Abstract
Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.
Highlights
Hepatitis delta virus (HDV) is a defective viroid-like agent which infects patients on the background of a newly acquired or an established infection with hepatitis B virus (HBV), in both cases aggravating liver disease
Chronic HBV/HDV infection presents a more severe liver disease than chronic HBV mono-infection and is manifested by an accelerated fibrosis progression, early decompensation in the settings of established cirrhosis, and an increased risk of hepatocellular carcinoma attributed to the rapid development of cirrhosis
Antigenomic HDV RNA is partially edited by dsRNA-adenosine deaminase 1 (ADAR) [8] that converts the UAG stop codon to an amber UIG codon
Summary
Hepatitis delta virus (HDV) is a defective viroid-like agent which infects patients on the background of a newly acquired or an established infection with hepatitis B virus (HBV) (co-, and super-infection, respectively), in both cases aggravating liver disease. Co-infection with HDV and HBV in 95%–98% cases resolves as acute hepatitis B, but can cause a severe fulminant hepatitis The latter results in a massive necrosis of hepatocytes, liver failure, and death in up to 80% of patients, if they cannot undergo liver transplantation. Antigenomic HDV RNA is partially edited by dsRNA-adenosine deaminase 1 (ADAR) [8] that converts the UAG stop codon to an amber UIG codon The latter results in the elongation of ORF which generates an extended 214 amino acid long protein referred to as the large HDV antigen (27 kDa; L-HDAg). Our goal was to fill this gap and generate antibodies against HDV antigens that could be used in a variety of HDV-specific immune assays. The level of antibodies directed against S-HDAg in rabbit serum was assessed by ELISA, and antibody affinity tested by Western blot and immunofluorescence/confocal microscopy
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