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

J Vet Diagn Invest. 2014 Jan;26(1):61-71. doi: 10.1177/1040638713511813. Epub 2013 Dec 6.

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.

Keywords: Anaplasma antibody; cattle; competitive enzyme-linked immunosorbent assay; diagnostic specificity and sensitivity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Bacterial Outer Membrane Proteins
  • DNA, Bacterial
  • Recombinant Proteins
  • major surface protein 5, Anaplasma
  • Glutathione Transferase