Quantitative detection of analytes on immunochromatographic strips

• US 5,569,608 A
• Filed: 01/30/1995
• Issued: 10/29/1996
• Est. Priority Date: 01/30/1995
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Bayer Corporation (Bayer AG)
• Inventors: Sommer, Ronald G.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): NGUYEN, BAO THUY L
• Application Number:
  US08/380,119
• Time in Patent Office: 638 Days
• Field of Search: 435/7.92, 435/169, 435/810, 435/805, 435/970, 436/523, 436/169, 436/525, 436/514, 436/810, 422/63, 422/56, 23/230 B
• US Class Current: 436/518
• CPC Class Codes: G01N 30/90 Plate chromatography, e.g. thin layer or paper chromatography
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

A method for determining the concentration of analyte in a test fluid by immunochromatography techniques which involves quantitatively determining the signals from captured analyte/labeled binding partner complex by an instrument, e.g. reflectance spectrometer. In a preferred embodiment, a reflectance reading is determined for the captured complex and uncomplexed labeled binding partner which is captured in a separate zone of the immunochromatographic strip and the ratio of these reflectances is used to provide additional quantitation to the assay method.

Claims

1. A method for determining the concentration of one or more analyte in a fluid test sample which comprises:

a) providing a test strip comprising a matrix through which the fluid test sample can flow by capillarity wherein a first region of the strip contains mobile specific binding partner for the analyte which bears a detectable label and can react with the analyte to form an analyte/labeled binding partner complex and at least one second region which contains immobilized analyte or analog thereof,

b) developing the strip by applying the fluid test sample suspected of containing the analyte thereto thereby allowing the same contact the mobile, labeled specific binding partner for the analyte whereby analyte present in the fluid test sample binds to the labeled specific binding partner to form the complex leaving excess, unreacted labeled binding partner free to further react whereby the fluid test sample carries the analyte/labeled binding partner complex and unreacted labeled binding partner along the strip by capillarity to the second region containing the immobilized analyte or analog thereof in which region unreacted labeled binding partner is bound to the inmlobilized analyte in inverse relationship to the concentration of analyte in the fluid test sample,

c) reading the developed strip on an instrument having a detector capable of measuring the signal from the detectable label to determine the concentration of the labeled binding partner in the second region, and

d) determining the concentration of analyte in the fluid test sample by comparing the signal from the detectable label measured in step c) with the measurements of the signal made in the same manner using fluid samples containing known concentrations of analyte.

2. The method of claim 1 wherein there is a third region of the strip which contains means for immobilizing the complex formed between the analyte and the labeled binding partner and measuring the signal from the detectable label immobilized in this third region and wherein the ratio of the signal from the labeled binding partner immobilized in the second region and the immobilized complex in the third region is determined.

3. The method of claim 1 wherein the instrument contains means for moving the strip or the detector relative to each other to give accurate quantitation for regions which may not have been precisely located on the instrument.

4. The method of claim 1 wherein there are two or more mobile labeled binding partners on the strip in the same or different first regions and two or more second regions containing immobilized analytes or analogs thereof, so that two or more analytes can be determined in a single test.

5. The method of claim 1 wherein the instrument is a reflectance spectrometer.

6. The method of claim 1 wherein the matrix is comprised of a material which is capable of non-bibulous lateral flow or a bibulous material.

7. The method of claim 1 wherein the analyte is human serum albumin.

8. The method of claim 1 wherein the detectable label is a colored species.

9. The method of claim 8 wherein the colored species is a gold sol.

10. The method of claim 1 wherein the labeled binding partner is an antibody.

11. A method for quantitatively determining the concentration of one or more analyte in a fluid test sample which comprises the steps of:

a) providing a test strip comprising a matrix through which the fluid test sample can flow by capillarity said strip having a first region wherein the first region contains mobile specific binding partner for the analyte which binding partner bears a detectable label and can react with the analyte to form an analyte/labeled binding partner complex, at least one second region which contains an immobilized analyte or analog thereof and at least one third region which contains means for immobilizing the complex formed between the analyte and labeled binding partner;

b) developing the strip by applying the fluid test sample suspected of containing the analyte thereto to allow the same to contact the mobile specific binding partner whereby analyte present in the fluid test sample binds to the labeled specific binding partner to form the complex leaving excess, unreacted labeled binding partner free to further react whereby the fluid test sample carries the analyte/labeled binding partner complex and unreacted labeled binding partner along the strip by capillarity to the second region containing the immobilized analyte or analog thereof in which region unreacted labeled binding partner is bound to the immobilized analyte in inverse relationship to the concentration of the analyte in the fluid test sample and the analyte/labeled binding partner complex is carried by capillarity to the third region where it is captured by the immobilization means;

c) reading the second zone of the developed strip on an instrument having a detector capable of measuring the signal from the detectable label to determine the concentration of the labeled binding partner in the second zone and reading the third zone of the developed strip in a similar manner to determine the signal from the labeled binding partner in the third zone of the strip;

d) determining the ratio of the signal from the labeled binding partner immobilized in the second region to the signal from the labeled binding partner in the third region; and

e) determining the concentration of the analyte in the fluid test sample by comparing the ratio of the signals measured in step c with the measurements of the ratios of the signals measured in the same manner for fluid samples containing known concentrations of the analyte.

12. The method of claim 11 wherein the measuring instrument is a reflectance spectrometer.

13. The method of claim 11 wherein the matrix is comprised of a material which is capable of non-bibulous lateral flow or a bibulous material.

14. The method of claim 11 wherein the analyte is human serum albumin.

15. The method of claim 11 wherein the detectable label is a gold sol.

16. The method of claim 11 wherein the labeled binding partner is an antibody.