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

Abstract

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.

Figure 1

Determination of Beads A volume required for immunocapture of American Type Culture Collection (ATCC) strain 19698 of Mycobacterium avium subsp. paratuberculosis (MAP). For each Beads A volume used, DNA was run undiluted (N) and at dilutions of 1:10 (−1) and 1:100 (−2). The bead volumes were as follows: lanes 1 to 3, 10 μL; lanes 4 to 6, 20 μL; lanes 7 to 9, 30 μL; lanes 10 to 12, 40 μL. Lane −C — negative water control; lane M — 1-kb molecular weight marker; bp — base pairs.

Figure 2A

Polymerase chain reaction (PCR) results with different concentrations of DNA extracted from ATCC 19698 MAP cells immunocaptured by 40 μL of Beads A incubated at room temperature (RT) for different times; DNA was run undiluted (N) and at dilutions of 1:10 (−1) and 1:100 (−2). The incubation times were as follows: lanes 1 to 3, 15 min; lanes 4 to 6, 30 min; lanes 7 to 9, 60 min. Lane +C — positive-control DNA (ATCC 19698); lane +IC — positive internal-control DNA; lane −C — negative water control; lane M — 1-kb molecular weight marker.

Figure 2B

Results of PCR with the use of 40 μL of Beads A to immunocapture MAP ATCC 19698 spiked in bovine feces (2 × 10⁴ cells/g) incubated at RT for 15 min. Lanes 1 and 2 — DNA diluted 1:10 and 1:100; lanes 3 and 4 — negative-control bovine feces with DNA diluted 1:10 and 1:100; lane 5 — positive-control DNA (ATCC 19698); lane 6 — negative water control; lane M — 1-kb molecular weight marker.

Figure 3

Determination of MagaCell-IgY beads volume required for immunocapture of MAP ATCC 19698 spiked in bovine feces (2 × 10⁴ cells/g) and incubated at RT for 15 min. For each MagaCell-IgY beads volume used, DNA was run undiluted (N) and at dilutions of 1:10 (−1) and 1:100 (−2). The bead volumes were as follows: lanes 1 to 3, 5 μL; lanes 4 to 6, 10 μL; lanes 7 to 9, 15 μL; lanes 10 to 12, 20 μL. Lane M — 1-kb molecular weight marker; lane +C — positive-control DNA (ATCC 19698); lane −C — negative water control.

Figure 4

Results of PCR with the use of 10 μL of MagaCell-IgY beads to immunocapture MAP strain ATCC 19698 spiked in bovine feces at concentrations of 2 × 10³ cells/g (lanes 1 to 3), 2 × 10⁴ cells/g (lanes 4 to 6), and 2 × 10⁵ cells/g (lanes 7 to 9). End-point titration was performed with extracted DNA run undiluted (N) and at dilutions of 1:10 (−1) and 1:100 (−2). Lane +C — positive-control DNA (ATCC 19698); lane +IC — positive internal-control DNA; lane −C — negative water control; lane M — 1-kb molecular weight marker.