title: Improved diagnostic performance of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5-glutathione S-transferase fusion protein as antigen.
abstract: The current study tested the hypothesis that removal of maltose binding protein (MBP) from recombinant antigen used for plate coating would improve the specificity of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay (cELISA). The number of 358 sera with significant MBP antibody binding (≥30%I) in Anaplasma-negative herds was 139 (38.8%) when tested using the recombinant major surface protein 5 (rMSP5)-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (<30%I) using the commercial rMSP5-MBP cELISA with MBP adsorption, resulting in 97.8% specificity. This specificity was higher than some previous reports, so to improve the specificity of the commercial cELISA, a new recombinant antigen designated rMSP5-glutathione S-transferase (GST) was developed, eliminating MBP from the antigen and obviating the need for MBP adsorption. Using the rMSP5-GST cELISA, only 1 of 358 Anaplasma-negative sera, which included the 139 sera with significant (≥30%I) MBP binding in the rMSP5-MBP cELISA without MBP adsorption, was positive. This resulted in an improved diagnostic specificity of 99.7%. The rMSP5-GST cELISA without MBP adsorption had comparable analytical sensitivity to the rMSP5-MBP cELISA with MBP adsorption and had 100% diagnostic sensitivity when tested with 135 positive sera defined by nested polymerase chain reaction. Further, the rMSP5-GST cELISA resolved 103 false-positive reactions from selected sera with possible false-positive reactions obtained using the rMSP5-MBP cELISA with MBP adsorption and improved the resolution of 29 of 31 other sera. In summary, the rMSP5-GST cELISA was a faster and simpler assay with higher specificity, comparable sensitivity, and improved resolution in comparison with the rMSP5-MBP cELISA with MBP adsorption.
authors: 1Chungwon Chung, VMRD Inc., 425 NW Albion Drive, Pullman, WA 99163. chungwon@vmrd.com.
authors: Chungwon
authors: C
authors: Chung
authors: Carey
authors: C
authors: Wilson
authors: Chandima-Bandara
authors: CB
authors: Bandaranayaka-Mudiyanselage
authors: Eunah
authors: E
authors: Kang
authors: D Scott
authors: DS
authors: Adams
authors: Lowell S
authors: LS
authors: Kappmeyer
authors: Donald P
authors: DP
authors: Knowles
authors: Terry F
authors: TF
authors: McElwain
authors: James F
authors: JF
authors: Evermann
authors: Massaro W
authors: MW
authors: Ueti
authors: Glen A
authors: GA
authors: Scoles
authors: Stephen S
authors: SS
authors: Lee
authors: Travis C
authors: TC
authors: McGuire
mesh_terms: D000709:Anaplasma / Q000235:genetics / Q000302:isolation & purification*; D000712:Anaplasmosis / Q000175:diagnosis / Q000382:microbiology*; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D015153:Blotting, Western / Q000662:veterinary; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards / Q000662:veterinary*; D005189:False Positive Reactions; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012372:ROC Curve; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C111578:major surface protein 5, Anaplasma; D005982:Glutathione Transferase
keywords: Anaplasma antibody; cattle; competitive enzyme-linked immunosorbent assay; diagnostic specificity and sensitivity