Detection and estimation of avian infectious bronchitis virus antigen by a novel indirect liquid-phase blocking enzyme-linked immunosorbent assay using chicken and rabbit affinity purified immunoglobulins.

An indirect liquid-phase blocking (LPB) enzyme-linked immunosorbent assay (ELISA) using chicken and rabbit affinity purified immunoglobulin G (IgG) has been developed to detect and estimate avian infectious bronchitis virus (IBV) antigen concentration directly in infected allantoic fluid. The method is based on the principle of binding of specific IgG to the test IBV antigen and the assay of unbound IgG on an antigen-coated ELISA plate. The immunoglobulins are chicken N-terminal S2 peplomeric protein-specific IgG isolated by immunoaffinity chromatography on synthetic peptide coupled to CNBr-activated Sepharose 4B or rabbit polyclonal IgG purified from the serum using Protein A Sepharose 4B. The assay detected all tested IBV strains and field isolates propagated in chicken embryos. Signal to noise ratios were calculated from LPB ELISA absorbance units and a diagnostic threshold was established from the signal to noise ratio frequency distribution of samples positive or negative for IBV by virus titration or reverse transcription polymerase chain reaction. The relative sensitivity of the test ranged between 10(5) and 10(6) median egg infectious doses (EID(50)) for chicken IgG and between 10(3) and 10(4) EID(50) for rabbit IgG, depending on the test strain. The assay is simple and takes less than 3 h to perform. It does not require expensive reagents and can be readily adapted to monitor the IBV antigen concentration in allantoic fluids during propagation of vaccine strains or in samples of freeze-dried, live-attenuated IBV vaccines.