Preparation of conjugate based on colloidal gold and recombinant g protein for diagnosis of brucellosis of animals
Journal Title: Бюлетень "Ветеринарна біотехнологія" - Year 2018, Vol 32, Issue
Abstract
Introduction. There is a threat of brucellosis in Ukraine due to the smuggling of animals, slaughter products, raw materials, dairy products from countries where this problem is relevant, and in the case of disease transmission from wild or animal-bacterial carriers. In comparison to classical methods of brucellosis diagnosis in animals, dot immunoassay (DIA) is simple, fast, highly sensitive and informative method. In membrane tests, colloidal gold is widely used for the preparation of conjugates in diagnostic kits for parasitic, viral and fungal diseases. The goal of the work. Preparation of conjugate of G protein with colloidal gold for the test kit based on the dot immunoassay designed for diagnosis of brucellosis. Materials and methods. The gold sol was prepared before the conjugation of protein G with colloidal gold. This was done using the Frances method in modification. Conjugation of colloidal gold with protein G of Streptococcus spp. by the optimized and adapted protocol of Hermanson G. T. et al. The quality of the prepared conjugate was assessed using dot immunoassay. Results of research and discussion. The conjugate was tested by the dot immunoassay using the panel of blood sera, which included positive and negative reference sera for brucellosis, OIE (UK) and blood sera from healthy animals. Conjugate of protein G with colloidal gold reacted with both strongly and low positive for brucellosis reference sera. It did not react with negative reference sera of healthy animals. In addition to the visual assessment, the results were verified by the software “Episkrin AB”. The resulting numerical values completely coincided with the visual estimate. Conclusions and perspectives of further investigations. According to the results of research, the method for preparation of the conjugate of G protein of Streptococcus spp. with colloidal gold was experimentally validated. It was used as a component of test kit based on DIA for the diagnosis of brucellosis in animals. The next step is determination of specificity, sensitivity and reproducibility of diagnostic test kit.
Authors and Affiliations
V. G. Khomenko, Yaroslav Khomenko
Keywords
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- EP ID EP407699
- DOI 10.31073/vet_biotech32(2)-73
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