sensitivity and 100% of specificity in cut-off value of 0.13. It was concluded that OMP 16 kDa could be acceptable alternative antigen for detecting Brucella IgG antibody with high accuracy.
Insights
A new indirect ELISA test using recombinant Brucella abortus outer membrane protein 16 kDa (rOMP16) shows high accuracy for detecting human brucellosis. This diagnostic tool offers a reliable method for identifying Brucella IgG antibodies, crucial for disease control.
Area of Science:
- Veterinary Medicine
- Immunology
- Infectious Diseases
Background:
- Brucellosis is a significant global zoonotic disease causing substantial medical and economic burdens, particularly in tropical regions.
- The lack of specific symptoms necessitates rapid and accurate diagnostic methods to prevent Brucella transmission and ensure effective treatment.
Purpose of the Study:
- To develop and evaluate a novel indirect ELISA for detecting human brucellosis.
- To utilize recombinant Brucella abortus outer membrane protein 16 kDa (rOMP16) as a key antigen in the diagnostic assay.
Main Methods:
- The OMP16 gene from B. abortus was synthesized, cloned into a pET-21d vector, and expressed in E. coli.
- Protein expression was verified using SDS-PAGE, western blotting, and dot blotting techniques.
- An indirect ELISA was performed on 70 human serum samples using purified rOMP16, with results compared against commercial ELISA kits and Rose Bengal plate agglutination tests.
Main Results:
- The developed OMP16 ELISA demonstrated 100% sensitivity and 95.00% specificity when compared to the Rose Bengal plate test, with good Fleiss kappa agreement.
- In comparison to a commercial ELISA kit, the OMP16 ELISA achieved 100% sensitivity and 100% specificity at a cut-off value of 0.13, showing very good kappa agreement.
Conclusions:
- Recombinant Brucella abortus OMP16 kDa is a highly accurate and acceptable alternative antigen for the detection of Brucella IgG antibodies.
- The developed indirect ELISA offers a promising tool for the accurate diagnosis of human brucellosis, aiding in disease surveillance and management.
