Liposome immunoassay reagent and method

Claim 2

An assay reagent for determination of an analyte in a lipid-vesicle immunoassay, said reagent comprising: a suspension of oligolamellar lipid vesicles containing encapsulated glucose-6-phosphate dehydrogenase at a specific activity of between about 1-10 units/μmole vesicle lipid, and glucose-6-phosphate, at a concentration of between about 2 and 50 mM, and carried on each vesicle, a surface array of ligand molecules adapted to bind specifically and with high affinity to analyte-related anti-ligand molecules.

Claim 3

The reagent of claim 1, wherein the encapsulated glucose-6-phosphate dehydrogenase has a specific activity of at least about 5 units/μmole lipid.

Claim 4

The reagent of claim 1, wherein the encapsulated glucose-6-phosphate has a concentration of between about 5 and 25 mM.

Claim 5

The reagent of claim 3, wherein the surface-bound ligand molecules are derivatized to a vesicle lipid component.

Claim 6

The reagent of claim 4, for determination of theophylline, wherein the surface-bound ligand molecules are derivatized to phosphatidylethanolamine.

Claim 7

The reagent of claim 1, for determination of an antigen analyte, wherein the surface-bound ligand molecules on said vesicles are antigen or antigen-like molecules.

Claim 8

A homogeneous enzyme immunoassay system for determination of an analyte comprising an assay reagent composed of a suspension of oligolamellar vesicles encapsulating glucose-6-phosphate dehydrogenase at a specific activity of between about 1-10 units/μmole vesicle lipid, and glucose-6-phosphate, at a concentration of between about 5 and 50 mM, and carried on each vesicle, a surface array of ligand molecules adapted to bind to anti-ligand analyte molecules, or to compete with soluble ligand-like analyte molecules for binding to soluble anti-ligand molecules, complement adapted to react with the vesicles in the presence of anti-ligand molecules, to effect, in proportion to the amount of ligand/anti-ligand binding on the vehicle surfaces, vesicle lysis and release of encapsulated glucose-6-phosphate dehydrogenase, and a reporter system adapted to react with released glucose-6-phosphate dehydrogenase, to produce a detectable spectral change, in proportion to the amount of enzyme released.

Claim 9

A homogeneous enzyme immunoassay system for determination of an analyte comprising an assay reagent composed of a suspension of oligolamellar vesicles encapsulating glucose-6-phosphate dehydrogenase at a specific activity of between about 1-10 units/μmole vesicle lipid, and glucose-6-phosphate, at a concentration of between about 5 and 50 mM, and carried on each vesicle, a surface array of ligand molecules adapted to bind to anti-ligand analyte molecules, or to compete with soluble ligand-like analyte molecules for binding to soluble anti-ligand molecules, complement adapted to react with the vesicles in the presence of anti-ligand molecules, to effect, in proportion to the amount of ligand/anti-ligand binding on the vehicle surfaces, vesicle lysis and release of encapsulated glucose-6-phosphate dehydrogenase, and a reporter system adapted to react with released glucose-6-phosphate dehydrogenase, to produce a detectable spectral change, in proportion to the amount of enzyme released.

Claim 10

The system of claim 7, wherein the encapsulated glucose-6-phosphate dehydrogenase has a specific activity of at least about 5 units/μmole vesicle lipid.

Claim 11

The system of claim 7 for determination of a ligand-like analyte, wherein the system includes such soluble anti-ligand molecules.

Claim 12

The system of claim 9, for determination of theophylline, wherein said anti-ligand molecules include anti-theophylline antibody and the ligand molecules are theophylline molecules derivatized with a vesicle lipid component.

Claim 13

The system of claim 7, wherein the said complement further includes small unilamellar vesicles added to reduce serum interference, in the absence of such ligand/anti-ligand binding on the vesicle surfaces.

Claim 14

The system of claim 7, wherein said reporter system includes glucose-6-phosphate, NAD, an electron carrier and a dye which undergoes a detectable color change upon reduction by NADH in the presence of such carrier.

Claim 15

The system of claim 12, wherein the dye includes dichloroindophenol.

Claim 18

The method of claim 14, wherein said providing includes preparing vesicles by reverse evaporation phase in the presence of glucose-6-phosphate, at an aqueous-phase concentration of at least 5 mM.

Claim 19

The method of claim 14, for use in determination of ligand-like analyte, wherein said mixing includes reacting the analyte and reagent in the presence of soluble anti-analyte antibody molecules.

Claim 20

The method of claim 16, for use in determination of theophylline, wherein the reagent provided includes surface-bound theophylline molecules and said mixing includes reacting the reagent with analyte and anti-theophylline antibodies.

Claim 21

The method of claim 14, wherein said adding further includes adding small unilamellar vesicles in an amount effective to reduce serum interference in the presence of complement.

Claim 22

The method of claim 14, wherein said determining includes forming NADH, by enzymatic oxidation of glucose-6-phosphate, and reducing a dye with the NADH formed, to produce a detectable color change in the dye.