Bead-based microfluidic immunoassay for diagnosis of Johne's disease

Journal Article · Sun Jan 01 04:00:00 EST 2012 · Journal of Immunological Methods

DOI:https://doi.org/10.1016/j.jim.2012.06.006· OSTI ID:1049822

Wadhwa, Ashutosh ^[1]; Foote, Robert ^[2]; Shaw, Robert W ^[2]; Eda, Shigetoshi ^[2]

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1. University of Tennessee, Center for Wildlife Health, Department of Forestry
2. ORNL

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne s disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types of SAB, and types of magnetic beads) were optimized for a great degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested by commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads by laser-induced fluorescence. Antigen-coated magnetic beads treated with bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples obtained with the system were compared to those obtained with the flow cytometer, a high level of correlation (linear regression, r2 = 0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD. Key Words: Mycobacterium avium ssp. paratuberculosis, Johne s disease, microfluidics, lab-on-a-chip.

• Research Organization: Oak Ridge National Laboratory (ORNL)
• Sponsoring Organization: ME USDOE - Office of Management, Budget, and Evaluation; ORNL work for others
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 1049822
• Journal Information: Journal of Immunological Methods, Journal Name: Journal of Immunological Methods Journal Issue: 1-2 Vol. 382; ISSN 0022-1759; ISSN JIMMBG
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ANTIBODIES
ANTIGENS
CATTLE
CELL FLOW SYSTEMS
DIAGNOSIS
DISEASES
FLUORESCENCE
GLASS
IMMUNOASSAY
INFECTIOUS DISEASES
Johne's disease
MYCOBACTERIUM