Validation of a Commercially Available Monoclonal Antibody-Based Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies toNeospora caninumin Cattle

Abstract

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.

Development of competitive ELISA for neosporosis by employing immunoproteomics

“…The results of present study indicated the possibility that C-ELISA would not produce false positive results. Sensitivity of C-ELISA with chicken IgY (83.3%) and Herd-chek ® ELISA (73.2%) demonstrated a value lower than the other reports published (15,21,22,(40)(41)(42). The cause of this relatively low sensitivity in ELISA seems to be related to the sensitivity on IFAT.…”

“…Anti-T. gondii serum, moreover, were produced by immunizing rabbits. Prior to inoculation, all the donors were confirmed as N. caninum and T. gondii negative by IFAT (15,16). To obtain high immune response similar with natural infection, a cow was intravenously inoculated with 1 ϫ 10 8 live tachyzoites of the KBA-2 isolate suspended in phosphate-buffered saline (PBS).…”

Factores de Riesgo Asociados a la Seroprevalencia de Neospora caninum en Bovinos Naturalmente Infectados en la Ceja de Selva de Oxapampa, Perú

“…Se calculó la prevalencia corregida según la sensibilidad (97.6%) y especificidad (98.6%) de la prueba (Baszler et al, 2001). Asimismo, se analizó la asociación entre la seroprevalencia a N. caninum y los potenciales factores de riesgo mediante regresión logística, considerando factores de riesgo aquellos con valores p<0.05 (Boubeta y Varela, 2008).…”

Evaluation of a SRS2 Sandwich Commercial Enzyme-Linked Immunosorbent Assay for the Detection ofAnti-Neospora CaninumAntibodies in Bovine and Canine Sera

“…?show "fnote_aff1"$^! "content-markup(./author-grp [1]/aff|./author-grp [1]/dept-list)> Neospora caninum (order Eucoccidiorida, suborder Eimeriorina, family Sarcocystidae) is an apicomplexan parasite responsible for abortion in cows and neuromuscular disease in dogs. This heteroxenous parasite uses the dog and the American coyote (Canis latrans) as definitive hosts.…”

“…23 ELISA validation can also be performed without reference method by using true positive and true negative sera previously confirmed by several methods. 1 Although it was not the main purpose of the current study, preliminary information about the potential impact of N. caninum in Algeria was collected for the first time. The seroprevalence in cattle (3.9%) was low compared to the authors' previous study conducted in Belgium (29%).…”