| The purpose of this study is to develop rapid, easy-to-perform, sensitive, and specific biosensors for the detection of polyvalent antigens, using dye-encapsulated antibody-conjugated liposomes as an analytical reagent. A model analyte for these assays is Escherichia coli O157:H7 (0157), a newly emerging foodborne pathogen.; Two conjugation strategies are used to prepare the immunoliposomes. The first is the conjugation of anti-O157 antibodies (Ab) thiolated by 2-iminothiolane to maleimide-lipid incorporated liposomes preformed by the reverse-phase evaporation method. Using this strategy, up to 45 Ab per liposome were conjugated without any detectable nonspecific aggregation. The second is the conjugation of the Ab thiolated by SATA (succinimidyl-S-acetylthioacetate) to maleimide-polyethylene glycol (2000)-lipid incorporated liposomes preformed by the same liposome preparation method. Using this strategy, up to 173 Ab per liposome were conjugated without any detectable aggregation.; The advantages of immunoliposomes, encapsulating dye were utilized in developing two rapid detection systems based on a direct sandwich assay format for the detection of the pathogen in foods. The first assay system, immunoliposome sandwich colorimetric (ILSC) assay, consists of a wicking reagent, immunoliposomes encapsulating a red dye, the test sample, and a plastic-backed nitrocellulose strip with two capture-measurement zones. The detection limit of the current assay with pure cultures of O157 is ca. 104 cells/mL. The assay can be completed in 8 min. O157-spiked ground beef samples at the levels from 0.7 to 102 CFU (colony forming unit)/g and O157-spiked apple cider samples at the levels from ca. 1 to 103 CFU/mL were detected by the assay after a 12-h incubation and a 8-h incubation, respectively. These results demonstrate that the assay is highly suitable for the detection of polyvalent antigens such as foodborne pathogens in the field, especially at sites lacking instruments for qualitative detection.; The second assay system, immunoliposome sandwich fluorometric (ILSF) assay, consists of immunoliposomes encapsulating a fluorphor, a microtiter plate, the test sample, a lysis solution, and a fluorescence plate-reader operated by a computer. The detection limit of the ILSF assay for pure cultures of O157 was determined to be ca. 104 cells/mL. The ILSF assay with pure cultures of O157 can be completed, from sample applications into the 96 wells of a microtiter plate to obtaining a dose-response curve, within 2 h 30 min. The assay also successfully differentiated O157-spiked apple cider samples from unspiked samples in spite of being affected by the sample matrix. The automated immunoliposome-based heterogeneous assay system will be useful for the routine screening of foodborne pathogens from a large number of food samples in a relatively short time. |
