Concanavalin A- Sandwich-ELISA for the detection of antibodies against Newcastle disease virus (NDV) in chicken

摘要

Concanavalin A (Con A), a lectin interacts with carbohydrate moieties of viruses and provide stable and sensitive detection when used as a capture agent. Indirect ELISA methods need purified Newcastle disease virus (NDV) or recombinant antigens for adsorption, whereas use of Con A as capture agent will enable the use of non-purified and non-concentrated virus as antigen replacing costly and time-consuming virus purification step. Con A based sandwich ELISA with non-purified NDV whole virus antigen with single serum dilution format has been developed in this study. The optimum concentrations of the capture agent, Con A and non-purified antigen preparations were determined by checker-board titration. Briefly, microplates were coated with predetermined optimum concentration of ConA (0.5 mg/ml; 50 mu g per well) and incubated for 18h at 4 degrees C. After washing, allantoic fluid with Newcastle disease virus (NDV) LaSota (HA titre, 2(10)) at a constant predetermined dilution (1:1; 50 mu l) was coated and incubated for 45 min at 37 degrees C, followed by blocking with 2 % bovine serum albumin for 45 min at 37 degrees C. The antigen coated plates were used in the detection of antibody titre against NDV in serum samples at single serum dilution of 1: 500. Then, wells were added with goat anti-chicken IgG horseradish peroxidase conjugate and incubated for 1h at 37 degrees C, followed by addition of TMB substrate and the plates were read spectrophotometrically at 450 nm. ELISA antibody titres were determined by standard serial dilution of positive sera and endpoints were calculated by a subtraction method. By using positive negative threshold curve (PNT), intercept and slope of the standard curve were calculated. Total of 271 random chicken serum samples were analyzed for antibodies against NDV by Haemagglutination inhibition assay (HI), indirect ELISA and compared with the Con A-S-ELISA developed in this study. The Con A-S-ELISA showed a high coefficient of correlation (r=0.85, n=271, P<0.01) and an agreement of kappa=0.99 with the commercially available Indirect-ELISA. The relative sensitivity and specificity were 98% and 85% respectively in comparison to HI test. Hence, the Con A-S-ELISA is a simple, easy and effective for monitoring serum antibody levels against NDV.