Microchip-based system for hiv diagnostics

• US 20060234209A1
• Filed: 02/05/2004
• Published: 10/19/2006
• Est. Priority Date: 02/05/2003
• Status: Abandoned Application

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.), Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Walker, Bruce D., Rodriguez, William R., McDevitt, John C.
• Application Number:
  US10/544,864
• Publication Number:
  US 20060234209A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/52 involving transaminase
• CPC Class Codes: G01N 33/537 with separation of immune complex from unbound antigen or antibody
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/56988 HIV or HTLV
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The invention relates to microchip-based assays to measure HIV-associated analytes of interest (e.g., CD4 lymphocytes, HIV RNA and liver enzymes) in a sample from a subject infected with the HIV virus. Methods of the present invention are optimal for use in monitoring HIV disease in resource-poor settings.

Claims

1. A method for detecting an HIV-associated analyte in a sample of blood comprising:

passing an HIV-associated analyte coupled to a fluorescence-emitting conjugate through a filter, wherein the analyte is immobilized on the filter thereby separating the analyte from the blood;

delivering light to the analyte at a wavelength suitable for excitation of the conjugate;

digitally imaging a conjugate emitted fluorescent signal using a detector.

2. The method of claim 1, wherein the filter is a polycarbonate porous membrane.

3. The method of claim 1, wherein the detector is a CCD digital camera or a CMOS detector.

5. The method of claim 1, wherein the analyte is lymphocyte.

6. The method of claim 1, wherein the lymphocyte is a CD4+ T cell, a CD8+ T cell, a CD3+ T cell, or mixtures thereof.

12. The method of claim 1, wherein the fluorescence-emitting conjugate comprises an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD3 antibody, or mixtures thereof.

13. The method of claim 1, wherein the fluorescence-emitting conjugate comprises an anti-CD4 antibody, and wherein the anti-CD4 antibody is an Alexa488-conjugated anti-CD4 antibody.

16. The method of claim 1, wherein the fluorescence-emitted conjugate comprises an anti-CD3 antibody, and wherein the fluorescence-emitting conjugate is an Alexa647 conjugated anti-CD3 antibody.

17. The method of claim 1, wherein the blood is obtained from a fingerstick sample of blood.

4. (canceled)

7-11. -11. (canceled)

14-15. -15. (canceled)

18. A method for determining the ratio of CD4+ T cells to CD8+ T cells in a sample of blood comprising:

passing CD4+ and CD8+ T lymphocytes through a filter, wherein the CD4+ and a CD8+ T lymphocytes are captured on the filter;

coupling the CD4+ and the CD8+lymphocytes to different fluorescence emitting conjugates;

delivering light to the lymphocytes at a wavelength suitable for excitation of each conjugate;

digitally imaging each conjugate emitted fluorescent signal using a detector; and

determining the ratio of CD4+ cells to CD8+ cells.

19. A method for detecting HIV-RNA in a sample of blood comprising:

passing a blood sample comprising HIV-RNA through a flow cell comprising one or more cavities, wherein one or more of the cavities contains a filter comprising a complementary nucleotide sequence coupled to a fluorescence-emitting compound;

forming a binding complex between the HIV-RNA and the complementary nucleotide sequence on the filter, whereby the signal emitted from the fluorescence-emitting compound is enhanced by formation of the binding complex;

delivering light to the complex at a wavelength suitable for excitation of the fluorescence-emitting compound; and

digitally imaging a fluorescent signal emitted by the complex using a detector.

20. A method for detecting an HIV-associated analyte in a sample of blood comprising:

passing an HIV-associated analyte coupled to a fluorescence-emitting conjugate through a flow cell comprising one or more cavities, wherein one or more of the cavities contains an agarose bead comprising a suitable biological agent having binding affinity for the analyte, such that the analyte is immobilized on the bead, thereby separating the analyte from the blood;

delivering light to the analyte at a wavelength suitable for excitation of the conjugate;

digitally imaging a conjugate emitted fluorescent signal using a detector.

21. The method of claim 20, wherein the detector is a CCD digital camera or a CMOS detector.

23. The method of claim 20, wherein the analyte is lymphocyte.

24. The method of claim 20, wherein the lymphocyte is a CD4+ T cell, a CD8+ T cell, a CD3+ T cell, or mixtures thereof.

27. The method of claim 20, wherein the analyte is a liver enzyme.

30. The method of claim 20, wherein the fluorescence-emitting conjugate comprises an anti-CD4 antibody, an anti-CD8 antibody an anti-CD3 antibody, or mixtures thereof.

31. The method of claim 20, wherein the fluorescence-emitting conjugate comprises an anti-CD4 antibody, and wherein the anti-CD4 antibody is an Alexa488-conjugated anti-CD4 antibody.

35. The method of claim 20, wherein the blood is obtained from a fingerstick sample of blood.

22. (canceled)

25-26. -26. (canceled)

28-29. -29. (canceled)

32-33. -33. (canceled)

36. A method for determining the ratio of CD4+ T cells to CD8+ T cells in a sample of blood comprising:

passing CD4+ and CD8+ T lymphocytes, each coupled to a different fluorescence-emitting conjugate, through a flow cell comprising one or more cavities, wherein one or more of the cavities contains an agarose bead comprising a biological agent having binding affinity for either CD4+ or CD8+ T lymphocytes, such that the lymphocytes are immobilized on the bead, thereby separating the analyte from the blood;

delivering light to the lymphocytes at a wavelength suitable for excitation of each conjugate;

digitally imaging each conjugate emitted fluorescent signal using a detector; and

determining the ratio of CD4+ cells to CD8+ cells.

37. The method of claim 36, wherein the biological agent is an antibody.

38. A method for detecting HIV-RNA in a sample of blood comprising:

passing a blood sample comprising HIV-RNA through a flow cell comprising one or more cavities, wherein one or more of the cavities contains an agarose bead comprising a complementary nucleotide sequence coupled to a fluorescence-emitting compound;

forming a binding complex between the HIV-RNA and the complementary nucleotide sequence on the bead, whereby the signal emitted from the fluorescence-emitting compound is enhanced by binding of the HIV-RNA to the complementary nucleotide sequence;

delivering light to the complex at a wavelength suitable for excitation of the fluorescence-emitting compound; and

digitally imaging a fluorescent signal emitted by the complex using a detector.