Concentrating zone method in heterogeneous immunoassays

Claim 2

An immunoassay method for determining an analyte which is a member of an immunological pair, defined as a mip, consisting of ligand and its homologous antiligand ; said method employing ih combination an assay device and a signal producing system; said assay device characterized by an immunosorbing zone comprising mip non-diffusively bound to at least a portion of a bibulous support serving as an inlet port for liquids into said device; and a liquid absorbing zone in liquid receiving relationship with said immunosorbing zone; and a signal producing system characterized by being capable of producing a detectible signal in said. immunosorbing zone and having one component conjugated to a mip to provide a signal label-mip conjugate, wherein the amount of signal label producing said detectible signal in said immunosorbing zone is related to the amount of analyte; said method comprising: contacting said assay device in a predetermined order with: a) a solution of a sample suspected of containing said analyte; and b) a solution of components of said signal producing system other than components bound to said assay device; whereby said solutions migrate through said immunosorbing zone into said liquid absorbing zone resulting in an amount of signal label-mip conjugate bound to said mip bound to said support in relation to the amount of analyte in said sample; and production of a detectable signal in said immunosorbing zone; and determining said detectable signal.

Claim 3

A method according to Claim 1, wherein said signal label-mip conjugate is contacted with said assay device in a solution not earlier than concurrently with said sample solution. -

Claim 4

3 . An immunoassay method for determining an analyte which is a member of an immunological pair, defined as a mip, consisting of ligand and its homologous antiligand, said method employing in combination an assay device and a signal producing system; said assay device characterized by an immunosorbing zone comprising mip non-diffusively bound to at least a portion of a bibulous support serving as an inlet port for liquids into said device; and a liquid absorbing zone in liquid receiving relationship with said immunosorbing zone and at least partially enclosed to prohibit contact with liquids except through said immunosorbing zone; said signal producing system characterized by being capable of producing a detectible signal in said immunosorbing zone and having (1) an enzyme as a signal label conjugated to a mip to provide an enzyme-mip conjugate, wherein the amount of enzyme producing said detectible signal in said immunosorbing zone is related to the amount of analyte, and (2) a signal generator precursor which is enzymatically transformed to a signal generator; said method comprising: contacting said assay device in a predetermined order with: a) a solution of a sample suspected of containing said analyte; b) a solution of enzyme-mip conjugate, except when said enzyme-mip conjugate is previously bound to said assay device through complexation with said mip bound to said support; and c) a solution of a signal generator precursor, whereby said solutions migrate through said immunosorbing zone into said liquid absorbing zone and said signal generator precursor is converted by said enzyme to a signal generator producing a detectible signal in said immunosorbing zone; and determining said detectable signal. ;

Claim 5

3 . An immunoassay method for determining an analyte which is a member of an immunological pair, defined as a mip, consisting of ligand and its homologous antiligand, said method employing in combination an assay device and a signal producing system; said assay device characterized by an immunosorbing zone comprising mip non-diffusively bound to at least a portion of a bibulous support serving as an inlet port for liquids into said device; and a liquid absorbing zone in liquid receiving relationship with said immunosorbing zone and at least partially enclosed to prohibit contact with liquids except through said immunosorbing zone; said signal producing system characterized by being capable of producing a detectible signal in said immunosorbing zone and having (1) an enzyme as a signal label conjugated to a mip to provide an enzyme-mip conjugate, wherein the amount of enzyme producing said detectible signal in said immunosorbing zone is related to the amount of analyte, and (2) a signal generator precursor which is enzymatically transformed to a signal generator; said method comprising: contacting said assay device in a predetermined order with: a) a solution of a sample suspected of containing said analyte; b) a solution of enzyme-mip conjugate, except when said enzyme-mip conjugate is previously bound to said assay device through complexation with said mip bound to said support; and c) a solution of a signal generator precursor, whereby said solutions migrate through said immunosorbing zone into said liquid absorbing zone and said signal generator precursor is converted by said enzyme to a signal generator producing a detectible signal in said immunosorbing zone; and determining said detectable signal. ;

Claim 6

A method according to Claim 3 , wherein a second enzyme is non-diffusively bound in said immunosorbing zone, wherein the product from one enzyme is a substrate for the other enzyme.

Claim 7

A method according to Claim 4 , wherein said second enzyme produces a product which is a substrate of said enzyme-mip conjugate.

Claim 8

6 . An immunoassay method for determining an analyte which is a member of an immunological pair, defined as a mip, consisting of ligand and its homologous antiligand, said method employing in combination an assay device and a signal producing system; said assay device characterized by an immunosorbing zone comprising mip non-diffusively bound to a bibulous support serving as a inlet port for liquids into said device; and a liquid absorbing zone in liquid receiving relationship with said immunosorbing zone and at least partially enclosed to prohibit contact with liquids except through said immunosorbing zone; said signal producing system characterized by being capable of producing a detectible signal in said immunosorbing zone and having a fluorescer as signal label conjugated to a mip to provide a fluorescer-mip conjugate, wherein the amount of fluorescer producing said detectible signal in said immunosorbing zone is related to the amount of analyte; said method comprising: contacting said assay device in a predetermined order with: a) a solution of a sample suspected of containing said analyte; b) a solution of fluorescer-mip conjugate, except when said fluorescer-mip conjugate is bound to said assay device prior to combining with said sample solution; and c) a wash solution, except when said fluorescer-mip conjugate is bound to said assay device prior to combining with said sample solution, whereby said solutions migrate through said immunosorbing zone into said liquid absorbing zone and said fluorescer-mip conjugate is bound in said immunosorbing zone in relation to the amount of analyte in said sample; and determining the fluorescence from said immunosorbing zone.

Claim 9

6 . An immunoassay method for determining an analyte which is a member of an immunological pair, defined as a mip, consisting of ligand and its homologous antiligand, said method employing in combination an assay device and a signal producing system; said assay device characterized by an immunosorbing zone comprising mip non-diffusively bound to a bibulous support serving as a inlet port for liquids into said device; and a liquid absorbing zone in liquid receiving relationship with said immunosorbing zone and at least partially enclosed to prohibit contact with liquids except through said immunosorbing zone; said signal producing system characterized by being capable of producing a detectible signal in said immunosorbing zone and having a fluorescer as signal label conjugated to a mip to provide a fluorescer-mip conjugate, wherein the amount of fluorescer producing said detectible signal in said immunosorbing zone is related to the amount of analyte; said method comprising: contacting said assay device in a predetermined order with: a) a solution of a sample suspected of containing said analyte; b) a solution of fluorescer-mip conjugate, except when said fluorescer-mip conjugate is bound to said assay device prior to combining with said sample solution; and c) a wash solution, except when said fluorescer-mip conjugate is bound to said assay device prior to combining with said sample solution, whereby said solutions migrate through said immunosorbing zone into said liquid absorbing zone and said fluorescer-mip conjugate is bound in said immunosorbing zone in relation to the amount of analyte in said sample; and determining the fluorescence from said immunosorbing zone.

Claim 10

7 .. An assay device for performing immunoassays which comprises: an immunosorbing member comprising a member of an immunological pair, defined as a mip, non-diffusively bound to at least a portion of a bibulous support; and a liquid absorbing member comprising a bibulous material in liquid receiving relationship with said immunosorbing member and extending transversely thereform, wherein at least the portion of said liquid absorbing member about said immunosorbing member is enclosed in an impermeable enclosure to inhibit contact with solutions except through said immunosorbing member and said immunosorbing member has a relatively small liquid holding capacity as compared to said liquid absorbing member.

Claim 11

7 .. An assay device for performing immunoassays which comprises: an immunosorbing member comprising a member of an immunological pair, defined as a mip, non-diffusively bound to at least a portion of a bibulous support; and a liquid absorbing member comprising a bibulous material in liquid receiving relationship with said immunosorbing member and extending transversely thereform, wherein at least the portion of said liquid absorbing member about said immunosorbing member is enclosed in an impermeable enclosure to inhibit contact with solutions except through said immunosorbing member and said immunosorbing member has a relatively small liquid holding capacity as compared to said liquid absorbing member.

Claim 12

8 . An assay device according to Claim 7 , wherein said immunosorbing member and said liquid absorbing member are a single strip, said immunosorbing member being at the end of said single strip.

Claim 13

9 . An assay device according to Claim 7 , wherein said immunosorbing member is a layer above a portion of said liquid absorbing member.

Claim 14

10 . An assay device for performing immunoassay comprising: (1) an immunosorbing member; (2) a liquid absorbing member; 4 (3) a non-permeable barrier; said immunosorbing member serving as a liquid entry port to said device and comprised of at least two zones; a first bibulous layer to which a member of an immunological pair, defined as mip, is non-diffusively bound, and a second zone between said bibulous member and said liquid absorbing member of different flow resistant characteristic from said bibulous layer; said liquid absorbing member comprised of a bibulous layer in liquid receiving relationship with said immunosorbing zone and extending transversely therefrom, wherein said immunosorbing member has relatively small liquid holding capacity as compared to said liquid absorbing member; and said non-permeable barrier comprising a non-permeable layer about said liquid absorbing and immunosorbing members to inhibit entry of liquid to said device except through said immunosorbing member.

Claim 15

10 . An assay device for performing immunoassay comprising: (1) an immunosorbing member; (2) a liquid absorbing member; 4 (3) a non-permeable barrier; said immunosorbing member serving as a liquid entry port to said device and comprised of at least two zones; a first bibulous layer to which a member of an immunological pair, defined as mip, is non-diffusively bound, and a second zone between said bibulous member and said liquid absorbing member of different flow resistant characteristic from said bibulous layer; said liquid absorbing member comprised of a bibulous layer in liquid receiving relationship with said immunosorbing zone and extending transversely therefrom, wherein said immunosorbing member has relatively small liquid holding capacity as compared to said liquid absorbing member; and said non-permeable barrier comprising a non-permeable layer about said liquid absorbing and immunosorbing members to inhibit entry of liquid to said device except through said immunosorbing member.

Claim 16

An immunoassay kit comprising an assay device according to Claims 7 , 8, 9 or 10 in combination with a labeled mip separate from said assay device.