Evaluation of a Monoclonal Blocking Enzyme-Linked Immunosorbent Assay for the Detection of Mycoplasma gallisepticum-Specific Antibodies

Abstract

A monoclonal blocking enzyme-linked immunosorbent assay (blocking-ELISA) was developed to detect antibodies to Mycoplasma gallisepticum (MG) in poultry sera with the help of a peroxidase-labeled monoclonal antibody (MAb) recognizing an epitope of a 56-kilodalton polypeptide (p56) of MG. Immunoglobulins from undiluted MG-positive sera prevent the MAb conjugate from attaching to its specific binding site on p56, which results in no color development. The opposite result--a strong color reaction--was obtained after incubation with MG-negative sera (or when no serum was added before the MAb conjugate). Results were expressed in percent inhibition or ELISA titers. The blocking-ELISA detected 84.7% positive chickens in an experimentally infected flock and 72.6% of chickens in naturally infected flocks, whereas the hemagglutination-inhibition (HI) test was positive only with 68.4% and 48.6% of these serum samples, respectively. All HI-positive serum samples reacted positively in blocking-ELISA. Of sera negative by the HI test, 51.6% and 46.8% proved to be positive when examined with the blocking-ELISA. Overall agreement between the ELISA and HI test was 76.8%. Infection with closely related M. synoviae did not induce any false-positive reactions in blocking-ELISA. There was a strong positive correlation between HI and blocking-ELISA titers (r = 0.83).