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Permanent link (DOI): https://doi.org/10.7939/R3D50G567

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

Descriptions

Other title
Subject/Keyword
Primary Biliary Cirrhosis
Human betaretrovirus
ELISA
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Kneteman, Mark S
Supervisor and department
Mason, Andrew (Medicine)
Examining committee member and department
Mason, Andrew (Medicine)
Houghton, Michael (Medical Microbiology & Immunology)
Schang, Luis (Biochemistry)
Abraldes, Juan (Medicine)
Evans, David (Medical Microbiology & Immunology)
Department
Department of Medicine
Specialization

Date accepted
2014-11-28T11:04:42Z
Graduation date
2015-06
Degree
Master of Science
Degree level
Master's
Abstract
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.
Language
English
DOI
doi:10.7939/R3D50G567
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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