title: Validation of an indirect ELISA to detect antibodies against BoHV-1 in bovine and guinea-pig serum samples using ISO/IEC 17025 standards.
abstract: Two ELISAs to quantify antibodies to BoHV-1 in the sera of cattle and immunized guinea pigs were developed and validated using ISO/IEC 17025 standards. The cut-off value of the assay was established at 20% positivity of a high positive control for screening of cattle. Using this threshold, the assay properly classified the OIE bovine reference sera EU1, EU2 and EU3. For vaccine potency testing, a cut-off of 40% was selected for both species. The reliability of the assays, given by their diagnostic sensitivity and specificity, using the threshold of 40% was 89.7% and 100%, respectively, for bovines and 94.9% and 100% for guinea pigs, respectively. There was almost perfect agreement between the ELISA and virus neutralization results. In addition, after vaccination, there was a good correlation between the neutralizing and ELISA antibody titers of the serum from the same bovine or guinea pig, sampled at 60 and 30 days post-vaccination, respectively (R(bovine)=0.88, R(guinea pig)=0.92; p<0.0001). A similar correlation was observed when analyzing the mean antibody titers of groups of vaccinated animals (R(bovine)=0.95 and R(guinea pig)=0.97; p<0.0001), indicating the relevance of the ELISAs for batch to batch vaccine potency testing in the target species and in the laboratory animal model. The intermediate precision of the assays expressed as the relative coefficient of variation (CV) of the positive control assayed over a 3-year period in the same laboratory was 22.2% for bovines and 23.1% for guinea pigs. The reproducibility of both techniques obtained in inter-laboratory assays was CV=12.4% for bovines and CV approximately 0 for guinea pigs, which met the requirements of the OIE (CV<30%). The validated ELISAs represent important methods for vaccine potency testing and for controlling BoHV-1 infections.
authors.affiliation: Instituto de Virología, Centro de Investigaciones en Ciencias Veterinarias y Agronomicas (CICV y A) - Instituto Nacional de Tecnologia Agropecuaria (INTA), Buenos Aires, Argentina; CONICET, Argentina. vparreno@cnia.inta.gov.ar
authors.forename: Viviana
authors.initials: V
authors.lastname: Parreño
authors.forename: S Alejandra
authors.initials: SA
authors.lastname: Romera
authors.forename: Lucia
authors.initials: L
authors.lastname: Makek
authors.forename: Daniela
authors.initials: D
authors.lastname: Rodriguez
authors.forename: Darío
authors.initials: D
authors.lastname: Malacari
authors.forename: Silvina
authors.initials: S
authors.lastname: Maidana
authors.forename: Diego
authors.initials: D
authors.lastname: Compaired
authors.forename: Gustavo
authors.initials: G
authors.lastname: Combessies
authors.forename: María Marta
authors.initials: MM
authors.lastname: Vena
authors.forename: Lorena
authors.initials: L
authors.lastname: Garaicoechea
authors.forename: Andrés
authors.initials: A
authors.lastname: Wigdorovitz
authors.forename: Laura
authors.initials: L
authors.lastname: Marangunich
authors.forename: Fernando
authors.initials: F
authors.lastname: Fernandez
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D006168:Guinea Pigs; D007242:Herpesvirus 1, Bovine / Q000276:immunology*; D022283:Herpesvirus Vaccines / Q000276:immunology; D009500:Neutralization Tests; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D000914:Antibodies, Viral; D022283:Herpesvirus Vaccines