Amplified assay for a ligand

Claim 2

An assay for an analyte, the assay comprising: contacting tracer and analyte with a binder for at least the analyte, the tracer comprising a ligand coupled to a sac lysing agent, the ligand portion of the tracer being bound by one of the analyte and binder, whereby a portion of the tracer is bound to the binder; contacting unbound tracer with sacs, at least some of the sacs including in the interior thereof a detectable marker and at least some of the sacs including a sac lysing agent in an inactive form and which is active when released from the sacs; and determining marker released from the sacs as a measure of analyte.

Claim 3

An assay according to Claim 1, wherein the sacs are liposomes.

Claim 4

An assay according to Claim 2, wherein the lysing agent is an enzyme and the enzyme in the interior of the sacs is in an inactive form.

Claim 5

An assay for an enzyme comprising: contacting tracer and analyte with a binder for at least the analyte, the tracer comprising a ligand coupled to active liposome lysing agent comprising an enzyme, the ligand portion of the tracer being bound by one of the binder and analyte, whereby a portion of the tracer is bound by the binder; contacting unbound tracer with liposomes containing in the interior therein a non-isotopic detectable marker and a liposome lysing agent comprising an enzyme in inactive form, the contacting with liposomes being effected in the presence of an activating agent for the liposome lysing agent within the liposomes whereby detectable marker is released from the lysed liposomes and liposome lysing agent released from the liposomes is active for lysing additional liposomes to provide a cascading effect; and determining released marker as a measure of analyte.

Claim 6

An assay for an enzyme comprising: contacting tracer and analyte with a binder for at least the analyte, the tracer comprising a ligand coupled to active liposome lysing agent comprising an enzyme, the ligand portion of the tracer being bound by one of the binder and analyte, whereby a portion of the tracer is bound by the binder; contacting unbound tracer with liposomes containing in the interior therein a non-isotopic detectable marker and a liposome lysing agent comprising an enzyme in inactive form, the contacting with liposomes being effected in the presence of an activating agent for the liposome lysing agent within the liposomes whereby detectable marker is released from the lysed liposomes and liposome lysing agent released from the liposomes is active for lysing additional liposomes to provide a cascading effect; and determining released marker as a measure of analyte.

Claim 7

An assay according to Claim 3 or 4, wherein the interior of the liposome includes a calcium dependent enzyme lysing agent and a calcium complexing agent to inactivate the enzyme lysing agent within the liposome, and the activating agent comprises calcium in an amount sufficient to activate released lysing agent.

Claim 8

An assay according to Claim 3, 4 or 5, wherein the interior of the liposome includes an apo-enzyme and the activating agent is a coenzyme.

Claim 9

An assay according to any preceding Claim, wherein the sacs comprise sacs which include both the detectable marker and the lysing agent.

Claim 10

An assay according to any preceding Claim, wherein the binder is supported on a solid support.

Claim 11

An assay according to any preceding Claim, wherein the binder is a binder for both the analyte and tracer.

Claim 12

An assay according to Claim 9, wherein bound and unbound tracer portions are not separated prior to release of the marker whereby the assay is a homogeneous assay.

Claim 13

An assay according to any preceding Claim wherein the detectable marker is a non-isotopic marker.

Claim 14

An assay according to any preceding Claim, wherein the analyte is a hapten.

Claim 15

An assay according to any preceding Claim, wherein tne analyte is in a sample of a body fluid.

Claim 16

A reagent kit for an assay for analyte. the kit comprising a package containing (a) a tracer comprising a ligand coupled to a sac lysing agent; and (b) sacs, at least some of the sacs containing in the interior thereof a sac lysing agent in an inactive form and at )least some of the sacs containing in the interior thereof a detectable marker.

Claim 17

A reagent kit for an assay for analyte. the kit comprising a package containing (a) a tracer comprising a ligand coupled to a sac lysing agent; and (b) sacs, at least some of the sacs containing in the interior thereof a sac lysing agent in an inactive form and at )least some of the sacs containing in the interior thereof a detectable marker.

Claim 18

A kit according to claim 14, wherein the sacs comprise sacs which contain both the sac lysing agent, in inactive form, and the detectable marker in the interior thereof, said detectable marker preferably being a non-isotopic marker.

Claim 19

A kit according to Claim 14 or 15, wherein the package further contains a binder for at least the analyte, the ligand portion of the tracer preferably being bound by the binder.

Claim 20

A kit according to any of Claims 14 to 16, wherein the package further contains standard solutions of the analyte and an activating buffer for the sac lysing agent in the interior of the sacs.

Claim 21

A kit according to any of Claims 14 to 17, wherein the lysing agent is an enzyme, and the enzyme in the interior of the sacs is in an inactive form.

Claim 22

A kit according to Claim 18, wherein the sacs are liposomes.

Claim 23

A kit according to Claim 19, wherein the enzyme lysing agent in the liposome is a calcium dependent enzyme and the interior of the liposome further contains a calcium complexing agent to inactivate the enzyme.