Methods and reagents for quantitation of HLA-DR and CD11b expression on peripheral blood cells

• US 6,423,505 B1
• Filed: 09/24/1999
• Issued: 07/23/2002
• 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:
  US09/406,013
• Time in Patent Office: 1,033 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 2333/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: G01N 2333/70553 Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/56977 HLA or MHC typing
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives

Abstract

Improved methods, reagents, and kits for quantitation of HLA-DR and/or CD11b expression on peripheral blood cells are presented. Inclusion of a lysosomotropic amine, such as chloroquine, during staining stabilizes HLA-DR and CD11b expression. 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 CD11b expression on the surface of human blood cells, comprising:

contacting a sample containing human blood cells with a lysosomotropic amine and an anti-CD11b antibody; and

then

detecting the binding of said anti-CD11b antibody to said cells.

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-CD11b 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.