Pan-Kinase Activation and Evaluation of Signaling Pathways

Abstract

Methods and reagents are provided for determining the activation state of a signal transduction pathway signaling protein. There exists a need in the art for methods that can monitor the efficacy of a signal transduction inhibitor in a patient. Other needs exist for detecting and monitoring certain disease or disorders that are associated with aberrant activation of a signal transduction pathway signaling protein. The present assay provides a highly sensitive assay that is also useful in patient populations in which obtaining a large cellular sample is difficult, for example, neonates.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application 61 / 094,304, filed Sep. 4, 2008, which is incorporated herein in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]Certain embodiments of the invention relate to methods for determining the activation state of a signal transduction pathway signaling protein. In at least some embodiments, methods are provided for monitoring the efficacy of a signal transduction inhibitor in a patient. The present invention further provides highly sensitive assays useful in patient populations in which obtaining a large cellular sample is difficult, for example, neonates. Certain embodiments of the invention relate to methods of identifying novel signal transduction pathway protein inhibitors. Certain embodiments of the invention relate to methods for detecting sepsis in a patient sample. Kits for practicing the methods of the invention are also provided.[0004]2. B...

AI Technical Summary

Benefits of technology

[0035]A significant advantage of the present invention is that the increased specificity and sensitivity allows for use of the methods with very small sample sizes. This is important where only a small patient sample is obtainable, for example, with neonates.

[0036]It should be noted that as used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural references unless the context clearly dictates otherwise.

[0037]Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the invention, the particular methods, devices, and materials are now described.Preparation of the Test Sample

[0038]The terms test sample and sample are used interchangeably herein. The test sample in the methods of the present invention can include any leukocyte-containing sample, for example, whole blood, blood plasma, bone marrow aspirates (or any cells obtained from bone marrow), urine, serum, saliva, cerebral spinal fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, body tissue extracts or cellular extracts. In certain embodiments, the sample is a blood sample. In a preferred embodiment, the blood sample is whole blood. The whole blood can be obtained from the individual or test subject using standard clinical procedures.

[0039]Obtaining a blood sample encompasses obtaining the blood sample directly from the patient, for example by a phlebotomist. Obtaining a blood sample also encompasses obtaining a blood sample that was previously obtained from a patient, for example a laboratory technician obtaining a patient's blood sample for analysis using the methods of the present invention. Plasma can be obtained from whole blood samples by centrifugation of anti-coagulated blood. Such a process provides a bottom layer of packed red blood cells, an intermediate buffy coat layer and a supernatant plasma layer.

[0040]Leukocytes can be isolated from whole blood samples by any of various techniques including buoyant density centrifugation and others known in the art. The leukocytes, i.e., white blood cells, comprise monocytes, lymphocytes, neutrophils, basophils, and eosinophils. Monocytes are especially preferred for use in the methods of the present invention.

Problems solved by technology

Current assays rely on immunoblotting techniques that cannot identify the actual cell-type generating the response in a mixed cell population.

Also, by having to remove the drug from the samples, these assays are cumbersome and are subject to a lack of sensitivity.

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