Veterynarna biotehnologija – Veterinary biotechnology, 2020, 36, 176-183 [in Ukrainian]. https://doi.org/10.31073/vet_biotech36-18
Institute of Veterinary Medicine NAAS
State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise
BRUCELLA ANTIGEN PURIFICATION FOR THE DIAGNOSIS OF ANIMAL BRUCELLOSIS WITH INDIRECT ELISA TEST
Introduction. Brucellosis is a zoonosis infection caused by Br. abortus and poses an important medical and veterinary problem, threatening the health of humans and animals. Brucellosis is practically eradicated in Ukraine. Only sporadic cases of the disease were registered among sheep population but the facts of circulation of the pathogen are confirmed in wild animals. Improvement of technology for brucellosis antigen purification and its usage for indirect ELISA test will help to solve the problem of brucellosis incidence in farm animals. ELISA is one of the diagnostic methods, which is widely used all around the world, and has high sensitivity and high specificity.
The goal of the work was to develop a protocol for brucellosis antigen purification and subsequent use of purified antigen as a component of the diagnostic test, which based on an indirect ELISA.
Materials and methods. The standardized inactivated culture of brucella from strain Brucella abortus 19 was used as a source of antigen. Brucellous lipolysaccharide (LPS) was prepared according to the OIE-recommendation in modification. The parameters of centrifugation, temperature regime, etc. were optimized, which made it possible to increase the production of brucellosis antigen. Polystyrene plates (SARSTEDT, USA) were coated with LPS diluted in a 0.05 M carbonate-bicarbonate buffer, pH 9.6. To determine the most optimal dilution for ELISA antigen was pre-titrated by double dilution. Next, quantity of antigen was tested using positive and negative for brucellosis animal sera, and also using blood sera of animals with yersiniosis.
Results of research and discussion. Antigen was previously purified using classical OIE protocol and was used for the indirect of ELISA test at 1:12 500 dilution. The new purification technology allowed to use LPS at 1:30 000 dilution. Moreover, test kits based on the antigen had a 2.4-fold higher diagnostic performance. The purified antigen interacted with brucellosis-positive serum samples and had no nonspecific interactions with the sera of healthy animals. The tested yersiniosis sera of the animals were also classified as negative for brucellosis.
Conclusions and prospects for further research. The obtained data allowed to use the purified antigen for the design of diagnostic test kits for animal brucellosis diagnostics.
Keywords: brucellosis, antigen, lipopolysaccharide, enzyme immunoassay.