| Abstract [eng] |
Domestic pigs, wild boars, rats, and other animals serve as natural reservoirs for the zoonotic hepatitis E virus (HEV). HEV can infect humans through the consumption of inadequately cooked meat, contaminated water, or direct contact with infected animals. Although most infections are asymptomatic, zoonotic HEV may cause acute hepatitis E, chronic hepatitis E in immunocompromised or immunosuppressed patients, and serious complications during pregnancy or after solid organ transplantation. In Europe, the predominant zoonotic genotype is HEV-3, while reports of rat HEV infections have risen in recent years. As the epidemiological importance of HEV continues to grow, there is an increasing need for new immunochemical methods capable of detecting HEV antigens or virus-specific antibodies. The aim of this master’s thesis is to develop immunochemical methods for identifying zoonotic HEV in human and wild-animal samples. The specific objectives are: to purify monoclonal antibodies (mAbs) directed against the capsid proteins of HEV-3 and rat HEV; to establish an immunoprecipitation (IP) assay for detecting HEV capsid proteins in wild-animal liver samples; to create a serological test for rat HEV-specific antibodies and to examine human and rat blood specimens. Four mAbs specific to HEV-3 , five mAbs specific to rat HEV, and three cross-reactive mAbs were purified from hybridoma growth medium. All of the mAbs were used in optimizing the immunoprecipitation method for recombinant HEV capsid proteins. During optimization, an effective wash buffer (TBS containing 0.1 % SDS and 2 mM DTT) was developed that eliminated most nonspecific interactions between HEV capsid proteins and Sepharose beads. With mAb CPB11, as little as 0.05 µg of HEV-3 protein could be detected in wild-boar liver lysate, while mAb CPH7 allowed detection of 0.4 µg of rat HEV protein in rat liver lysate. By blocking HEV-3-specific and cross-reactive antibodies with recombinant HEV-3 protein, rat HEV-specific antibodies were identified in human serum and rat CCF samples. Among 50 wild-rat CCF samples, rat HEV-specific antibodies were found in 24 (48 %), whereas none were detected in 15 HEV-positive human serum samples. |
