Liquid single reagent for air enzyme complementation assay

Claim 1

A method for using a preformed liquid single reagent in an assay of an analyte, wherein said analyte is a member of a specific binding pair comprising said analyte and a binding protein, wherein said liquid single reagent comprises (1) a β-galactosidase enzyme donor-analyte conjugate, referred to herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA, (2) said binding protein, and (3) an amount of a surfactant sufficient to inhibit binding between said analyte and said binding protein, said method comprising: combining said liquid single reagent with an amount of a cyclodextrin sufficient to overcome inhibition of binding of said binding protein to said analyte, thereby allowing formation of said complex.

Claim 2

A method for using a preformed liquid single reagent in an assay of an analyte, wherein said analyte is a member of a specific binding pair comprising said analyte and a binding protein, wherein said liquid single reagent comprises (1) a β-galactosidase enzyme donor-analyte conjugate, referred to herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA, (2) said binding protein, and (3) an amount of a surfactant sufficient to inhibit binding between said analyte and said binding protein, said method comprising: combining said liquid single reagent with an amount of a cyclodextrin sufficient to overcome inhibition of binding of said binding protein to said analyte, thereby allowing formation of said complex.

Claim 4

A method for determining the amount of analyte present in a sample, wherein said analyte is a member of a specific binding pair comprising said analyte and a binding protein, said method comprising: (a) combining to form an assay medium (1) a β-galactosidase enzyme donor/analyte conjugate, referred to herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EDA, (2) said binding protein, (3) said β-galactosidase enzyme acceptor, (4) enzyme substrate reactive with β-galactosidase, (5) an amount of surfactant sufficient to inhibit formation of a complex between said analyte and said binding protein, (6) said sample, and (7) a cyclodextrin which is added in an amount sufficient to reverse inhibition of binding between said analyte and said binding protein, with the provisos that (1) EA and Ed-analyte conjugate are combined in said assay medium in the presence of said surfactant and (2) cyclodextrin is not added to said assay medium prior to addition of said sample to said assay medium; and (b) determining rate of β-galactosidase activity on said substrate as indicative of the amount of analyte present in said sample.

Claim 5

A method for determining the amount of analyte present in a sample, wherein said analyte is a member of a specific binding pair comprising said analyte and a binding protein, said method comprising: (a) combining to form an assay medium (1) a β-galactosidase enzyme donor/analyte conjugate, referred to herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EDA, (2) said binding protein, (3) said β-galactosidase enzyme acceptor, (4) enzyme substrate reactive with β-galactosidase, (5) an amount of surfactant sufficient to inhibit formation of a complex between said analyte and said binding protein, (6) said sample, and (7) a cyclodextrin which is added in an amount sufficient to reverse inhibition of binding between said analyte and said binding protein, with the provisos that (1) EA and Ed-analyte conjugate are combined in said assay medium in the presence of said surfactant and (2) cyclodextrin is not added to said assay medium prior to addition of said sample to said assay medium; and (b) determining rate of β-galactosidase activity on said substrate as indicative of the amount of analyte present in said sample.

Claim 13

A method for determining the amount of analyte present in a serum sample, comprising: (a) combining the following reagents in the order given: (i) a liquid single reagent comprising a β-galactosidase enzyme donor/analyte conjugate, referred to herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA; an analyte receptor; and a surfactant in an amount sufficient o inhibit binding between said analyte and said analyte receptor; (ii) said β-galactosidase enzyme acceptor; (iii) a β-galactosidase enzyme substrate; (iv) said sample; and (v) a sufficient amount of a cyclodextrin to reverse inhibition of binding between said analyte receptor and said analyte; and (b) determining a change in light absorption or emission by a product of β-galactosidase-catalyzed reaction of said substrate as indicative of the amount of analyte present in said sample.

Claim 14

A method for determining the amount of analyte present in a serum sample, comprising: (a) combining the following reagents in the order given: (i) a liquid single reagent comprising a β-galactosidase enzyme donor/analyte conjugate, referred to herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA; an analyte receptor; and a surfactant in an amount sufficient o inhibit binding between said analyte and said analyte receptor; (ii) said β-galactosidase enzyme acceptor; (iii) a β-galactosidase enzyme substrate; (iv) said sample; and (v) a sufficient amount of a cyclodextrin to reverse inhibition of binding between said analyte receptor and said analyte; and (b) determining a change in light absorption or emission by a product of β-galactosidase-catalyzed reaction of said substrate as indicative of the amount of analyte present in said sample.

Claim 17

A method for determining the amount of analyte present in a serum sample, comprising: (a) combining the following substances in the order given: (i) a liquid single reagent comprising a β-galactosidase enzyme donor/analyte conjugate, referred herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA; an analyte receptor; said β-galactosidase enzyme acceptor; a β-galactosidase enzyme substrate; and a surfactant in an amount sufficient to inhibit binding between said analyte and said analyte receptor; (ii) said sample; and (iii) a sufficient amount of a cyclodextrin to reverse inhibition of binding between said analyte receptor and said analyte; and (b) determining change in light absorption or emission by a product of a β-galactosidase-catalyzed reaction of said substrate as indicative of the amount of analyte present in said sample.

Claim 18

A method for determining the amount of analyte present in a serum sample, comprising: (a) combining the following substances in the order given: (i) a liquid single reagent comprising a β-galactosidase enzyme donor/analyte conjugate, referred herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA; an analyte receptor; said β-galactosidase enzyme acceptor; a β-galactosidase enzyme substrate; and a surfactant in an amount sufficient to inhibit binding between said analyte and said analyte receptor; (ii) said sample; and (iii) a sufficient amount of a cyclodextrin to reverse inhibition of binding between said analyte receptor and said analyte; and (b) determining change in light absorption or emission by a product of a β-galactosidase-catalyzed reaction of said substrate as indicative of the amount of analyte present in said sample.

Claim 21

A method for determining the amount of thyroxine, referred to herein as T4, present in a serum sample comprising in the following order: (a) incubating a solution comprising from about 10 mM to about 25 mM of a β-galactosidase enzyme donor-T4 conjugate, referred to herein as ED-T4 conjugate, wherein said ED-T4 conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA, from about 1.0 μM to 2.0 μM EA, from about 0.2% to bout 0.5% (w/w) of a polyoxylene p-t-octyl phenol, anti-T4 antibody, and from about 1 mg/ml to about 2 mg/ml O-nitrophenyl-β-galactoside with said sample for about 10 to 30 min. at 37° C. to form an assay medium; (b) adding a cyclodextrin solution with said assay medium to a final concentration of from about 0.5% to 1.2% (w/w); (c) incubating said assay medium for about 1 min. to about 3 min. at about 37° C.; (d) determining the optical density of said assay medium; (e) incubating said assay medium for about 3 min. to about 6 min., at about 37° C.; (f) determining the optical density of said assay medium; (g) comparing the difference in the optical density of steps (d) and (f) to the difference in optical density of a sample having a known concentration of T4.

Claim 22

A method for determining the amount of thyroxine, referred to herein as T4, present in a serum sample comprising in the following order: (a) incubating a solution comprising from about 10 mM to about 25 mM of a β-galactosidase enzyme donor-T4 conjugate, referred to herein as ED-T4 conjugate, wherein said ED-T4 conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA, from about 1.0 μM to 2.0 μM EA, from about 0.2% to bout 0.5% (w/w) of a polyoxylene p-t-octyl phenol, anti-T4 antibody, and from about 1 mg/ml to about 2 mg/ml O-nitrophenyl-β-galactoside with said sample for about 10 to 30 min. at 37° C. to form an assay medium; (b) adding a cyclodextrin solution with said assay medium to a final concentration of from about 0.5% to 1.2% (w/w); (c) incubating said assay medium for about 1 min. to about 3 min. at about 37° C.; (d) determining the optical density of said assay medium; (e) incubating said assay medium for about 3 min. to about 6 min., at about 37° C.; (f) determining the optical density of said assay medium; (g) comparing the difference in the optical density of steps (d) and (f) to the difference in optical density of a sample having a known concentration of T4.

Claim 23

A method for determining the amount of an analyte present in a serum sample comprising in the following order: (a) incubating a solution comprising from about 5 nM to about 50 nM of a β-galactosidase enzyme donor-analyte conjugate, referred to herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA, from about 1 μM to about 2 μM EA, from about 0.2% to bout 1.0% (w/w) of a polyoxylene-p-t-octyl phenol, analyte receptor and from about 0.5 mg/ml to about 1.5 mg/ml O-nitrophenyl-β-galactoside with said sample and a cyclodextrin in a concentration of from about 0.5% to about 2% (w/w) to form an assay medium; (b) incubating said assay medium for about 1 min. to about 4 min. at about 37° C; (c) determining the optical density of said assay medium; (d) incubating said assay medium for about 3 min. to about 8 min. at about 37° C.; (e) determining the optical density of said assay medium; (f) comparing the difference in the optical density of steps (c) and (e) to the difference in optical density of a sample having a known concentration of analyte.

Claim 24

A method for determining the amount of an analyte present in a serum sample comprising in the following order: (a) incubating a solution comprising from about 5 nM to about 50 nM of a β-galactosidase enzyme donor-analyte conjugate, referred to herein as ED-analyte conjugate, wherein said ED-analyte conjugate is capable of forming an active enzyme having β-galactosidase activity when combined with a β-galactosidase enzyme acceptor, referred to herein as EA, from about 1 μM to about 2 μM EA, from about 0.2% to bout 1.0% (w/w) of a polyoxylene-p-t-octyl phenol, analyte receptor and from about 0.5 mg/ml to about 1.5 mg/ml O-nitrophenyl-β-galactoside with said sample and a cyclodextrin in a concentration of from about 0.5% to about 2% (w/w) to form an assay medium; (b) incubating said assay medium for about 1 min. to about 4 min. at about 37° C; (c) determining the optical density of said assay medium; (d) incubating said assay medium for about 3 min. to about 8 min. at about 37° C.; (e) determining the optical density of said assay medium; (f) comparing the difference in the optical density of steps (c) and (e) to the difference in optical density of a sample having a known concentration of analyte.