Development of a mast cell-based biosensor.
A mast cell-based biosensor has been developed to enable the use of these cells in numerous applications including pharmaceutical screening, environmental monitoring, clinical diagnosis and homeland security. Rat basophilic leukemia (RBL) mast cells offer excellent potential for biosensor applications because they are robust and undergo a dramatic exocytotic response within minutes of antigen addition. To monitor mast cell activation, fluorescent dyes were loaded into the cells and used as indicators of alkalinization of secretory granules, calcium fluxes or generation of reactive oxygen species. These fluorescence assays efficiently measure activation of antigen-stimulated RBL mast cells, detecting the antigen with picomolar sensitivity. To demonstrate the utility of this mast cell-based biosensor for detection of microbial pathogens, an IgE chimeric protein was created by fusing the Fc region of the IgE antibody to CD14, a receptor for lipopolysaccharide. This chimeric protein has the capacity to bind to Escherichia coli and Listeria monocytogenes and also to IgE receptors on the mast cells, thereby stimulating a signaling response to bacteria. RBL mast cells labeled with the calcium indicator Fluo-4 are shown to be responsive to E. coli, only when sensitized with the chimeric protein, thus demonstrating a highly versatile biosensor for bacterial contamination.
Agave BioSystems, Ithaca, NY 14850, USA. curtist@cortland.edu
Theresa
T
Curtis
Rose Mary Z G
RM
Naal
Carl
C
Batt
Joel
J
Tabb
David
D
Holowka
D000818:Animals; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002460:Cell Line; D015169:Colony Count, Microbial / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000302:isolation & purification*; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D008407:Mast Cells / Q000382:microbiology*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods