Buoyant-Antigen-Magnetic (BAM) ImmunoSeparation, isolation, and detection of Specific Pathogenic Bacterial Cells

Abstract

Separating, collecting, and detecting specific cells and molecules from complex and heterogeneous samples is key for many biomedical, environmental, and food safety applications. Antibodies are widely used to specifically bind cells and molecules to a solid support such as a column or suspended bead to separate, wash, and concentrate them. However, nonspecific binding of non-target cells and molecules to the support limits the specificity and resulting purity of traditional separation methods. Herein we describe a buoyant-antigen-magnetic (BAM) separation approach to increase specificity using two distinct separation processes with "AND" logic. Buoyant silica microbubbles and magnetic microbeads, each with their own antibody, bind to the target cell forming a BAM sandwich complex which can be separated from non-targeted cells (which are neither magnetic nor buoyant), as well as from cells bound to only single buoyant or magnetic microparticles but not both. Escherichia coli O157:H7, a pathogenic bacteria that can contaminate food products, was used as a model organism to establish a nearly 100-fold reduction in the non-specific carryover of non-target Escherichia coli bacteria in comparison to simple magnetic separation. Beads with no attached analyte were also removed. Furthermore, the BAM complexes could be visualized by eye (or camera) providing a rapid and sensitive method for pathogenic bacterial detection. Bacterial concentrations as low as 5 x 104 CFU/ml were evident by eye and could easily be distinguished from non-target S. aureus bacteria even at much higher concentrations (5x107 cfu/mL). This approach enabled simple user-friendly measurement for on-site detection and isolation without expensive equipment. HIGHLIGHTS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/517249v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@194c382org.highwire.dtl.DTLVardef@1afcd3dorg.highwire.dtl.DTLVardef@cba148org.highwire.dtl.DTLVardef@c9b6cb_HPS_FORMAT_FIGEXP M_FIG C_FIG