title: Validation of a blocking enzyme-linked immunosorbent assay for detection of antibodies against porcine reproductive and respiratory syndrome virus.
abstract: Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most significant diseases of swine. IDEXX HerdChek PRRS, a commercially available enzyme-linked immunosorbent assay (ELISA), has become the industry standard for the detection of antibodies against PRRS virus (PRRSV). The need to accurately determine the PRRSV serostatus of herds and individual animals has prompted the development of several follow-up assay methods. A highly specific and repeatable blocking ELISA (bELISA) was developed on the basis of the use of an expressed PRRSV nucleocapsid (N) protein as the antigen and a biotinylated monoclonal antibody. Validation of the bELISA used sera from 316 animals experimentally and naturally infected with North American PRRSV and sera from 370 uninfected animals. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 97.8% and a diagnostic specificity of 100%. The between-run coefficient of variation of an internal quality control serum was 4.24%. The bELISA was able to detect seroconversion as well as the IDEXX ELISA and the indirect fluorescent antibody (IFA) assay; kappa values were 0.94 and 0.96, respectively. A collection of 133 serum samples with unexpected positive IDEXX ELISA results was obtained from 4,038 diagnostic samples submitted from farms from which PRRS-negative results were expected. The bELISA identified 97% of the samples as PRRS seronegative, while the IFA identified 100% as seronegative. The anticipated use of the bELISA is as a follow-up test to the IDEXX ELISA for determining the PRRSV serostatus of individual animals with unexpected positive test results from swine herds from which negative results are expected.
authors.affiliation: Department of Veterinary Science, P.O. Box 2175, North Campus Dr., South Dakota State University, Brookings, SD 57007, USA. neal_ferrin@sdstate.edu
authors.forename: Neal H
authors.initials: NH
authors.lastname: Ferrin
authors.forename: Ying
authors.initials: Y
authors.lastname: Fang
authors.forename: Craig R
authors.initials: CR
authors.lastname: Johnson
authors.forename: Michael P
authors.initials: MP
authors.lastname: Murtaugh
authors.forename: Dale D
authors.initials: DD
authors.lastname: Polson
authors.forename: Montserrat
authors.initials: M
authors.lastname: Torremorell
authors.forename: Marie L
authors.initials: ML
authors.lastname: Gramer
authors.forename: Eric A
authors.initials: EA
authors.lastname: Nelson
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood* / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D019921:Biotinylation; D015153:Blotting, Western; D016001:Confidence Intervals; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000706:statistics & numerical data / Q000662:veterinary; D005189:False Positive Reactions; D007239:Infections / Q000276:immunology; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology; D019316:Porcine respiratory and reproductive syndrome virus / Q000276:immunology*; D011237:Predictive Value of Tests; D012372:ROC Curve; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D014611:Vaccination
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins