Methods and reagents for quantitation of HLA-DR expression on peripheral blood cells

• US 6,376,202 B1
• Filed: 08/24/2000
• Issued: 04/23/2002
• Est. Priority Date: 12/03/1998
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Davis, Kenneth A.
• Primary Examiner(s): SAUNDERS, DAVID A
• Application Number:
  US09/645,966
• Time in Patent Office: 607 Days
• Field of Search: 435/7.24, 436/800
• US Class Current: 435/7.24
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: G01N 33/56977 HLA or MHC typing
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Improved methods, reagents, and kits for quantitation of HLA-DR expression on peripheral blood cells, particularly peripheral blood monocytes, are presented. Inclusion of a lysosomotropic amine, such as chloroquine, during staining stabilizes HLA-DR expression, and use of a novel anti-CD14 conjugate, anti-CD14-PerCP/CY5.5, permits the ready discrimination of monocytes. The improved methods, reagents, and kits may be used to assess immune competence, and to direct and monitor immunostimulatory therapies in septic patients exhibiting monocyte deactivation.

Claims

1. A method of measuring a level of HLA-DR expression on the surface of human blood cells, comprising:

contacting a sample containing human blood cells with a lysosomotropic amine and an antibody specific for HLA-DR; and

then

detecting binding of said antibody to said cells whereby a measured level of antibody binding per cell indicates the level of HLA-DR expression.

2. The method of claim 1, wherein said sample is unfractionated peripheral blood.

3. The method of either claim 1 or claim 2, wherein said lysosomotropic amine is selected from the group consisting of chloroquine, hydroxychloroquine, primaquine, and methylamine.

4. The method of claim 3, wherein said lysosomotropic amine is chloroquine.

5. The method of claim 3, wherein said lysosomotropic amine is hydroxychloroquine.

6. The method of either claim 1 or claim 2, wherein said anti-HLA-DR antibody is labeled with a fluorophore.

7. The method of claim 6, wherein said fluorophore is selected from the group consisting of PE, APC, FITC, and PerCP.

8. The method of claim 7, wherein said fluorophore is PE.

9. The method of claim 8, wherein said fluorophore is conjugated to said anti-HLA-DR antibody at a defined molar ratio.

10. The method of claim 9, wherein said ratio is 1:

1.

11. The method of either claim 1 or claim 2, wherein said antibody binding is detected flow cytometrically.

12. The method of claim 11, wherein said lysosomotropic amine is chloroquine and said anti-HLA-DR antibody is conjugated to PE.

13. The method of claim 12, wherein said anti-HLA-DR antibody is conjugated to PE at a molar ratio of 1:

1.

14. The method of either claim 1 or claim 2, wherein said detecting step further comprises detecting the binding of said anti-HLA-DR antibody to monocytes in said sample.

15. The method of claim 14, wherein said contacting step further comprises contacting with a monocyte-distinguishing antibody and said detecting step measures the binding of said anti-HLA-DR antibody to cells binding said monocyte-distinguishing antibody.

16. The method of claim 15, wherein said monocyte-distinguishing antibody is specific for CD14.

17. The method of claim 16, wherein said anti-CD14 antibody is conjugated to the PerCP moiety of a PerCP/CY5.5 tandem fluorophore.

18. The method of claim 17, wherein said anti-HLA-DR antibody is conjugated to a fluorophore whose emission spectrum is flow cytometrically distinguishable from PerCP/CY5.5.

19. The method of claim 18, wherein said anti-HLA-DR antibody is conjugated to PE.

20. The method of claim 19, wherein said anti-HLA-DR antibody is conjugated to PE at a defined molar ratio.

21. The method of claim 20, wherein said ratio is 1:

1.

22. The method of claim 19, wherein said lysosomotropic amine is chloroquine.

23. The method of claim 19, wherein said lysosomotropic amine is hydroxychloroquine.

24. The method of claim 22, wherein said antibody binding is detected flow cytometrically.

25. The method of claim 2, further comprising the step, after said contacting step and before said detecting step, of:

lysing erythrocytes.

26. The method of claim 24, further comprising the step, after said contacting step and before said detecting step, of:

lysing erythrocytes in said sample.

27. The method of claim 25, further comprising the step, after said lysing step and before said detecting step, of removing lysis debris.

28. The method of claim 26, further comprising the step, after said lysing step and before said detecting step, of removing lysis debris.

29. In a method of measuring an amount of HLA-DR surface expression per cell on human peripheral blood monocytes, the improvement comprising:

contacting said monocytes with chloroquine prior to or concurrently with contacting said monocytes with an anti-HLA-DR antibody.

30. In a method of measuring HLA-DR surface expression on human peripheral blood monocytes by contacting said monocytes with an anti-HLA-DR antibody, the improvement comprising:

contacting said monocytes with an anti-CD14 antibody conjugated to the PerCP moiety of a PerCP/CY5.5 tandem dye molecule.

31. A method of assessing the immune status of a human patient, comprising:

contacting a sample containing said patient'"'"'s blood cells with a lysosomotropic amine and an antibody specific for HLA-DR;

detecting the binding of said antibody to the monocytes in said sample; and

then

comparing the level of binding so detected with that so detected from human controls.

32. The method of claim 31, wherein said lysosomotropic amine is selected from the group consisting of chloroquine and hydroxychloroquine.

33. The method of claim 32, wherein said contacting step further comprises contacting said sample with a monocyte-distinguishing antibody, and said detecting step measures the binding of said anti-HLA-DR antibody to cells binding said monocyte-distinguishing antibody.

34. The method of claim 33, wherein said monocytedistinguishing antibody is an anti-CD14 antibody conjugated to the PerCP moiety of a PerCP/CY5.5 tandem dye molecule.

35. A method of determining the suitability of immunostimulatory therapy in a patient with sepsis, comprising:

contacting a sample containing said patient'"'"'s blood cells with a lysosomotropic amine and an antibody specific for HLA-DR;

detecting the binding of said antibody to the monocytes in said sample;

comparing the level of binding so detected with that detected from normal controls, wherein patients with binding lower than control are determined to be suitable for said therapy.

36. A method of determining the suitability of immunostimulatory therapy in a patient with sepsis, comprising:

contacting a sample containing said patient'"'"'s blood cells with a lysosomotropic amine and an antibody specific for HLA-DR;

quantitating the binding of said antibody to the monocytes in said sample;

wherein patients averaging fewer than 5000 anti-HLA-DR antibodies per monocytes are determined to be suitable for said therapy.

37. The method of claim 36, wherein patients averaging fewer than 3000 anti-HLA-DR antibodies per monocyte are determined to be suitable for said treatment.

38. The method of claim 37, wherein patients averaging fewer than 3000 anti-HLA-DR antibodies per monocyte over two consecutive days are determined to be suitable for said treatment.