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Journal of Clinical Microbiology, November 2001, p. 3851-3857, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.3851-3857.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Validation of a Commercially Available Monoclonal Antibody-Based Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to Neospora caninum in Cattle

Timothy V. Baszler,1,2,* Scott Adams,3 John Vander-Schalie,2 Bruce A. Mathison,1 and Miladin Kostovic3

Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-70401; Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, Washington 99165-20342; and VMRD Inc., Pullman, Washington 991633

Received 16 March 2001/Returned for modification 17 June 2001/Accepted 20 August 2001

A previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody to Neospora caninum, a major cause of bovine abortion. Modifications to the cELISA included capturing native N. caninum antigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a "gold standard" set of 184 cow sera defined by fetal histopathology and N. caninum immunohistochemistry and by maternal N. caninum indirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknown N. caninum status. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknown N. caninum status revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modified N. caninum cELISA provided a simple, rapid, and versatile method to accurately identify N. caninum infection status in cattle using a single cutoff value.

* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Bustad Hall, Washington State University, Pullman, WA 99164-7040. Phone: (509) 335-6047. Fax: (509) 335-8529. E-mail: baszlert{at}vetmed.wsu.edu.

Journal of Clinical Microbiology, November 2001, p. 3851-3857, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.3851-3857.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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