Development of an enzyme‐linked immunosorbent assay for the detection of humoral antibody topasteurella anatipestifer

Abstract

SUMMARYAn enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Pasteurella anatipestifer in duck sera is described. As part of the initial assay development, micro-titration plates from different manufacturers were assayed for their suitability to bind P. anatipestifer antigen. The Nunc Immunopiate II was chosen, on account of its overall reproducibility (5.5% coefficient of variation) and the absence of an edge effect. Optimum concentrations of reagents were determined and the inclusion of 1.0M NaCl in the wash buffer was found to reduce non-specific binding and increase assay sensitivity. The assay is specific in that antibodies were detected only in those ducks either exposed to or following vaccination with P. anatipestifer; sera from ducks immunised with other heterologous bacterial antigens, and having agglutinating antibodies to them, gave no detectable response in the ELISA. Between-assay coefficients of variation for the quality control serum pools representing high, medium and low levels of antibodies to P. anatipestifer were 6.8%, 8.3% and 8.6% respectively. A precision-dose profile was derived. A graph of absorbance versus log 2 serum end point titre showed a linear relationship (r = 0.99) over the range investigated. The derived regression line (P<0.001) was used to transform the absorbance measurement obtained for a single 1:100 dilution of serum into a log 2 titre value.It was demonstrated that the ELISA is a much superior method to rapid slide agglutination and agar gel precipitin tests in measuring antibody responses to exposure against P. anatipestifer type 2.

Pathogenesis ofRiemerella anatipestiferin turkeys after experimental mono-infection via respiratory routes or dual infection together with the avian metapneumovirus

“…Serum antibody response to RA was examined by an IgG-ELISA system following protocols previously developed for detection of RA antibodies in ducks (Hatfield et al, 1987;Lobbedey & Schlatterer, 2003). In contrast to the published protocol, we used commercially available dilution buffer, ABTS substrate, and stop solution and the anti-turkey-IgG conjugate instead of anti-duck-IgG.…”

ELISAs and the serological analysis of salmonella infections in poultry: a review

“…The use of ELISAs Recent advances in ELISA (enzyme-linked immunosorbent assay) technology, including miniaturization and mechanization together with availability of commercially-prepared high quality reagents, has resulted in the application of the technique to the detection of infection caused by several avian pathogens such as infectious bronchitis virus [7], turkey rhinotracheitis virus [8], Pasteurella rnultocida [9] and P. anatipestifer [10]. ELISAs based on lipopolysaccharide and Vi antigens have been used to detect S. typhirnuriurn and S. typhi infections in rodents and man respectively [11,12,13].…”

Antibodies to Myelin P0 and Ceramide Perpetuate Neuropathy in Long Standing Treated Leprosy Patients

“…Indirect ELISA [14] technique was standardized for myelin P0 and the optimal conditions for the assay are as follows. The protein was suspended in the Phosphate buffered saline (PBS) pH 9.6 (137 mM NaCl, 2.6 mM KCl, 10 mM Na 2 HPO 4 and 1.7 mM KH 2 PO 4 ) at a concentration of 100 ng/100ul per well and was coated on the Immulon-II flat bottom ELISA plates and incubated overnight at 37°C.…”