Desenvolvimento e prototipagem de métodos de diagnóstico para hepatite D

Abstract

Hepatitis D affects 15 to 20 million people worldwide, with South American, especially the Amazon basin, being one of the regions with the highest prevalence of cases. Hepatitis D is caused by the hepatitis D virus (HDV) and is responsible for the most severe form of viral hepatitis as it has rapid progression to cirrhosis and liver decompensation. This increases the need for early diagnosis, making the development of new forms of diagnosis for this disease very important. In order to develop and prototype a serological method for the diagnosis of hepatitis D, two recombinant proteins derived from the HDV delta antigen, DTH10.1 and DTH10, were expressed. Both proteins were expressed by the E. coli BL21 strain (DE3) system and purified by metal affinity chromatography with nickel columns due to the presence of a histidine tail in the C-terminal region of the proteins. Both recombinant proteins were used in the development and prototyping of two diagnostic methodologies for hepatitis D, namely ELISA and lateral flow immunochromatographic test. In ELISA both DTH10.1 and DTH10 were shown to be able to detect anti-HDV IgG antibodies in the serum of HDV-infected patients while not reacting with hepatitis C, rheumatoid factor and healthy samples, reacting with only a hepatitis B sample. The DTH10.1 ELISA showed 75.7% double-entry sensitivity and 99% specificity, while DTH10 showed 77.3% and 99%. In addition to being able to generate reproducible results and excellent stability of the solid phase under conditions of aggression to the system. Through the immunochromatographic test, DTH10.1 was able to reproduce the ELISA results with this protein, generating a sensitivity of 77.5% and specificity of 100%. It follows that both developed methodologies are promising methods for the diagnosis of hepatitis D.