Enzymatic poly-reactant channeling binding assay

Claim 2

A channelling assay method for detecting the presence of an analyte in a sample suspected of containing said analyte, when said analyte is a member of a specific binding pair consisting of first and second binding members ("FBM" and "SBM", respectively), characterised by said method employing a channeling signal producing system comprising an enzyme, enzyme , conjugated to an SBM to provide enzyme -SBM conjugate, an enzyme substrate and a Final Reactant, wherein said Final Reactant is (1) another enzyme, enzyme , where the two enzymes are related by the substrate of one enzyme being the product of the other enzyme, or (2) a compound which reacts with the product of enzyme , wherein the reactions of said enzyme + in conjunction with enzyme or said compound results in a change in an observable signal in relation to the amount of analyte in said sample; and linking system providing for the polymerization of said Final Reactant and the incorporation of enzyme within the polymer as a function of the binding of enzyme + -SBM conjugate to FBM so as to form a polymeric channeling aggregate; said method comprising: combining in an aqueous assay medium; (a) said sample; (b) said enzyme -SBM conjugate, and FBM when said analyte is a SEM, so as to form a complex between said FBM and said enzyme + -SBM conjugate in relation to the amount of analyte present; (c) any members of said linking system, whereby is formed a polymeric channeling aggregate of said Final Feactant and a complex of enzyme + -SBM conjugate with FBM; and (d) remaining members of a signal producing system, whereby a Final Product is produced as a result of channeling of said members of said signal producing system in said polymeric channeling aggregate, which results in a change in a detectable signal; and comparing said detectable signal to the detectable signal observed in an assay medium having a known amount of analyte.

Claim 3

A method according to Claim 1, said method employing (1) enzyme channeling where two enzymes, enzyme and enzyme , which are members of a signal producing system are employed, where the product of one enzyme is the substrate of the other enzyme, and enzyme is conjugated to a SBM to provide enzyme + -SBM conjugate; (2) a linking system providing for the polymerization of enzyme and the incorporation of enzyme through the binding of a complex of enzyme -SBM conjugate to FBM to form a polymeric channeling aggregate; and (3) the remaining members of the signal producing system comprising substrates, including cofactors, of said enzymes, which results in a change in a detectable signal as a result of the formation of said polymeric channeling aggregate in relation to the amount of analyte present; said method comprising: combining in an aqueous assay medium; (a) said sample; (b) said enzyme+ - SBM conjugate, and FBM wherein said analyte is a SBM, so as to form a complex between said FBM and said enzyme + -SBM conjugate in relation to the amount of analyte present; (c) members of said linking system, whereby is formed a polymeric channeling aggregate of enzyme and the complex of enzyme + -SBM conjugate with FBM; and (d) members of the signal producing system, whereby a final product is produced as a result of enzyme channeling of said members of said signal producing system in said polymeric channeling aggregate, which results in a change in a detectable signal; and comparing said detectable signal to the detectable signal observed in an assay medium having a known amount of analyte.

Claim 4

A method according to Claim 2, wherein said FBM is a ligand and said SBM is a receptor.

Claim 5

A method according to Claim 3, wherein said SBM is an antibody.

Claim 8

A method according to Claim 2, wherein said enzymes are oxidoreductases.

Claim 9

A method according to Claim 2, wherein said assay medium is transferred to a porous support prior to the addition of said signal producing system to said polymeric channeling aggregates.

Claim 10

A method according to Claim 2, wherein said sample and said enzyme -SBM conjugate are incubated together in said assay medium before the addition of a non-enzymatic member of said linking system.

Claim 11

A method according to Claim 1, wherein said analyte is a member of a specific binding pair consisting of ligand and receptor; said method employing (1) enzyme channeling where two oxidoreductase enzymes, enzyme and enzyme , are employed, which are members of a signal producing system; where the product of one enzyme is the substrate of the other enzyme; and where enzyme is present conjugated to a first receptor, enzyme -first receptor conjugate wherein first receptors bind to ligand; (2) a linking system providing for the polymerization of enzyme , comprising enzyme conjugated to first receptor, enzyme -first receptor conjugate, wherein said * enzyme -first receptor conjugate has naturally occurring determinant sites or such sites are synthetically introduced, and a polyvalent second receptor for said determinant sites, whereby polymerization of enzyme and the incorporation of enzyme through the binding of a complex of enzyme -first receptor conjugate to ligand occurs to form a polymeric channeling aggregate; and (3) a signal producing system comprising substrates, including cofactors, of said enzymes, which results in the production of a final product which provides a change in a detectable signal as a result of the formation of said polymeric channeling aggregate in relation to the amount of analyte present; said method comprising: combining in an aqueous medium; (a) said sample; (b) said enzyme -first receptor conjugate, and ligand when said analyte is a receptor, to form an enzymes-first receptor and ligand complex; (c) enzyme -first receptor and second receptor * * for polymerizing enzyme , whereby enzyme is polymerized and incorporates enzyme as part of said complex; (d) members of the signal producing system, whereby said members in conjunction with the channeling of said enzymes produce a final product providing a detectable signal in an amount related to the amount of analyte present; and comparing said detectable signal to the detectable signal observed in an assay medium having a known amount of analyte.

Claim 12

A method according to Claim 10, wherein * additional enzyme is added as a member of said linking system.

Claim 14

A kit comprising enzyme + -SBM conjugate and a linking system including as one component, enzyme , where enzyme + and enzyme * are related by the substrate of one being the product of the other, and * means for polymerizing enzyme .

Claim 15

A kit comprising enzyme + -SBM conjugate and a linking system including as one component, enzyme , where enzyme + and enzyme * are related by the substrate of one being the product of the other, and * means for polymerizing enzyme .

Claim 16

A kit according to Claim 13, wherein said * linking system comprises enzyme -receptor conjugate and * * receptor for enzyme or a ligand bound to enzyme .