Validation of a commercially available monoclonal antibody-based competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to Neospora caninum in cattle.
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.
Baszler
T V
TV
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040, USA. baszlert@vetmed.wsu.edu
Adams
S
S
Vander-Schalie
J
J
Mathison
B A
BA
Kostovic
M
M
D000034:Abortion, Veterinary / Q000469:parasitology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000913:Antibodies, Protozoan / Q000097:blood*; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D003048:Coccidiosis / Q000469:parasitology / Q000662:veterinary*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D008807:Mice, Inbred BALB C; D018688:Neospora / Q000254:growth & development / Q000276:immunology*; D011247:Pregnancy; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan