RAPID DETECTION OF MYCOPLASMA INFECTIONS IN CHICKENS WITH A FLUORESCENCE CONCENTRATION IMMUNOASSAY 1

Abstract

ABSTRACTMycoplasma gallisepticum infection in chickens was detected with a fluorescence concentration immunoassay (FCIA) using intact mycoplasmal cells as the reacting particles. The antigen and test chicken antisera were directly pipetted into the wells of a 96‐well fluoricon assay plate and incubated 30 min at ambient temperature in the chamber of a fluorescence concentration analyzer. The antigen‐antibody bound complexes and free components automatically were separated by vacuum filtrations and washings through the membrane filter equipped at the base of the wells. Fluorescein isothiocyanate (FITC) was used as the tracer via FITC‐goat‐anti‐chicken IgG which was incubated with the above formed antigen‐antibody complexes in the wells for another 30 min. After the vacuum filtrations and washings, the fluorescent signal was read at a wavelength of 485/535 nm and was reported in a numerical arbitrary fluorescence unit (AFU). The positive cutoff AFU value was calculated by adding 2 standard deviations to the average AFU of the negative control. Samples with AFU above the cut‐off value were scored positive indicating the infection. It was found the FCIA method was comparable in sensitivity to the Agritech ELISA method and more sensitive than the serum plate agglutination (SPA) test or hemagglutination‐inhibition (HI) test. Forty‐seven/51 sera from M. gallisepticum inoculated chickens were scored positive using FCIA or ELISA methods, while 36/51 and 40/51 were indicated positive by HI and SPA tests respectively. The specificity of the FCIA assay using sera from M. gallisepticum inoculated chickens was shown to be comparable to the HI test. Cross‐reactivity occurred at 35%, 8% and 11% in SPA, HI and FCIA tests respectively. The FCIA assay can be completed in less than 2 h with a total of 24 samples including one negative control and one positive control sera assayed in one plate.