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

• US 6,200,766 B1
• Filed: 12/03/1998
• Issued: 03/13/2001
• 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:
  US09/204,860
• Time in Patent Office: 831 Days
• Field of Search: 435/7.24, 435/975
• 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 composition for flow cytometric measurement of HLA-DR on human peripheral blood cells, comprising:

a fluorophore-conjugated anti-HLA-DR antibody, and

a lysosomotropic amine.

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

3. The composition of claim 2, wherein said lysosomotropic amine is chloroquine.

4. The composition of claim 2, wherein said lysosomotropic amine is hydroxychloroquine.

5. The composition of claim 1, wherein said fluorophore is PE.

6. The composition of claim 5, wherein said PE fluorophore and said antibody are conjugated at a defined molar ratio.

7. The composition of claim 6, wherein said ratio is 1:

1.

8. A kit for flow cytometric measurement of HLA-DR on the surface of peripheral blood cells, comprising:

a composition according to claim 1; and

an erythrocyte lysing composition.

9. The kit according to claim 8, further comprising:

pelletized beads conjugated with defined levels of PE.