Quantitative immunochromatographic assays

• US 5,753,517 A
• Filed: 03/29/1996
• Issued: 05/19/1998
• Est. Priority Date: 03/29/1996
• Status: Expired due to Term

• Current Assignee: British Columbia Cancer Agency Branch (Province of British Columbia)
• Original Assignee: University of British Columbia
• Inventors: Devine, Dana, Brooks, Donald Elliott
• Primary Examiner(s): Achutamurthy, Ponnathapura
• Assistant Examiner(s): Nelson, Brett L.
• Application Number:
  US08/625,048
• Time in Patent Office: 781 Days
• Field of Search: 436/514, 436/534, 435/7.1, 422/61
• US Class Current: 436/514
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387

Abstract

Quantitative immunochromatographic assays for measuring the amount of an analyte, and an apparatus for use in the assays, are disclosed. The assays involve obtaining a fluid sample which contains the analyte; supplying a RAMP™ apparatus which includes a membrane having an application point, a detection zone, and a contact region, where the contact region is between the application point and the detection zone, and has antibody-coated particles imbedded within it; contacting the application point with the fluid sample; maintaining the RAMP™ apparatus under conditions sufficient to allow fluid to transport analyte by capillary action to the contact region, where the analyte binds to the antibody-coated particles; further maintaining the apparatus under conditions sufficient to allow fluid to transport analyte-bound antibody-coated particles to the detection zone, where they interact with a detection reagent; and detecting the amount of analyte-bound antibody-coated particles which have reacted with the detection reagent. Alternatively, the fluid sample can be contacted with the detection zone of the apparatus, and the antibody-coated particles are mobilized by addition of fluid to the application point. The RAMP™ apparatus can additionally include one or more of the following features: a wicking pad; an application pad covering the application point; a contact pad covering the contact region; a separator pad between the membrane and the contact pad; and an internal control.

Claims

1. A method for quantitatively measuring the amount of an analyte of interest in a sample, comprising the steps of:

a. obtaining a fluid sample to be assayed for the analyte of interest;

b. supplying a rapid antigen measurement platform apparatus, the apparatus comprising a membrane strip, the membrane strip comprising an application point, a contact region, a control reaction zone and a detection zone, wherein the contact region is between the application point and the detection zone;

c. contacting the application point of the membrane strip with the fluid sample;

d. maintaining the membrane strip under conditions which are sufficient to allow fluid to transport analyte of interest in the fluid sample by capillary action through the strip to the contact region, the contact region having a population of antibody-coated particles imbedded therein, wherein the antibody is an antibody to the analyte of interest, and a population of internal control particles imbedded therein, wherein the internal control particles are coated with an antibody to a control detection reagent;

e. further maintaining the membrane strip under conditions which are sufficient to allow analyte of interest to bind to the antibody-coated particles, thereby generating contacted antibody-coated particles;

to allow the fluid in the sample to transport the contacted antibody-coated particles by capillary action through the strip to the detection zone, the detection zone having the analyte of interest immobilized thereon, and to allow the fluid in the sample to transport the internal control particles by capillary action through the strip to the control reaction zone, the control reaction zone having the control detection reagent immobilized thereon;

to allow the immobilized analyte of interest in the detection zone to bind to contacted antibody-coated particles which are insufficiently coated with the analyte of interest in the fluid sample, and to allow the internal control particles to bind to the control detection reagent; and

f. detecting the amount of contacted antibody-coated particles bound to the immobilized analyte of interest in the detection zone, and the amount of internal control particles bound to the control detection reagent in the control reaction zone,

wherein the amount of analyte of interest in the fluid sample is inversely related to a ratio of the amount of antibody-coated particles bound to the immobilized analyte of interest in the detection zone, to the amount of internal control particles bound to the control detection reagent in the control reaction zone.

2. The method of claim 1, wherein the membrane strip is made of cellulose nitrate or glass fiber.

3. The method of claim 1, wherein the particles are latex beads.

4. The method of claim 1, wherein the particles are labeled.

5. The method of claim 4, wherein the label is selected from the group consisting of:

colorimetric, fluorescent, and luminescent.

6. The method of claim 1, wherein the analyte of interest is thrombospondin, and the fluid sample is blood or a platelet-rich plasma sample.

7. The method of claim 6, wherein the detection reagent is thrombospondin.

8. The method of claim 1, wherein the analyte of interest is myoglobin, and the fluid sample is selected from the group consisting of:

whole blood, plasma, and serum.

9. The method of claim 1, wherein the analyte of interest is urinary albumin, and the fluid sample is urine.

10. A method for quantitatively measuring the amount of an analyte of interest in a sample, comprising the steps of:

a. obtaining a fluid sample to be assayed for the analyte of interest;

b. supplying a rapid antigen measurement platform apparatus, the apparatus comprising a membrane strip, the membrane strip comprising an application point, a contact region, a control reaction zone and a detection zone, wherein the contact region is between the application point and the detection zone;

c. contacting the application point of the membrane strip with the fluid sample;

d. maintaining the membrane strip under conditions which are sufficient to allow fluid to transport analyte of interest in the fluid sample by capillary action through the strip to the contact region, the contact region having a population of antibody-coated particles imbedded therein, wherein the antibody is an antibody to the analyte of interest, and a population of internal control particles imbedded therein, wherein the internal control particles are coated with an antibody to a control detection reagent;

e. further maintaining the membrane strip under conditions which are sufficient to allow analyte of interest to bind to the antibody-coated particles, thereby generating contacted antibody-coated particles;

to allow the fluid in the sample to transport the contacted antibody-coated particles by capillary action through the strip to the detection zone, the detection zone having immobilized thereon an antibody selected from the group consisting of;

an antibody directed against the same epitope as the antibody on the antibody-coated particles, and an antibody directed against a different epitope as the antibody on the antibody-coated particles; and

to allow the fluid in the sample to transport the internal control particles by capillary action through the strip to the control reaction zone, the control reaction zone having the control detection reagent immobilized thereon;

to allow the immobilized antibody in the detection zone to bind to analyte bound to the contacted antibody-coated particles, and to allow the internal control particles to bind to the control detection reagent; and

f. detecting the amount of contacted antibody-coated particles bound to the immobilized antibody in the detection zone, and the amount of internal control particles bound to the control detection reagent in the control reaction zone,

wherein the amount of analyte of interest in the fluid sample is directly related to a ratio of the amount of contacted antibody-coated particles bound to the immobilized antibody in the detection zone, to the amount of internal control particles bound to the control detection reagent in the control reaction zone.

11. The method of claim 10, wherein the membrane strip is made of cellulose nitrate or glass fiber.

12. The method of claim 10, wherein the particles are latex beads.

13. The method of claim 10, wherein the particles are labeled.

14. The method of claim 13, wherein the label is selected from the group consisting of:

colorimetric, fluorescent, and luminescent.

15. The method of claim 10, wherein the analyte of interest is thrombospondin, and the fluid sample is blood or a platelet-rich plasma sample.

16. The method of claim 15, wherein the detection reagent is an antibody to thrombospondin.

17. The method of claim 10, wherein the analyte of interest is myoglobin, and the fluid sample is selected from the group consisting of:

whole blood, plasma, and serum.

18. The method of claim 10, wherein the analyte of interest is urinary albumin, and the fluid sample is urine.

19. A method for measuring the amount of an analyte of interest in a sample, comprising the steps of:

a. obtaining a fluid sample to be assayed for the analyte of interest;

b. supplying a rapid antigen measurement platform apparatus, the apparatus comprising a membrane strip, the membrane strip comprising an application point, a contact region, a control reaction zone and a detection zone, wherein the contact region is between the application point and the detection zone;

c. contacting the application point of the membrane strip with the fluid sample;

d. maintaining the membrane strip under conditions which are sufficient to allow fluid to transport analyte of interest in the fluid sample by capillary action through the strip to the contact region, the contact region having a population of particles imbedded therein, wherein the particles are coated with an agent that specifically binds to the analyte, and a population of internal control particles imbedded therein, wherein the internal control particles are coated with an antibody to a control detection reagent;

e. further maintaining the membrane strip under conditions which are sufficient to allow analyte of interest to bind to the coated particles, thereby generating contacted coated particles;

to allow the fluid in the sample to transport the contacted coated particles by capillary action through the strip to the detection zone, the detection zone having a detection reagent immobilized thereon, and to allow the fluid in the sample to transport the internal control particles by capillary action through the strip to the control reaction zone, the control reaction zone having the control detection reagent immobilized thereon;

to allow the detection reagent to interact with contacted coated particles, thereby generating arrested detection-reagent-particle complexes; and

to allow the internal control particles to bind to the control detection reagent; and

f. detecting the amount of arrested detection-reagent-particle complexes in the detection zone and the amount of internal control particles bound to the control detection reagent in the control reaction zone,

wherein the amount of analyte of interest in the fluid sample is related to a ratio of the amount of arrested detection-reagent-particle complexes in the detection zone, to the amount of internal control particles bound to the control detection reagent in the control reaction zone.

20. A method for measuring the amount of an analyte of interest in a sample, comprising the steps of:

a. obtaining a fluid sample to be assayed for the analyte of interest;

b. supplying a rapid antigen measurement platform apparatus, the apparatus comprising a membrane strip, the membrane strip comprising an application point, a contact region, a control reaction zone and a detection zone, wherein the contact region is between the application point ant the detection zone;

c. contacting the detection zone of the membrane strip with the fluid sample, the detection zone having an antibody to the analyte of interest immobilized thereon, and maintaining the membrane strip under conditions which are sufficient to allow analyte of interest to bind to the antibody in the detection zone, thereby generating immobilized analyte;

d. contacting the application point of the membrane strip with a buffer, and maintaining the membrane strip under conditions which are sufficient

     1) to allow buffer to transport a population of antibody-coated particles imbedded in the contact region, wherein the antibody is an antibody to the analyte, by capillary action to the detection zone, and

     2) to allow buffer to transport a population of internal control particles imbedded in the contact region, wherein the internal control particles are coated with an agent that specifically binds to a control detection reagent, by capillary action through the strip to the control reaction zone, the control reaction zone having the control detection reagent immobilized thereon;

e. further maintaining the membrane strip under conditions which are sufficient

     1) to allow immobilized analyte to interact with the antibody-coated particles, thereby generating immobilized analyte-particle complexes, and

     2) to allow the internal control particles to bind to the control detection reagent; and

f. detecting the amount of immobilized analyte-particle complexes in the detection zone, and the amount of internal control particles bound to the control detection reagent in the control reaction zone,

wherein the amount of analyte of interest in the fluid sample is related to a ratio of the amount of immobilized analyte-particle complexes in the detection zone, to the amount of internal control particles bound to the control detection reagent in the control reaction zone.

21. A method for quantitatively measuring the amount of an analyte of interest in a sample, comprising the steps of:

a. obtaining a fluid sample to be assayed for the analyte of interest;

b. supplying a rapid antigen measurement platform apparatus, the apparatus comprising a membrane strip, the membrane strip comprising an application point, a contact region, a control reaction zone and a detection zone, wherein the contact region is between the application point and the detection zone;

c. contacting the application point of the membrane strip with the fluid sample;

d. maintaining the membrane strip under conditions which are sufficient to allow fluid to transport analyte of interest in the fluid sample by capillary action through the strip to the contact region, the contact region having imbedded therein a population of particles coated with an agent that specifically binds to the analyte of interest, and also imbedded therein a population of internal control particles, wherein the internal control particles are coated with an agent that specifically binds to a control detection reagent;

e. further maintaining the membrane strip under conditions which are sufficient to allow analyte of interest to bind to the particles coated with the agent that specifically binds to the analyte of interest, thereby generating contacted, coated particles;

to allow the fluid in the sample to transport the contacted, coated particles by capillary action through the strip to the detection zone, the detection zone having a detection reagent immobilized thereon;

to allow the fluid in the sample to transport the internal control particles by capillary action through the strip to the control reaction zone, the control reaction zone having the control detection reagent immobilized thereon;

to allow the contacted, coated particles to bind to the detection reagent in the detection zone; and

to allow the internal control particles to bind to the control detection reagent; and

f. detecting the amount of contacted, coated particles bound to the detection reagent in the detection zone, and the amount of internal control particles bound to the control detection reagent in the control reaction zone,

wherein the amount of analyte of interest in the fluid sample is related to a ratio of the amount of contacted, coated particles bound to the detection reagent in the detection zone, to the amount of internal control particles bound to the control detection reagent in the control reaction zone.