Liposome based homogeneous immunoassay for diagnostic tests

Details Liposome based homogeneous immunoassay for diagnostic tests Liposome based homogeneous immunoassay for diagnostic tests

G - Physics

01

N

167/139, 167/44

G01N 33/544 (2006.01) G01N 33/543 (2006.01) G01N 33/58 (2006.01) G01N 33/68 (2006.01) G01N 33/76 (2006.01)

Patent

CA 1309344

"LIPOSOME BASED HOMOGENEOUS IMMUNOASSAY FOR DIAGNOSTIC TESTS" ABSTRACT The present invention provides for novel homogeneous immunoassay systems involving complement- mediated lysis of marker-encapsulating lipid vesicles (liposomes) for detection of analyte in a fluid sample. These systems do not require the separation of unbound antigens and/or antibody conjugates yet provide highly sensitive procedures for analyte detection. Liposomes containing a marker, are coupled to antibody fragments in a way which confers the liposomes with immunological specificity yet avoids sensitizing the liposomes to complement mediated lysis in the absence of analyte. Antibody sensitized liposomes (the first reagent) are sequentially incubated with an analyte- containing sample, and optionally "dummy" liposomes, which do not contain encapsulated marker, a second antibody (the second reagent), and finally with a complement source such as plasma. Complement is activated by the liposome-antibody-antigen-second antibody complex causing liposome lysis and a concomitant release of marker. Antibody of the first reagent may be an anti-analyte F(ab')2 antibody fragment, or an anti-analyte Fab' antibody fragment. Antibody of the second reagent may be provided in either soluble form, or in insoluble form e.g., bound onto carboxylated polystyrene particles or coupled to a third antibody in the form of a "double antibody" immune precipitate. Where the antibody of the second reagent is provided in an insoluble form, the analyte-containing sample is preferably incubated with the second reagent, to form an analyte-second antibody complex, prior to incubation with the first reagent. Where the second reagent consists of soluble antibodies, the first reagent is incubated with the analyte containing sample prior to incubation with the second reagent. Also provided are methods for preparing antibody sensitized liposomes in the presence of a polysaccharide capable of forming a reversible gel and methods for preparing derivatized Fab' antibody fragments for coupling to lipid vesicles.

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