Studies on avian infectious bronchitis: II. Standardization of an indirect enzyme-linked immunosorbent assay (ELISA) for antibody measurement

Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to avain infections bronchitis virus (IBV) were standardied. By adopting the optimized conditions an equation calculating ELISA antibody titers from the observations at single serum dilution was formulated. The purified antigen of IBV lMass-41 strain was dispensed into polystyrene microplate wells at a concentration of 300ng per well (100 micr l) and the plates were coated by completey drying at 37deg C. Diluted chicken serum and horseradish peroxidase conjugated goat anti-chicken IgG were added in order in 100 micro l volumes per well and allowed to react for 30 minutes each at room temperature. Just before use and after each reaction the plates were washed three times with distilled water. Finally o-phenylenediamine solution was added as an enzyme substrate. After incubation for another 15 minutes at room temperature absorbances were read at 492 nm. Hyperimmune serum against Mass-41 strain was used as internal reference positive (IRP)serum. After repeated titration of IRP and negative sera, a constant titer of IRP was determined. Serum titrations were carried out for various sample sera together with IRP and negative sera and the observed titers of sample sera were corrected by reflecting the ratio between observed and constant titers of IRP serum. These corrected titers of the sample sera were plotted against sample/positive (S/P)OD ratios. All the OD's measured in the serum titrations were also corrected by substracting negative serum OD. The following equation was formulated from the above data; Log10 ELISA titer = 5.568 (log10 S/P) + 4.161. Thus it was possible to calculate ELISA titer by measuring absorbance at 1/400 single serum dilution. Titer measured by cross ELISA tests employing Mass-41 strain and three local IBV isolates were similar. These results suggest that the ELISA tests standardized in this study can be used for evaluating not only vaccinal immunity but also for infection status against fields IBV's