Development of an immunocapture-polymerase chain reaction assay using IgY to detect Mycobacterium avium subsp....

A diagnostic assay using immunomagnetic separation was developed to capture Mycobacterium avium subsp. paratuberculosis (MAP) from bovine feces by means of IgY derived from chicken eggs. The antibody was coupled directly onto the surface of MagaCell cellulose/iron oxide beads or indirectly by being mixed with MagaBeads and a rabbit IgG linker against chicken antigen. Optimization parameters for the immunocapture included incubation time, temperature, volume, and type of immunocapture beads. Analytical sensitivity and specificity were determined by extracting DNA from the captured bacteria and amplifying it by polymerase chain reaction (PCR). The 2 bead preparations had the same analytical sensitivity, and the detection level of MAP cells in spiked bovine feces was 2 x 10(4) cells/g. No PCR inhibition was observed with DNA from the organisms captured with use of the MagaCell-IgY beads.