title: Enhanced sensitivity of an antibody competitive blocking enzyme-linked immunosorbent assay using Equine arteritis virus purified by anion-exchange membrane chromatography.
abstract: In an effort to improve a competitive blocking enzyme-linked immunosorbent assay (cELISA) for antibody detection to Equine arteritis virus (EAV), antigen purified by anion-exchange membrane chromatography capsule (AEC) was evaluated. Virus purification by the AEC method was rapid and easily scalable. A comparison was made between virus purified by the AEC method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of EAV glycoprotein 5 (GP5) containing the epitope defined by monoclonal antibody 17B7, and 2) the relative sensitivity of a commercial antibody cELISA with the only change being the 2 purified antigens. On evaluation by Western blot using GP5-specific monoclonal antibody 17B7, the AEC-purified EAV contained 86% GP5 monomer whereas the differentially centrifuged EAV contained <29% of the monomer. Improvement of analytical sensitivity without sacrifice of analytical specificity was clearly evident when cELISAs prepared with EAV antigen by each purification method were evaluated using 7 sensitivity and specificity check sets. Furthermore, the AEC-purified EAV-based cELISA had 30-40% higher agreement with the virus neutralization (VN) test than the cELISA prepared with differentially centrifuged EAV based on testing 40 borderline EAV-seropositive samples as defined by the VN test. In addition, the AEC-purified cELISA had highly significant (P = 0.001) robustness indicated by intra-laboratory repeatability and interlaboratory reproducibility when evaluated with the sensitivity check sets. Thus, use of AEC-purified EAV in the cELISA should lead to closer harmonization of the cELISA with the World Organization for Animal Health-prescribed VN test.
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee) chungwon@vmrd.com.
authors.forename: Chungwon J
authors.initials: CJ
authors.lastname: Chung
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee).
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Grimm
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee).
authors.forename: Carey L
authors.initials: CL
authors.lastname: Wilson
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee).
authors.forename: Udeni B R
authors.initials: UB
authors.lastname: Balasuriya
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee).
authors.forename: Grace
authors.initials: G
authors.lastname: Chung
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee).
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Timoney
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee).
authors.forename: Chandima-Bandara
authors.initials: CB
authors.lastname: Bandaranayaka-Mudiyanselage
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee).
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Lee
authors.affiliation: VMRD Inc., Pullman, WA (Chung, Grimm, Wilson, Chung, Bandaranayaka-Mudiyanselage, McGuire)Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Balasuriya, Timoney)University of Idaho, Moscow, ID (Lee).
authors.forename: Travis C
authors.initials: TC
authors.lastname: McGuire
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D000914:Antibodies, Viral / Q000032:analysis; D018174:Arterivirus Infections / Q000175:diagnosis / Q000662:veterinary*; D002852:Chromatography, Ion Exchange / Q000379:methods / Q000662:veterinary*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D007240:Equartevirus / Q000276:immunology / Q000302:isolation & purification*; D006734:Horse Diseases / Q000175:diagnosis*; D006736:Horses; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral
keywords: Anion-exchange chromatography; Equine arteritis virus; competitive blocking enzyme-linked immunosorbent assay; glycoprotein 5; purification