The Detection of Antibodies against Human Betaretrovirus Surface Protein in Patients with Primary Biliary Cirrhosis

• Author / Creator

  Kneteman, Mark S

• Introduction: Primary biliary cirrhosis is an autoimmune liver disease in which cellular and humoral immune responses towards cholangiocytes cause progressive intrahepatic bile duct loss. Cholestasis, liver fibrosis, and eventually cirrhosis result, with liver transplantation or death occurring in end stage disease. The etiology of the disease is unknown, but is thought to involve a permissive genetic predisposition and an environmental insult. Plausible environmental insults include xenobiotic exposure, and bacterial or viral infections. Previous work in our group suggests infection by human betaretrovirus, a virus with close similarity to mouse mammary tumor virus, may be associated with PBC. We are developing an ELISA based assay with the goal of establishing whether patients with primary biliary cirrhosis produce antibodies to human betaretrovirus. We also seek to produce a diagnostic indirect ELISA for detecting prevalence of human betaretrovirus in patients with primary biliary cirrhosis and the general population. Methods: Human betaretrovirus Surface (gp52 Su) protein was expressed as a recombinant antigen from both E. coli and human cells. Affinity tags co-expressed with the antigens, GST for E.coli antigens and polyhistidine residues for mammalian antigens, were utilized to purify expressed antigen. Western blot was used to confirm successful antigen expression. ELISAs were developed and optimized for both E. coli and mammalian expressed antigens. These ELISAs were utilized to probe healthy control and PBC patient serum samples for antibodies against human betaretrovirus surface protein. Western blot was used to validate the findings from the mammalian antigen ELISA. Results: Three of four E. coli Human betaretrovirus Surface antigens were expressed as well as the mammalian antigen. While preliminary results from the E. coli antigen ELISA were promising, larger studies failed to reach significance. A preliminary ELISA utilizing mammalian expressed protein, the human betaretrovirus gp52 Su ELISA version 1, produced significant results with 20% of PBC patients exhibiting seroreactivity compared to 4.5% of healthy controls. A second version of the ELISA produced by InBios from the protocol of the first ELISA also produced significant results, and was a larger study with approximately 200 samples from healthy controls, PBC, primary sclerosing cholangitis and non-biliary liver disease patients. In this study, 20% of PBC patients had seroreactivity versus 3% of controls. In contrast, the HBRV gp52 Su ELISA version 3 detected reactivity in 4% of PBC patients versus 2% of controls. Western blot studies of selected samples (n=42) only demonstrated reactivity in one control and one PBC patient. Of these patients, none were positive samples by the HBRV gp52 Su ELISA version 3. Therefore, Western blot results did not validate the ELISA results. Discussion: The results of the first two versions of the HBRV gp52 Su ELISA suggest that a portion of PBC patients produce anti-HBRV humoral immunity. However, these results must be validated by a secondary immunoassay that preserves conformational epitopes, and must be replicated with further ELISA studies. Preliminary Western blot results suggest some PBC patients and controls produce anti-HBRV gp52 Su antibodies to linear epitopes of HBRV Su. The sensitivity of the HBRV Su ELISA was clearly insufficient to act as a diagnostic assay as the two ELISAs with significant findings were only 20% sensitive for the detection of anti-HBRV Su antibodies in PBC patient samples. Further ELISA development is required to establish a relationship between PBC and anti-HBRV reactivity.

• Subjects / Keywords

  • Primary Biliary Cirrhosis
  • Human betaretrovirus
  • ELISA

• Graduation date

  Spring 2015

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3D50G567

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.