Simultaneous calibration heterogeneous immunoassay

• US 4,849,338 A
• Filed: 05/21/1985
• Issued: 07/18/1989
• Est. Priority Date: 07/16/1982
• Status: Expired due to Term

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Syntec Incorporated (Atos SE)
• Inventors: Ullman, Edwin F., Litman, David J.
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/736,485
• Time in Patent Office: 1,519 Days
• Field of Search: 435/4, 435/7, 435/188, 435/805, 435/810, 436/524, 436/525, 436/527-529, 436/531, 436/546, 436/808, 436/810, 436/824, 436/501, 436/518, 422/55, 422/56, 422/61
• US Class Current: 435/7.91
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

An assay method and compositions are provided for determining the presence of an analyte in a sample. The analyte is a member of an immunological pair (mip) comprising ligand and receptor. By providing a first measurement surface capable of specifically binding a labelled reagent in an amount depending upon the presence of analyte in the sample and a second calibration surface capable of binding a second labeled reagent in a manner unaffected by the presence of analyte in the sample, calibration of individual tests can be accomplished simultaneously with the performance of the test itself. A signal producing system includes an enzyme, a catalyst usually bonded to a mip which defines the first labeled reagent for binding to the measurement surface and the same catalyst conjugated to a ligand capable of binding to the calibration surface. Preferably, both labeled reagents have the same composition and the calibration surface include anti-(first catalyst).

Claims

1. In a method for determining the presence in a sample of an analyte, which analyte is a member of a specific binding pair consisting of ligand and receptor, ("antiligand"),

said method employing a labeled mip, a signal producing system and a measurement first surface, where the amount of labeled mip which binds to said first surface as a result of mip complex formation is related to the amount of analyte in said assay medium,

said method comprising the steps of contacting said measurement surface with said sample in a aqueous assay medium and simultaneously or successively contacting said measurement surface with members of said signal producing system including at least said labeled mip which provides an amount of signal generating compound at said first surface related to the amount of analyte in said assay medium,

the improvement which comprises:

having in contact with said assay medium a calibration second surface to which said labeled mip binds as the result of interaction with a specific binding pair member wherein said specific binding pair member binds to a determinant site on said label or a determinant site on said labeled mip other than the determinant site involved in the binding of the analyte to the specific binding pair member homologous to the analyte, whereby the intensity of the signal at said second surface compared to the intensity of signal at said first surface defines the amount of analyte in said sample substantially independent of non-specific factors.

2. A method according to claim 1, wherein said labelled mip has a hub nucleus to which at least one mip is bound in a plurality of chemiluminescer or fluorescer molecules are bound.

3. A method according to claim 2, wherein a plurality of fluorescer molecules are bound to said hub nucleus.

4. In a method for determining the presence in a sample of an analyte which analyte is a member of a specific binding pair ("mip") consisting of ligand and receptor, ("antiligand")

said method employing at least one catalyst including a catalyst bound to a mip ("catalyst-bound-mip") and a solute which is catalytically transformed by a catalyst bound to a mip-containing measurement first surface to produce a change in a detectable signal at said first surface in proportion to the amount of catalyst-bound-mip bound to said first surface, wherein contacting of said first surface with said sample and said catalyst-bound-mip results in binding of said catalyst-bound-mip to said first surface in proportion to the amount of analyte in said sample,

the improvement which comprises:

having in said assay medium a calibration second surface to which said catalyst is directly bound in an amount to produce a level of a signal at said second surface corresponding to a predetermined amount of analyte and said analyte in said sample is determined substantially independent of non-specific factors by comparing the intensity of the signal at said first surface to the intensity of the signal at said second surface.

5. A method according to claim 4, wherein said catalyst is an enzyme.

7. A method according to any of claims 4, 5, or 6, wherein said signal producing system includes two enzymes as catalysts, wherein the product of one enzyme is the substrate of the other enzyme, and one of the enzymes is bound to each of said first and second surfaces prior to said contacting.

8. A method according to claim 7, wherein said solute is a dye precursor which undergoes an enzyme catalyzed reaction to produce an insoluble dye which binds to said surface.

9. A method according to claim 8, wherein said catalyst-bound-mip is enzyme-bound-antiligand.

10. A method according to claim 8, wherein said catalyst-bound-mip is enzyme-bound-ligand.

6. In a method for determining the presence in a sample of an analyte, which analyte is a member of a specific binding pair consisting of ligand and receptor,

said method employing at least one catalyst including a catalyst bound to a mip and a solute which is catalytically transformed by a catalyst bound to a mip-containing measurement first surface to produce a change in a detectable signal at said first surface in proportion to the amount of catalyst-bound-mip bound to said first surface, wherein contacting of said first surface with said sample and said catalyst-bound-mip results in binding of said catalysts-bound-mip to said first surface in proportion to the amount of analyte in said sample,

the improvement which comprises:

having in said assay medium a calibration second surface to which said catalyst binds in a predetermined amount by means of mip complex formation involving predetermined amount by means of mip complex formation involving a specific binding pair member, wherein said specific binding pair member binds to a determinant site on said catalyst or to a determinant site on a catalyst-bound mip other than the determinant site involved in the binding of the analyte to specific binding pair member homologous to the analyte, whereby the intensity of the signal at said second surface to the intensity of signal at said first surface defines the amount of analyte in said sample independent of non-specific factors.

11. In method for determining the presence in a sample of an analyte, which analyte is a member of a specific binding pair consisting of ligand and receptor, ("antiligand"),

said method employing a signal producing system having at least two enzymes, including one enzyme bound to a mip ("enzyme-bound-mip"), and a solute dye precursor which is catalytically transformed to an insoluble dye by one of said enzymes bound to a mip-containing measurement first surface, said insoluble dye producing a change in a detectable signal at said first surface in proportion to the amount of analyte, where said mip at said first surface provides for binding of enzyme-bound-mip through mip complex formation to said first surface, wherein contacting of said first surface with said sample and said enzyme-bound-mip results in binding of said enzyme-bound-mip to said first surface in proportion to the amount of analyte in said sample,

the improvement which comprises:

during said contacting, having adjacent to said first surface, a calibration second surface to which enzyme of said enzyme-bound-mip becomes bound through mip complex formation involving a specific binding pair wherein the specific binding pair member binds to a determinant site on said enzyme or a determinant site on said enzyme-bound-mip other than the determinant site involved in the binding of said enzyme-bound-mip to the specific binding pair member homologous to the analyte in a predetermined amount, whereby the intensity of the signal at said second surface compared to the intensity of signal at said first surface defines the amount of analyte in said sample substantially independent of non-specific factors.

12. A method according to claim 11, wherein anti-enzyme receptor is bound to said second surface in a predetermined amount.

13. A method according to claim 11, wherein a second ligand other than the ligand analyte is bonded to said enzyme of said enzyme-bound-mip and anti receptor is bound to said second surface.

14. A method according to claim 13, wherein said second ligand is conjugated to an enzyme molecule other that enzyme-bound-mip.

15. A method according to claim 13, wherein said second ligand is conjugated to the same enzyme molecule as said enzyme-bound-mip.

16. An internally calibrated diagnostic apparatus comprising a support which contains a measurement first surface of a porous material and a calibration second surface of a porous material in close proximity to said first surface;

a member of a specific binding pair non-diffusively bound to said first surface; and

a member of a specific binding pair non-diffusively bound to said second surface wherein said member of said specific binding pair of said second surface binds to a determinant site on a member of a signal producing system or a determinant site on a conjugate of a member of a signal producing system and a member of a specific binding pair other than the determinant site involved in the binding of said analyte to the member of a specific binding pair of said first surface.

17. A device according to claim 16, wherein the member of the signal producing system is an enzyme.

18. A kit for use in a diagnostic assay comprising a device according to claim 17 and a conjugate of an enzyme and a member of a specific of a specific binding pair.