|MADSEN-BOUTERSE, SALLY - Washington State University|
|DASSANAYAKE, ROHANA - Washington State University|
|KUMPULA-MCWHIRTER, NANCY - Washington State University|
|O'ROURKE, KATHERINE - Retired ARS Employee|
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2015
Publication Date: 5/3/2015
Citation: Madsen-Bouterse, S.A., Schneider, D.A., Dassanayake, R.P., Truscott, T.C., Zhuang, D., Kumpula-Mcwhirter, N., O'Rourke, K.I. 2015. PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay. Journal of Veterinary Diagnostic Investigation. 27(3):332-343.
Interpretive Summary: Immunoassays, such as immunohistochemistry and western blot, are widely used in the diagnosis of disease. Critical to immunoassays are the antibodies used to detect disease-associated proteins in tissue samples. Such proteins may be detected by several different antibodies, each binding to a unique region on that protein. Natural genetic variation in the structure of a protein may thus affect the binding of one antibody but not others. In the United States, diagnosis of scrapie infection relies heavily on immunohistochemistry with a single antibody (F99/97.6.1) for detection of PrPSc, a disease-associated form of prion protein that accumulates in the brain and other tissues. In goats, however, a genetic variation in the region bound by F99/97.6.1 reduces detection of PrP-Sc. This study demonstrates that other anti-PrP-Sc antibodies that bind to regions different from that bound by F99/97.6.1 are not adversely affected by this genetic variant. The effects of additional genetic variants known to occur in goats were also assessed. In summary, immunoassay detection of PrP-Sc in goats with specific known genetic variations in the prion protein may be reduced with certain antibodies, including F99/97.6.1. In the absence of genetic testing, use of a multi-antibody approach may provide more robust detection of PrP-Sc in infected tissues from goats.
Technical Abstract: Immunoassays are extensively utilized in disease diagnostics with monoclonal antibodies serving as critical tools within the assay. Detection of scrapie in sheep and goats relies heavily on immunoassays including immunohistochemistry, western blotting, and ELISA. In the United States, regulatory testing has primarily utilized a single antibody, typically F99/97.6.1, for immunohistochemical analysis of PrP-Sc (a disease-associated, misfolded isoform of normal cellular prion protein, PrP-C) in postmortem brain and ante-mortem lymphoid biopsy samples. The epitope bound by antibody F99/97.6.1 (amino acids 220-225; QYQERS) appears to be highly conserved in sheep, but allelic variation in goats at the epitope binding site results in decreased sensitivity. This study demonstrates that additional allelic variants in goats in the epitope binding site of another anti-prion antibody (F89/160.1.5; epitope amino acids 142-145, IHFG) results in similar reductions in detection or PrP-C by western blotting and PrP-Sc in goats with clinical disease by immunohistochemistry. Decreasing antibody titers of F89/160.1.5 were used to demonstrate the impact of antibody sensitivity in the detection of PrP-Sc in pre-clinical goats or what could be observed with F99/97.6.1 in the presence of an alternative allele within its epitope binding region. Due to the presence of a number of variations in caprine prion protein, these results suggest that a multi-antibody approach may provide a more robust assessment of scrapie in goats relative to the current single antibody approach.