Simultaneous assay of target and target-drug binding

• US 8,828,671 B2
• Filed: 02/05/2013
• Issued: 09/09/2014
• Est. Priority Date: 05/24/2004
• Status: Active Grant

• Current Assignee: Laboratory Corporation Of America Holdings
• Original Assignee: Laboratory Corporation Of America Holdings
• Inventors: Purvis, Norman B., Stelzer, Gregory T.
• Primary Examiner(s): Gabel, Gail R
• Application Number:
  US13/759,158
• Publication Number:
  US 20130177926A1
• Time in Patent Office: 581 Days
• Field of Search: 435/7.2, 435/7.21, 435/7.23, 435/7.24, 435/40.51, 435/7.1, 435/7.94, 436/517, 436/522, 436/538, 436/10, 436/63, 436/64, 436/172, 436/175, 436/540, 436/546, 436/548, 424/9.1, 424/9.2
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2333/70514 CD4
• CPC Class Codes: G01N 2333/70553 Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• CPC Class Codes: G01N 2333/70596 Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
• CPC Class Codes: G01N 2333/7158 for chemokines
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]

Abstract

Whole cell, simultaneous target and drug-target assay using differentially labeled antibodies and flow cytometry. First antibody binds to total target and second antibody binds to the drug binding site of the target, thus drug binding will competitively inhibit the second antibody allowing for a competitive inhibition assay of drug-target binding. The assay allows for whole cell analysis and even analysis of mixed populations of cells, yet provides detailed kinetic assessment of drug activity.

Claims

1. A method of determining drug binding for a drug administered to a subject, comprising:

(a) isolating target-expressing cells from a biological sample obtained from a subject to whom a known amount of a drug has been administered, wherein the drug interacts with the target expressed by the target-expressing cells;

(b) incubating the target-expressing cells with an anti-Total antibody and an anti-Free-Site antibody, wherein the anti-Total antibody and the anti-Free-Site antibody are monoclonal antibodies that bind specifically to different epitopes of the same target;

(c) detecting the amount of the anti-Total antibody bound to the target-expressing cells of step (b), wherein the anti-Total antibody binds to an epitope on the target regardless of whether the target is also interacting with the drug;

(d) detecting the amount of the anti-Free-Site antibody bound to the target expressing cells of step (b), wherein the anti-Free-Site antibody recognizes and binds to an epitope on the target when the drug is not interacting with the target but is competitively inhibited by the interaction of the drug with the target,

wherein the amount of the anti-Total antibody detected in step (c) corresponds to the total amount of target expressed by the target-expressing cells and the amount of the anti-Free-Site antibody detected in step (d) corresponds to the total amount of drug-free target, and

wherein the amount of drug-bound target is proportional to the amount of the anti-Total antibody detected in step (c) less the amount of the anti-Free-Site antibody detected in step (d).

2. The method of claim 1, wherein step (c) and step (d) are performed simultaneously.

3. The method of claim 1, further comprising permeabilizing the target-expressing cells of step (a) prior to step (b).

4. The method of claim 1, further comprising fixing the target-expressing cells of step (b) prior to step (c).

5. The method of claim 1, wherein the anti-Free-Site antibody and the anti-Total antibody are differentially labeled.

6. The method of claim 1, wherein the anti-Free-Site antibody and the anti-Total antibody are each conjugated to a different dye or a different fluorescent label.

7. The method of claim 1, further comprising combining the isolated target-expressing cells of step (b) with one or more cell type-specific antibodies.

8. The method of claim 7, further comprising identifying one or more cell types in the biological sample by identifying cellular components in the biological sample bound to the one or more cell type-specific antibodies.

9. The method of claim 7, wherein the anti-Total antibody, the anti-Free-Site antibody and the one or more cell type-specific antibodies are differentially labeled.

10. The method of claim 7, wherein the anti-Total antibody, the anti-Free-Site antibody and the one or more cell type-specific antibodies are each conjugated to a different dye or a different fluorescent label.

11. The method of claim 1, wherein the anti-Total antibody comprises an anti-human CD11a antibody, an anti-human CD11b antibody, an anti-human CD18 antibody, an anti-human CD22 antibody, an anti-human CD25 antibody, an anti-human CD20 antibody, an anti-human CD52 antibody, an anti-human CD33 antibody, an anti-human CD4 antibody, an anti-human CD3 antibody, an anti-human CD14 antibody, an anti-human CD30 antibody, an anti-human human leukocyte antigen (HLA) antibody, an anti-human mitogen-activated protein (MAP) kinase antibody, an anti-human RAF-1 antibody, an anti-human AKT antibody, an anti-human B cell lymphoma 2 (BCL-2) antibody, an anti-human chemokine receptor antibody, or an anti-human estrogen receptor alpha (ESRA) antibody.

12. The method of claim 1, wherein the anti-Free-Site Antibody comprises an anti-human CD11a antibody, an anti-human CD11b antibody, an anti-human CD18 antibody, an anti-human CD22 antibody, an anti-human CD25 antibody, an anti-human CD20 antibody, an anti-human CD52 antibody, an anti-human CD33 antibody, an anti-human CD4 antibody, an anti-human CD3 antibody, an anti-human CD14 antibody, an anti-human CD30 antibody, an anti-human human leukocyte antigen (HLA) antibody, an anti-human mitogen-activated protein (MAP) kinase antibody, an anti-human RAF-1 antibody, an anti-human AKT antibody, an anti-human B cell lymphoma 2 (BCL-2) antibody, an anti-human chemokine receptor antibody, or an anti-human estrogen receptor alpha (ESRA) antibody.

13. The method of claim 1, wherein the biological sample comprises a biological fluid comprising.

14. The method of claim 1, wherein the biological sample comprises at least one of blood, plasma, or bone marrow.