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Clinical and Vaccine Immunology, November 2006, p. 1212-1216, Vol. 13, No. 11
1071-412X/06/$08.00+0     doi:10.1128/CVI.00196-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Babesia bovis

Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington 99164-6630,¹ Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly, Queensland, 4105, Australia,² Veterinary Clinic, Route 349 KM1.0 Interior, Mayaguez, Puerto Rico 00680,³ Department de Parasitologie, Institut Agronomique et Veterinaire Hassan II, 6202 Rabat, Morocco,⁴ Division of Clinical Veterinary Medicine, Department of Animal Health and Welfare, Faculty of Veterinary Medicine—University of Bari, Strada per Casamassima km 3-70010 Valenzano (Ba.), Italy,⁵ VMRD, Inc., Pullman, Washington 99163,⁶ Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-7040,⁷ Washington Animal Disease Diagnostic Laboratory, Pullman, Washington 99163-2037⁸

Received 30 May 2006/ Returned for modification 11 August 2006/ Accepted 28 August 2006

A previously developed competitive enzyme-linked immunosorbent assay (cELISA) based on a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of rhoptry-associated protein 1 of Babesia bovis was refined and validated for use internationally. Receiver operating characteristic analysis revealed an assay with a specificity and positive predictive value of 100% and a sensitivity of 91.1%, with various negative predictive values depending on the level of disease prevalence. The cELISA was distributed to four different laboratories, along with a reference set of 100 defined bovine sera, including known-positive, known-negative, and field samples. Pairwise concordances among the four laboratories ranged from 94% to 88%. Analysis of variance of the resulting optical densities and a test of homogeneity indicated no significant difference among the laboratories. Overall, the cELISA appears to have the attributes necessary for international application.

Published ahead of print on 6 September 2006.

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