Rapid diagnostic assay

• US 7,531,362 B2
• Filed: 06/06/2002
• Issued: 05/12/2009
• Est. Priority Date: 06/07/2001
• Status: Expired due to Term

• Current Assignee: MedMira Incorporated (MedMira Holding AG)
• Original Assignee: MedMira Incorporated (MedMira Holding AG)
• Inventors: Chan, Hermes K. W.
• Primary Examiner(s): Alexander; Lyle A
• Application Number:
  US10/163,675
• Publication Number:
  US 20030049857A1
• Time in Patent Office: 2,532 Days
• Field of Search: 422/56, 422/58, 422/61, 422/68.1, 436/63, 436/164, 436/166, 436/169
• US Class Current: 436/164
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

An improved rapid diagnostic device, assay and multifunctional buffer reagent are provided for the detection of a target analyte in a fluid test sample. The 2-step assay utilizes a dual component flow-through device comprising a test unit and a post-filter unit capable of receiving the fluid sample and multifunctional buffer, respectively. The test unit comprises a reaction zone containing immobilized capture reagent that can specifically bind to the target analyte, an absorbent zone supporting the reaction zone, and optionally, a blood separation zone in lateral fluid communication with the reaction zone. The post-filter unit comprises a label zone permeated with a dried indicator reagent which is capable of being placed in transient fluid communication with the reaction zone of the test unit during the assay procedure. The rapid diagnostic assay system reduces the number of assay reagents, method steps and time required for performance compared to other conventional assays.

Claims

1. A method for determining the presence or absence of a target analyte in a fluid test sample, the method comprising the steps of:

applying the fluid test sample to a reaction zone of a test unit, whereby the fluid test sample flows downwardly or vertically through the reaction zone, the reaction zone containing an immobilized capture reagent that binds the target analyte in the deposited fluid test sample to form a two-membered complex of a specific binding interaction;

allowing the fluid test sample to flow downwardly or vertically through the reaction zone into an absorbent zone in vertical communication with the reaction zone, the absorbent zone comprising an absorbent material positioned underneath the reaction zone for facilitating the downward or vertical flow of the fluid test sample through the reaction zone so as to concentrate the two-membered complex in the reaction zone;

affixing a post filter unit to the test unit, such that a label zone of the post-filter unit and the reaction zone of the test unit are proximally disposed, so as to be in transient fluid communication with one another to thereby allow direct downward or vertical fluid flow of a resolubilized dried indicator agent in the label zone to the reaction zone;

applying a buffer reagent to the post-filter unit to resolubilize the dried indicator reagent after the fluid test sample is applied to the reaction zone and after the post-filter unit is affixed to the test unit;

allowing the resolubilized indicator reagent to flow downwardly or vertically through the reaction zone and into the absorbent zone to bind with the two-membered complex concentrated in the reaction zone, with any unbound reactants being washed from the reaction zone into the absorbent zone; and

removing the post-filter unit from the test unit subsequent to the application of the buffer reagent to the label zone and the binding of the resolubilized indicator reagent with the two-membered complex to observe a test result depicted by a presence or absence of a visually detectable signal on the reaction zone.

2. The method according to claim 1, wherein the buffer reagent is a multifunctional buffer comprising:

a biological buffer to maintain the pH between 7.0 to 10.0;

at least one surfactant to reduce non-specific binding of assay reagents while simultaneously avoiding inhibition of a specific binding interaction;

a high molecular weight polymer as a dispersing and suspending reagent having a molecular weight in a range of from about 2×

10² to about 2×

10⁶ D;

a pH stabilizer to maintain the pH of the multifunctional buffer within a range of about pH 7.0 to 10.0;

an ionic salt to reduce non-specific binding of antibodies;

at least one preservative to reduce bacterial and microbial growth; and

a calcium chelator to prevent a whole blood test sample from clotting;

wherein the biological buffer, the surfactant, the high molecular weight polymer, the pH stabilizer, the ionic salt, the preservative and the calcium chelator are all in effective concentrations.

3. The method according to claim 1, wherein the specific binding interaction is an antibody-antigen interaction.

4. The method according to claim 1, wherein the indicator reagent is capable of binding to a target analyte at a site which does not interfere with the specific binding interaction between the target analyte and the capture reagent.

5. The method according to claim 1, wherein the indicator reagent is capable of binding to the capture reagent at a site which interferes with the specific binding interaction between the target analyte and the capture reagent.

6. The method according to claim 3, wherein the target analyte is an antigen and the capture reagent is a monoclonal antibody or an affinity purified polyclonal antibody for the antigen.

7. The method according to claim 1, wherein the reaction zone is comprised of a material which has a pore size permitting separation and filtration of unbound components from the fluid test sample and a thickness which permits an adequate amount of capture reagent to be immobilized thereto.

8. The method according to claim 7, wherein the material has a pore size ranging from about 0.1 to 12.0 microns.

9. The method according to claim 8, wherein the material has a pore size ranging from about 0.2 to 0.8 microns.

10. The method according to claim 7, wherein the thickness of the material ranges from about 0.05 mm to about 30 mm.

11. The method according to claim 10, wherein the thickness of the material ranges from about 0.1 to about 1.0 mm.

12. The method according to claim 7, wherein the material is a nitrocellulose membrane.

13. The method according to claim 1, wherein the reaction zone contains two or more different capture reagents immobilized thereto in discernable and separate areas so that multiple target analytes in a single fluid test sample can be analyzed simultaneously.

14. The method according to claim 1, wherein the reaction zone further comprises an immobilized control reagent in a discernable and separate area from the capture reagent.

15. The method according to claim 1, wherein the absorbent zone is separated from the reaction zone by an intervening spacer layer having one or more openings defined therein to permit fluid communication between the reaction zone and the absorbent zone.

16. The method according to claim 15, wherein the spacer layer is a rigid or semi-rigid fluid-resistant material.

17. The method according to claim 1, wherein the absorbent zone comprises one or more layers of a material which is capable of wicking fluid by capillary action and absorbing a substantial volume of fluid.

18. The method according to claim 17, wherein two or more layers comprise identical or different materials.

19. The method according to claim 17, wherein the material is cellulose acetate.

20. The method according to claim 1, wherein the label zone comprises a filter material having a pore size capable of allowing dried indicator reagent to be effectively resolubilized by buffer reagent and transferred to the reaction zone by laminar fluid flow.

21. The method according to claim 20, wherein the filter material is glass fiber material.

22. The method according to claim 1, wherein the indicator reagent comprises a direct label.

23. The method according to claim 22, wherein the direct label is colloidal gold.