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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
Journal article   Open access   Peer reviewed

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

Chungwon Chung, Carey Wilson, Chandima-Bandara Bandaranayaka-Mudiyanselage, Eunah Kang, D Scott Adams, Lowell S Kappmeyer, Donald P Knowles, Terry F McElwain, James F Evermann, Massaro W Ueti, …
Journal of veterinary diagnostic investigation, Vol.26(1), pp.61-71
01/2014
DOI: https://doi.org/10.1177/1040638713511813
Handle:
https://hdl.handle.net/2376/116471
PMID: 24318928
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Abstract

Enzyme-Linked Immunosorbent Assay - methods Cattle Diseases - microbiology Anaplasmosis - diagnosis Anaplasma - isolation & purification DNA, Bacterial - chemistry Recombinant Proteins - genetics Blotting, Western - veterinary False Positive Reactions Enzyme-Linked Immunosorbent Assay - standards Anaplasmosis - microbiology Animals DNA, Bacterial - genetics Glutathione Transferase - genetics Polymerase Chain Reaction - veterinary Anaplasma - genetics Cattle Sensitivity and Specificity Female ROC Curve Enzyme-Linked Immunosorbent Assay - veterinary Cattle Diseases - diagnosis Bacterial Outer Membrane Proteins - genetics
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.

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