Immunoliposome sandwich assay for the detection of Escherichia coli O157:H7.
We describe the development of a field-portable colorimetric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating dye as an analytical reagent. Antibodies (anti-E. coli O157:H7) thiolated by 2-iminothiolane were coupled to malemide-tagged liposomes encapsulating the marker dye, sulforhodamine B. Transmission electron microscopy showed that the immunoliposomes bound only to the serotype without any cross-reactivity with tested negative controls. A wicking reagent containing immunoliposomes and the test sample and a plastic-backed nitrocellulose strip with a measurement zone were used in a sandwich (noncompetitive) assay format. During the capillary migration of the wicking reagent, E. coli, with surface-bound immunoliposomes, was captured at the measurement zone on which antibodies to E. coli O157:H7 were immobilized. The color density of the measurement zone was directly proportional to the amount of E. coli O157:H7 in the sample. The detection limit of the current assay with pure cultures of the serotype was ca. 10(4) colony-forming units (CFU)/mL. The assay, which does not need washing and incubation steps, can be completed in 8 min. These results demonstrate the feasibility of using dye-encapsulating immunoliposomes in microporous membranes for the rapid detection of molecules with multivalent antigenic sites.
Park
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S
Department of Food Science and Technology, Cornell University, Geneva, New York 14456-0462, USA.
Durst
R A
RA
D000907:Antibodies, Bacterial / Q000276:immunology; D003124:Colorimetry / Q000379:methods; D003429:Cross Reactions; D019453:Escherichia coli O157 / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure; D008081:Liposomes / Q000648:ultrastructure; D008854:Microscopy, Electron; D012680:Sensitivity and Specificity
D000907:Antibodies, Bacterial; D008081:Liposomes