Method for amplification of the response signal in a sandwich immunoassay

• US 5,876,944 A
• Filed: 06/10/1996
• Issued: 03/02/1999
• Est. Priority Date: 06/10/1996
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Bayer Corporation (Bayer AG)
• Inventors: Kuo, Hai-Hang
• Primary Examiner(s): Hutzell, Paula K.
• Assistant Examiner(s): BAKALYAR, HEATHER
• Application Number:
  US08/660,923
• Time in Patent Office: 995 Days
• Field of Search: 435/7.1, 436/510, 436/814, 436/65, 436/88, 436/170, 422/56, 422/60
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: Y10S 436/814 Pregnancy

Abstract

Disclosed is an improvement to a sandwich type immunoassay in which there is immobilized to a solid support an antibody which is specific to an epitope of the analyte whose presence or concentration is being sought and a first labeled antibody which is specific to another epitope of the analyte. The improvement involves providing a second labeled antibody which is specific to the first labeled antibody to thereby form a chain of two or more labeled antibodies which results in amplification of the signal generated upon formation of the sandwich.

Claims

1. In a sandwich type immunoassay in which there is immobilized to a first region of a strip of porous material through which a test fluid suspected of containing an analyte can flow by capillarity a first antibody which is specific for a first epitope of the analyte whose presence or concentration in a liquid test medium is being sought by applying the liquid test medium to the strip and in a second region of the strip there is contained a particulate label which is visible when bound to the strip without further treatment as primary signal generator which has directly bound to it multiple second antibodies which are specific to a second epitope of the analyte, the improvement which comprises including in the second region of the strip a secondary signal generator which is also a visible particulate label having attached to it multiple third antibodies which are specific for the second antibody attached to the primary signal generator but not specific for the parts of the second antibody which comprise the analyte binding sites which third antibody specifically binds to the second antibody to form signal amplifying complexes, and allowing the second labeled antibody and third labeled antibody to interact with each other and the analyte to form signal amplifying complexes to amplify the signal generated when the first immobilized antibody specifically binds with the first epitope of the analyte when the liquid test medium containing the signal amplifying complex of the second antibody, third antibody and analyte flow into the first region of the strip by capillarity to thereby bind the signal amplifying complex to the first region of the strip to thereby provide an amplified detectable signal in the first region of the strip.

2. The improvement of claim 1 wherein the primary and secondary signal generators are the same.

3. The improvement of claim 2 wherein the primary and secondary signal generators are colored, colloidal sized particles.

4. The improvement of claim 3 wherein the colloidal sized particles comprise a metal sol.

5. The improvement of claim 4 wherein the metal is gold.

6. The improvement of claim 1 wherein the antibody attached to the secondary signal generator is specific to the heavy chain of the antibody which is attached to the primary signal generator.

7. The improvement of claim 1 wherein the test fluid is urine and the analyte is hCG.

8. The improvement of claim 1 wherein one or more of the antibodies is a complete antibody.

9. The improvement of claim 1 wherein one or more of the antibodies is a Fab or F(ab)₂ fragment.

10. A test strip for the detection of an analyte in a test fluid which strip is made of an absorbant material through which the test fluid can flow by capillarity which strip has immobilized to a first region thereof a first antibody specific for a first epitope of the analyte and which strip has absorbed therein in a second region distinct from the first region a second labeled antibody which is specific to an epitope of the analyte different than that to which the immobilized antibody is specific and a third labeled antibody which is specific to the second labeled antibody but not specific for the parts of the second antibody which comprise the analyte binding sites and wherein the label is a particulate label which is visible when bound to the strip without further treatment which particulate labels have respectively multiple second and third antibodies bound to them, so that when the test fluid is applied to the strip it flows along the strip by capillarity and analyte contained in the test fluid binds with the second labeled antibody and third labeled antibody to form a signal amplifying complex which complex flows by capillarity to the first region of the strip and binds with the first immobilized antibody to form a sandwich comprising the analyte bound between the immobilized antibody and the second labeled antibody which sandwich provides an amplified detectable signal due to the the formation of the signal amplifying complex in the second region of the strip.

11. The strip of claim 10 wherein the first and second labeled antibody are labeled with colored colloidal sized particles.

12. The strip of claim 11 wherein the colloidal sized particles comprise a metal sol.

13. The strip of claim 12 wherein the metal is gold.

14. The strip of claim 10 wherein the third labeled antibody is specific to the heavy chain of the second labeled antibody.

15. The strip of claim 10 wherein the analyte to which the second labeled antibody and the immobilized antibody are specific is human serum albumin.

16. The test strip of claim 10 wherein the analyte to which the second labeled antibody and the immobilized antibody are specific is hCG.

17. The strip of claim 10 wherein one or more of the antibodies is a complete antibody.

18. The strip of claim 10 wherein one or more of the antibodies is a Fab or F(ab)₂ fragment.

19. A test strip for the detection of an analyte in a test fluid which comprises an absorbant carrier material through which the test fluid can flow by capillarity which strip comprises:

a) a reagent region containing a second labeled antibody which is specific to an epitope of the analyte;

b) a second reagent region containing a third labeled antibody which is specific to the second labeled antibody but not specific to the parts of the second antibody which comprise the analyte binding sites and wherein the label on the second and third antibodies is a particulate label which is visible when bound to the strip without further treatment which particulate labels have respectively multiple second and third antibodies bound to them so that the second labeled antibody, third labeled antibody and analyte in the test fluid can form a signal amplifying complex upon application of an analyte containing test fluid to the strip; and

c) a capture zone into which the signal amplifying complex can flow by capillarity in which there is immobilized a first antibody specific to an epitope of the analyte different than that to which the second labeled antibody is specific so that when the signal amplifying complex flows into the capture zone, it is captured by the first antibody to thereby provide an amplified signal in the capture zone.

20. The strip of claim 19 which also contains a wicking region adjacent to the first reagent region for application of the test fluid .

21. The strip of claim 19 which also contains a positive control zone located downstream from the capture zone which contains an immobilized specific binding partner for the second or third labeled antibody.

22. The strip of claim 19 wherein one or more of the antibodies is a complete antibody.

23. The strip of claim 19 wherein one or more of the antibodies is a Fab or F(ab)₂ fragment.

24. The strip of claim 21 wherein the first and second reagent zones overlap so that the second and third labeled antibodies are intermixed in the dry strip.