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

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Abstract
An 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 log2 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 log2 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.