Method of assay having calibration within the assay

• US 5,726,064 A
• Filed: 09/21/1994
• Issued: 03/10/1998
• Est. Priority Date: 11/22/1990
• Status: Expired due to Fees

• Current Assignee: Laboratoires Serono SA
• Original Assignee: Applied Research Systems ARS Holding NV (Merck & Co., Inc.)
• Inventors: Robinson, Grenville Arthur, Attridge, John Worthington, Daniels, Phelim Brinley, Thomson, Aileen Margaret, Love, Colin Andrew, Deacon, Julie Karen
• Primary Examiner(s): Spiegel, Carol A.
• Application Number:
  US08/309,796
• Time in Patent Office: 1,266 Days
• Field of Search: 436/514, 436/518, 436/527, 436/529, 436/808, 422/56, 422/57, 422/82.07, 422/82.08, 422/82.11, 435/7.1, 435/967, 435/968, 435/975, 435/7.92-7.95
• US Class Current: 436/514
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

A method of assay for a ligand in a sample is described in which calibration occurs within the assay. This is achieved utilizing a measurement region and one ore more calibration regions. In at least one of the calibration regions a non-zero signal results, either because of the presence of a calibration reagent or as a result of a binding reaction analogous to that which takes place in the measurement region.

Claims

1. A method of manufacturing a plurality of capillary-fill devices, each device for assay of at least one ligand in a liquid sample comprising the steps of

(a) forming an array of patches for each ligand comprising at least two mutually separated zones I and II wherein each zone comprises a releasable layer of a first specific binding reagent selected from the group consisting of the ligand, an analogue of the ligand, and a binding partner capable of specifically binding with the ligand and the analogue, if present, disposed on a surface of a sheet material which is to provide a part of each of said plurality of the devices such that each first specific binding reagent does not mix with the other upon release during performance of the assay,

(b) forming a second array of patches comprising at least two zones IV and V corresponding in orientation to the aforementioned zones I and II respectively, each of zones IV and V comprising a corresponding immobilized layer of a second specific binding reagent selected from the group consisting of said first specific binding reagent and an auxiliary specific binding reagent capable of specifically binding with said first specific binding reagent on a surface of an additional structure, said additional structure together with the sheet material providing for each of the plurality of the devices a cavity of capillary dimensions for collecting and retaining a volume of the liquid sample in contact with the layers of the releasable reagents,

(c) combining the sheet material and the additional structure to form said plurality of the devices, and separating the plurality of devices into portions each portion providing said each device.

2. A competitive specific binding method for determining a ligand in a liquid sample, comprising:

(A) contacting the liquid sample to a capillary-fill device, said device comprising a measurement zone and at least one spatially distinct calibration zone wherein

(1) the measurement zone comprises (a) a known amount of a releasable first ancillary specific binding reagent and (b) a measurement surface comprising an immobilized measurement specific binding reagent capable of specifically binding with at least one of the ligand and the first ancillary specific binding reagent, wherein the first ancillary specific binding reagent is provided either on a surface of the measurement zone separate from the measurement surface or prebound to the immobilized measurement specific binding reagent, and is either (i) labelled ligand analogue or (ii) labelled specific binding partner for the ligand or (iii) a combination of the labelled ligand analogue and specific binding partner for the ligand,

(2) the calibration zone comprises

(c) a known amount of a releasable second ancillary specific binding reagent and a calibration surface comprising an immobilized calibration specific binding reagent having binding sites identical in structure to those of the immobilized measurement specific binding reagent and capable of specifically binding with at least one of the ligand and the second ancillary specific binding reagent or

(d) a calibration surface comprising a known amount of immobilized label, wherein the second ancillary specific binding reagent is provided either on a surface of the calibration zone separate from the calibration surface or prebound to the immobilized calibration specific binding reagent, and is (i) said labelled ligand analogue or (ii) said labelled specific binding partner for the ligand or (iii) said combination of the labelled ligand analogue and specific binding partner for the ligand, and

(3) the releasable reagents remain in their respective zones without mixing with each other in the method following either sequential or simultaneous contact with the liquid sample (a) to form a labelled measurement complex immobilized on the measurement surface in an amount dependent on the amount of the ligand present in the liquid sample and (b) to provide a calibration signal by (c) forming either a labelled calibration complex immobilized on the calibration surface in an amount either dependent on or independent of the amount of the ligand present in the liquid sample or (d) due to said known amount of immobilized label on the calibration surface; and

(B) measuring and comparing the amount of label on the measurement and calibration surfaces to determine the presence or amount of the ligand in the liquid sample.

3. The method as claimed in claim 2 wherein the label is a fluorogenic, phosphorogenic or luminogenic label.

4. The method as claimed in claim 2 wherein the method is an antigen-antibody immunoassay.

5. The method as claimed in claim 2, wherein the capillary-fill device further comprises a spatially distinct first auxiliary calibration zone comprising

(e) either a known amount of a releasable third ancillary specific binding reagent and a first auxiliary calibration surface comprising an immobilized first auxiliary calibration specific binding reagent having binding sites identical in structure to those of the immobilized measurement specific binding reagent and capable of specifically binding to at least one of the ligand and the third ancillary specific binding reagent, or

(f) a first auxiliary calibration surface comprising an immobilized first auxiliary calibration nonspecific binding reagent, wherein the third ancillary specific binding reagent is provided either on a surface of the first auxiliary calibration zone separate from the first auxiliary calibration surface or prebound to the immobilized first auxiliary calibration specific binding reagent, and is (i) said labelled ligand analogue or (ii) said combination of the labelled ligand analogue and specific binding partner for the ligand or (iii) an optionally labelled nonspecific binding ligand which binds to the immobilized first auxiliary calibration nonspecific binding reagent,

wherein step (A) further comprises (e) forming either a labelled first auxiliary calibration complex immobilized on the first auxiliary calibration surface in an amount either dependent on or independent of the amount of the ligand present in the liquid sample, or (f) forming an immobilized nonspecific binding complex in an amount dependent upon nonspecific binding background, and

wherein step (B) further comprises measuring and comparing the amount of label on the immobilized first auxiliary calibration surface.

6. A direct specific binding method for determining a ligand in a liquid sample, comprising:

(a) contacting the liquid sample to a capillary-fill device, said device comprising (1) a measurement zone, (2) at least one spatially distinct calibration zone and (3) at least one spatially distinct auxiliary calibration zone wherein

(1) the measurement zone comprises (a) a known amount of a releasable first ancillary specific binding reagent and (b) a measurement surface comprising an immobilized measurement specific binding reagent capable of specifically binding with at least one of the ligand and the first ancillary specific binding reagent, wherein the first ancillary specific binding reagent is provided either on a surface of the measurement zone separate from the measurement surface or prebound to the immobilized measurement specific binding partner reagent, and is a specific binding partner for the ligand,

(2) the calibration zone comprises

(c) a known amount of at least one releasable second ancillary specific binding reagent and a calibration surface comprising an immobilized calibration specific binding reagent having binding sites identical in structure to the those of the immobilized measurement specific binding reagent and capable of specifically binding with at least one of the ligand and the second ancillary specific binding reagent, or

(d) a calibration surface comprising a known amount of an immobilized calibration complex comprising the immobilized calibration specific binding reagent and either the ligand or the second ancillary specific binding reagent, wherein the second ancillary specific binding reagent is provided either on a surface of the calibration zone separate from the calibration surface or prebound to the immobilized calibration specific binding reagent, and is (i) said ligand analogue or (ii) said specific binding partner for the ligand, and

(3) the auxiliary calibration zone comprises

(e) either a known amount of at least one releasable third ancillary specific binding reagent and a first auxiliary calibration surface comprising an immobilized first auxiliary calibration specific binding reagent having binding sites identical in structure to those of the immobilized measurement specific binding reagent and capable of specifically binding to a least one of the ligand and the third ancillary specific binding reagent, or

(f) a first auxiliary calibration surface comprising an immobilized first auxiliary nonspecific binding reagent, wherein the third ancillary specific binding reagent is provided either on a surface of the first auxiliary calibration zone separate from the first auxiliary calibration surface or prebound to the immobilized first auxiliary calibration specific binding reagent, and is (i) said ligand analogue or (ii) said specific binding partner for the ligand or (iii) a nonspecific binding ligand which binds to the immobilized first auxiliary nonspecific binding reagent,

(4) the releasable reagents remain in their respective zones without mixing with each other in the method following either sequential or simultaneous contact with the liquid sample (a) to form an unlabelled measurement complex immobilized on the measurement surface in an amount dependent on the amount of the ligand present in the liquid sample, (b) to provide an unlabelled calibration signal by (c) forming either an unlabelled calibration complex immobilized on the calibration surface in an amount either dependent on or independent of the amount of the ligand present in the liquid sample or (d) due to said known amount of an immobilized calibration complex comprising the immobilized calibration specific binding reagent and either the ligand or the second ancillary specific binding reagent, and to provide an unlabelled auxiliary calibration signal by (d) forming either an unlabelled first auxiliary calibration complex immobilized on the first auxiliary calibration surface in an amount either dependent on or independent of the amount of the ligand present in the liquid sample or (f) forming an unlabelled nonspecific binding complex in an amount dependent upon nonspecific binding background, and

(B) measuring and comparing a change in property of the measurement, calibration and auxiliary calibration surfaces due to any change in mass on the measurement, calibration and auxiliary calibration surfaces due to the formation of said unlabelled measurement, calibration and first auxiliary calibration complexes to determine the presence or amount of the ligand in the liquid sample.

7. A sandwich specific binding method for determining a ligand in a liquid sample, comprising:

(A) contacting the liquid sample to a capillary-fill device, said device comprising a measurement zone and at least one spatially distinct calibration zone wherein;

(1) the measurement zone comprises (a) a known amount of a releasable first ancillary specific binding reagent and (b) a measurement surface comprising an immobilized measurement specific binding reagent capable of specifically binding to at least one of the ligand and the first ancillary specific binding reagent, wherein the first ancillary specific binding reagent is provided either on a surface of the measurement zone separate from the measurement surface or prebound to the immobilized measurement specific binding reagent, is labelled or unlabelled, and specifically binds the ligand at an epitope different from an epitope to which the measurement specific binding reagent or said first ancillary specific binding reagent specifically binds to, and

(2) the calibration zone comprises

(c) a known amount of a releasable second ancillary specific binding reagent and a calibration surface comprising an immobilized calibration specific binding reagent having binding sites identical in structure to those of the immobilized measurement specific binding reagent and which is capable of specifically binding with at least one of (i) an unknown amount of the ligand, (ii) a known amount of the ligand and (iii) the second ancillary specific binding reagent, or

(d) a calibration surface comprising a known amount of immobilized label, wherein the second ancillary specific binding reagent is provided either on a surface of the calibration zone separate from the calibration surface or prebound to the immobilized calibration specific binding reagent, and is (i) a labelled or unlabelled specific binding partner for the ligand or (ii) a known amount of the ligand prebound to a labelled specific binding partner for said ligand, and

(3) the releasable reagents remain in their respective zones without mixing with each other in the method following either sequential or simultaneous contact with the liquid sample (a) to form a labelled measurement complex immobilized on the measurement surface in an amount dependent on the amount of the ligand present in the liquid sample and (b) to provide a calibration signal by (c) forming either a labelled calibration complex immobilized on the calibration surface in an amount either dependent on or independent of the amount of the ligand present in the liquid sample or (d) due to said known amount of immobilized label on the calibration surface; and

(B) measuring and comparing the amount of label on the measurement and calibration surfaces to determine the presence or amount of the ligand in the liquid sample.

8. The method as claimed in claim 7 wherein the label is a fluorogenic, phosphorogenic or luminogenic label.

9. The method as claimed in claim 8 wherein the method is an antigen-antibody immunoassay.

10. The method as claimed in claim 7, wherein the method is an antigen-antibody immunoassay.

11. The method as claimed in claim 7, the capillary-fill device further comprises a spatially distinct first auxiliary calibration zone comprising

(e) either a known amount of a releasable third ancillary specific binding reagent and a first auxiliary calibration surface comprising an immobilized first auxiliary calibration specific binding reagent having binding sites identical in structure to those of the immobilized measurement specific binding reagent and capable of specifically binding to at least one of an unknown amount of the ligand, a known amount of the ligand, and the third ancillary specific binding reagent, or

(f) a first auxiliary calibration surface comprising an immobilized first auxiliary calibration nonspecific binding reagent, wherein the third ancillary specific binding reagent is provided either on a surface of the first auxiliary calibration zone separate from the first auxiliary calibration surface or prebound to the immobilized first auxiliary calibration specific binding reagent, and is (i) a labelled specific binding partner for said ligand, (ii) a known amount of the ligand prebound to said specific binding partner for said ligand, (iii) a labelled or unlabelled nonspecific binding ligand which binds to the immobilized first auxiliary calibration nonspecific binding reagent, or (iv) a combination of said nonspecific binding ligand and a labelled or unlabelled nonspecific binding reagent therefore, wherein step (A) further comprises (e) forming either a labelled first auxiliary calibration complex immobilized on the first auxiliary calibration surface in an amount either dependent on or independent of the amount of the ligand present in the liquid sample, or (f) forming an immobilized nonspecific binding complex in an amount dependent upon nonspecific binding background, and

wherein step (B) further comprises measuring and comparing the amount of label on the immobilized first auxiliary calibration surface.

12. A capillary-fill biosensor device suitable for use in assaying a ligand in a liquid sample, said device comprising (1) a measurement zone, (2) at least one spatially distinct calibration zone and, optionally, (3) at least one spatially distinct auxiliary calibration zone wherein

(1) the measurement zone comprises (a) a known amount of a releasable first ancillary specific binding reagent and (b) a measurement surface comprising an immobilized measurement specific binding reagent capable of specifically binding with at least one of the ligand, the first ancillary specific binding reagent, or a first binding partner which specifically binds to said ligand and said first ancillary specific binding reagent, wherein the first ancillary specific binding reagent is provided either on a surface of the measurement zone separate from the measurement surface or prebound to said immobilized measurement specific binding partner reagent,

(2) the calibration zone comprises

(c) a known amount of at least one releasable second ancillary specific binding reagent and a calibration surface comprising an immobilized calibration specific binding reagent having binding sites identical in structure to those of the immobilized measurement specific binding reagent and capable of specifically binding with at least one of the ligand, the second ancillary specific binding partner and a second binding partner which specifically binds to said ligand and said second ancillary specific binding reagent, and, optionally,

(3) the auxiliary calibration zone comprises

(d) an auxiliary calibration surface comprising an immobilized first auxiliary nonspecific binding reagent, wherein the releasable reagents remain in their respective zones without mixing with each other in the assay following either sequential or simultaneous contact with the liquid sample to provide (a) a specific binding complex immobilized on the measurement surface which is correlative of the presence or amount of the ligand in the liquid sample, (b) a calibration specific binding complex immobilized on the calibration surface as a result of an identical specific binding reaction as occurs on the measurement surface so as to provide a measurement correlative of a known amount of said ligand and, optionally, (c) a nonspecific binding complex immobilized on the auxiliary calibration zone to provide a measurement for correcting nonspecific background binding.

13. The device as claimed in claim 12 wherein said measurement, calibration and auxiliary calibration zones are provided as a transparent capillary comprising an upper portion and a lower portion, wherein the releasable reagents are provided on an inner surface of said upper portion and the measurement, calibration and auxiliary calibration surfaces are provided on an inner surface of said bottom portion, said transparent capillary being a light-transmissive waveguide.

14. The device as claimed in claim 13 further comprising a layer of light-absorbing or opaque material coating an outer surface of said bottom portion of said capillary opposite the measurement, calibration and auxiliary calibration surfaces.

15. The device as claimed in claim 13 further comprising said auxiliary calibration zone.

16. The device as claimed in claim 12 further comprising a detection means for measuring the specific binding complex immobilized on the measurement surface, the calibration specific binding complex immobilized on the calibration surface and, optionally, the nonspecific binding complex immobilized on the auxiliary calibration zone.

17. The device as claimed in claim 16 wherein the detection means is a fluorescence monitor, the releasable reagents are labeled with a fluorogenic label, and wherein the device further comprises a radiation source for exciting said fluorogenic label.

18. A specific binding method for determining a ligand in a liquid sample comprising:

(a) contacting the liquid sample with the capillary-fill biosensor device as claimed in claim 12 and

(b) measuring and comparing the amount of the specific binding complex immobilized on the measurement surface, the amount of the calibration specific binding complex immobilized on the calibration surface as a result of an identical specific binding reaction as occurs on the measurement surface so as to provide a measurement correlative of a known amount of said ligand and, optionally, the amount of the nonspecific binding complex immobilized on the auxiliary calibration zone to correct for nonspecific background binding in order to determine the presence or amount of the ligand in the liquid sample.

19. The method as claimed in claim 18 wherein the releasable reagents in the capillary-fill biosensor device are labeled with a fluorogenic, phosphorogenic or luminogenic label.

20. The method as claimed in claim 19 wherein the method is an antigen-antibody immunoassay.

21. The method as claimed in claim 18 wherein the method is an antigen-antibody immunoassay.