Visual discrimination qualitative enzyme complementation assay

Claim 1

A method for determining the presence of an analyte in a sample wherein said analyte is a member of a specific binding pair, said method comprising: (a) combining said sample with a solution comprising a hydrophobic β-galactosidase enzyme donor- (ED-) analyte conjugate and the complementary specific binding pair member for a time period sufficient for analyte conjugate and any analyte to react with said complementary specific binding pair member to form a specific binding pair complex-containing assay medium; (b) applying a volume of said assay medium sufficient for wicking from an applicator to a predetermined application site area of a bibulous solid support comprising a β-galactosidase enzyme acceptor (EA) affixed to said solid support and capable of forming with said (ED)-analyte conjugate an active enzyme complex having β-galactosidase activity; wherein said conjugate and said support are sufficiently hydrophobic so that said conjugate will remain substantially in the area applied; (c) incubating said solid support for a time sufficient to form solid support-affixed β-galactosidase; and (d) incubating said solid support with an enzyme substrate which forms a visually detectable product upon reaction with β-galactosidase; whereby a substantially larger product area extending from said application site area is detectable when the concentration of said analyte is below a threshold concentration as compared to a detectable product area extending from said application site area when said analyte is above said threshold concentration.

Claim 2

A method for determining the presence of an analyte in a sample wherein said analyte is a member of a specific binding pair, said method comprising: (a) combining said sample with a solution comprising a hydrophobic β-galactosidase enzyme donor- (ED-) analyte conjugate and the complementary specific binding pair member for a time period sufficient for analyte conjugate and any analyte to react with said complementary specific binding pair member to form a specific binding pair complex-containing assay medium; (b) applying a volume of said assay medium sufficient for wicking from an applicator to a predetermined application site area of a bibulous solid support comprising a β-galactosidase enzyme acceptor (EA) affixed to said solid support and capable of forming with said (ED)-analyte conjugate an active enzyme complex having β-galactosidase activity; wherein said conjugate and said support are sufficiently hydrophobic so that said conjugate will remain substantially in the area applied; (c) incubating said solid support for a time sufficient to form solid support-affixed β-galactosidase; and (d) incubating said solid support with an enzyme substrate which forms a visually detectable product upon reaction with β-galactosidase; whereby a substantially larger product area extending from said application site area is detectable when the concentration of said analyte is below a threshold concentration as compared to a detectable product area extending from said application site area when said analyte is above said threshold concentration.

Claim 8

A method for determining the presence of an analyte in a sample wherein said analyte is a member of a specific binding pair, said method comprising: (a) combining said sample with a solution comprising a hydrophobic β-galactosidase enzyme donor- (ED-) analyte conjugate and the complementary specific binding pair member for a time period sufficient for analyte conjugate and any analyte to react with said complementary specific binding pair member to form a specific binding pair complex-containing assay medium; (b) applying a volume of said assay medium sufficient for wicking from an applicator to a predetermined application site area of a bibulous solid support comprising a β-galactosidase enzyme acceptor (EA) affixed to said solid support and capable of forming with said (ED)-analyte conjugate an active enzyme complex having β-galactosidase activity; wherein said conjugate and said support are sufficiently hydrophobic for said conjugate to remain in substantially the area applied; (c) incubating said solid support for a time period sufficient to form solid support-affixed β-galactosidase; and (d) incubating said solid support with an enzyme substrate which forms a visually detectable product upon reaction with β-galactosidase whereby a substantially larger product area extending from said application site area is detectable when the concentration of said analyte is below a threshold concentration as compared to a detectable product area extending from said application site area when said analyte is above said threshold concentration.

Claim 9

A method for determining the presence of an analyte in a sample wherein said analyte is a member of a specific binding pair, said method comprising: (a) combining said sample with a solution comprising a hydrophobic β-galactosidase enzyme donor- (ED-) analyte conjugate and the complementary specific binding pair member for a time period sufficient for analyte conjugate and any analyte to react with said complementary specific binding pair member to form a specific binding pair complex-containing assay medium; (b) applying a volume of said assay medium sufficient for wicking from an applicator to a predetermined application site area of a bibulous solid support comprising a β-galactosidase enzyme acceptor (EA) affixed to said solid support and capable of forming with said (ED)-analyte conjugate an active enzyme complex having β-galactosidase activity; wherein said conjugate and said support are sufficiently hydrophobic for said conjugate to remain in substantially the area applied; (c) incubating said solid support for a time period sufficient to form solid support-affixed β-galactosidase; and (d) incubating said solid support with an enzyme substrate which forms a visually detectable product upon reaction with β-galactosidase whereby a substantially larger product area extending from said application site area is detectable when the concentration of said analyte is below a threshold concentration as compared to a detectable product area extending from said application site area when said analyte is above said threshold concentration.

Claim 18

A method for determining the presence of a drug in a serum or urine sample comprising: (a) combining said sample with a solution comprising a hydrophobic β-galactosidase ED-drug-conjugate and a receptor specific for said drug for a time sufficient for ED-drug-conjugate and any drug to react with said receptor to form an ED-drug-conjugate- and drug-receptor complex-containing assay medium; (b) applying a volume of said complex-containing assay medium to a predetermined application site area of a noncellulosic membrane comprising a membrane affixed β-galactosidase enzyme acceptor (EA); wherein said conjugate and said support are sufficiently hydrophobic for said conjugate to remain in substantially the area applied; (c) incubating for a period of time sufficient for EA to react with ED to form membrane-affixed β-galactosidase; and (d) combining said membrane affixed β-galactosidase with an enzyme substrate solution which forms a detectable colored product upon reaction with β-galactosidase; whereby a substantially larger colored product area extending from said application site area is detectable when the concentration of said drug is below a threshold concentration as compared to a detectable colored product area extending from said application site area when said drug is above said threshold concentration.

Claim 19

A method for determining the presence of a drug in a serum or urine sample comprising: (a) combining said sample with a solution comprising a hydrophobic β-galactosidase ED-drug-conjugate and a receptor specific for said drug for a time sufficient for ED-drug-conjugate and any drug to react with said receptor to form an ED-drug-conjugate- and drug-receptor complex-containing assay medium; (b) applying a volume of said complex-containing assay medium to a predetermined application site area of a noncellulosic membrane comprising a membrane affixed β-galactosidase enzyme acceptor (EA); wherein said conjugate and said support are sufficiently hydrophobic for said conjugate to remain in substantially the area applied; (c) incubating for a period of time sufficient for EA to react with ED to form membrane-affixed β-galactosidase; and (d) combining said membrane affixed β-galactosidase with an enzyme substrate solution which forms a detectable colored product upon reaction with β-galactosidase; whereby a substantially larger colored product area extending from said application site area is detectable when the concentration of said drug is below a threshold concentration as compared to a detectable colored product area extending from said application site area when said drug is above said threshold concentration.

Claim 20

A method for determining the presence of T3 in a serum sample comprising: (a) incubating said sample with a solution comprising from about 1 to about 10 nM β-galactosidase ED-T3 conjugate and anti-T3 antibody for about 30 to about 60 min. at about 37° C. to form an immune complex-containing assay medium; (b) applying a small volume of said assay medium to β-galactosidase EA affixed at a uniform concentration to an application site area of a nylon membrane; (c) incubating for about 10 to about 30 min. at about 37° C. to form membrane-affixed β-galactosidase; (d) combining said membrane-affixed β-galactosidase with 0.5 to 2 mM CPRG solution to form a detectable colored reaction product whereby a substantially larger product area extending from said application site area is detectable when the concentration of T3 is below a threshold concentration as compared to a detectable product area extending from said application site area when T3 is above said threshold concentration.

Claim 21

A method for determining the presence of T3 in a serum sample comprising: (a) incubating said sample with a solution comprising from about 1 to about 10 nM β-galactosidase ED-T3 conjugate and anti-T3 antibody for about 30 to about 60 min. at about 37° C. to form an immune complex-containing assay medium; (b) applying a small volume of said assay medium to β-galactosidase EA affixed at a uniform concentration to an application site area of a nylon membrane; (c) incubating for about 10 to about 30 min. at about 37° C. to form membrane-affixed β-galactosidase; (d) combining said membrane-affixed β-galactosidase with 0.5 to 2 mM CPRG solution to form a detectable colored reaction product whereby a substantially larger product area extending from said application site area is detectable when the concentration of T3 is below a threshold concentration as compared to a detectable product area extending from said application site area when T3 is above said threshold concentration.