Development of an immunomagnetic assay system for rapid detection of bacteria and leukocytes in body fluids

Abstract

Immunomagnetic (IM) separation and concentration of specific target ligands or particles, such as bacteria or leukocytes, from complex mixtures, such as bone marrow, blood and other body fluids, is now a widely accepted technique. IM methodologies require high affinity antibodies or other receptors, but are potentially as effective as density gradient separations. Thus, a computer-controlled first-generation immunomagnetic assay system (IMAS) biodetector is being developed for clinical diagnostics. This system is fully automated and affords the advantage of rapid flow-through capture of all types of magnetic beads (MBs) and obviates operator contact with body fluid samples during the collection and analysis phases. In the present work, biotinylated capture antibodies were bound to streptavidin-coated MBs for capture of E. coli O157:H7, T cells and T cell subsets. Samples were automatically vortex mixed with antibody-coated MBs, stained with an acridine dye or fluorescent antibody and collected in a specially designed flow cell containing multiple steel pins, which concentrate external magnetic field lines. IM complexes were rapidly (within minutes), separated from their media in the magnetic field. Magnetically captured particles were automatically rinsed in the flow cell to remove unwanted materials and detection was achieved via a flow-through fluorimeter. Samples can be subsequently captured on a microbiological filter for microscopic visualization and image analysis. Preliminary results demonstrate that rapid detection of target bacteria and leukocytes at low concentrations in body fluids is possible with a total assay time under 1 h. This IM technology has many other potential clinical, industrial and environmental monitoring applications.