PRODUCTION OF HYPERIMMUNE SERUM AGAINST RECOMBINANT PROTEINS SODC AND OMP16 OF BRUCELLA SPP. FOR THE DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TEST SYSTEM

Brucellosis is a widespread zoonotic disease transmitted from domestic animals to humans and is endemic in many regions of the world. There is a need for highly specific serological tests for brucellosis based on recombinant proteins to reduce cross-reactivity with other Gram-negative bacteria and to differentiate between vaccinated and infected individuals. The aim of the present study was to obtain hyperimmune sera against Brucella spp. antigens in laboratory animals. Rabbits and guinea pigs were used as experimental animals. For immunization, recombinant Brucella spp. proteins Omp16 and SodC were used in combination with adjuvants, including aluminum hydroxide (Al(OH)₃), as well as complete and incomplete Freund’s adjuvant (CFA/IFA). Immunization was performed according to a standard protocol, followed by monitoring of antibody levels in blood sera using enzyme-linked immunosorbent assay (ELISA) and agar gel precipitation test (AGPT). For protein expression, producer strains were cultured in LB medium supplemented with kanamycin. Protein synthesis was induced with 0.1 mM IPTG. Purification was performed using immobilized metal affinity chromatography (IMAC). As a result of the study, hyperimmune sera with a high level of specific antibodies were obtained. It was demonstrated that the use of adjuvants promotes the development of a pronounced humoral immune response in immunized animals. The highest antibody titers were observed in animals immunized with CFA/IFA adjuvants, where antibody titers reached 1:6400 in rabbits and 1:1600 in guinea pigs. When aluminum hydroxide was used, antibody titers were 1:3200 and 1:800 in rabbits and guinea pigs, respectively. The obtained results confirm the effectiveness of the proposed approach for producing hyperimmune sera and demonstrate their potential for use as reference positive controls in the development and standardization of serological methods for brucellosis diagnostics.