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Proximity-mediated assays for detecting oncogenic fusion proteins

A fusion protein, protein technology, applied in biological testing, material testing products, drug combinations, etc., can solve problems such as lack of specificity and sensitivity

Inactive Publication Date: 2012-09-12
SOC DES PROD NESTLE SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods lack the specificity and sensitivity required to determine the presence or level of BCR-ABL activity in a sample because they are single antibody assays that rely on the detection of surrogate protein phosphorylation

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  • Proximity-mediated assays for detecting oncogenic fusion proteins
  • Proximity-mediated assays for detecting oncogenic fusion proteins
  • Proximity-mediated assays for detecting oncogenic fusion proteins

Examples

Experimental program
Comparison scheme
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specific Embodiment approach

[0103] The present invention provides antibody-based arrays for detecting the activation state and / or total amount of one or more oncogenic fusion proteins and / or signal transduction molecules in a biological sample, such as a cell extract or lysate. The invention also provides methods of using the arrays to aid in cancer prognosis and diagnosis, prediction or identification of resistance to drug therapy, and design of personalized targeted therapy. In a specific embodiment, the compositions and methods of the invention preferably identify patients who are resistant to treatment with a tyrosine kinase inhibitor, such as imatinib, due to Mutation, non-compliance with treatment regimen, and / or administration of suboptimal drug doses.

[0104] In a specific embodiment, the present invention provides assays such as immunoassays for real-time detection of expression levels and / or Degree of activation (eg, phosphorylation). Accordingly, the present invention preferably provides be...

Embodiment 1

[0475] Example 1. Detection of single cells by a proximity dual detection microarray ELISA with tyramide signal amplification.

[0476] This example describes a multiplex high-throughput proximity dual-detection microarray sandwich ELISA with superior dynamic range, suitable for analyzing the activation status of fusion proteins and signal transduction molecules in cell extracts prepared from isolated cells. In a specific embodiment, said proximity assay is in the form of an addressable microarray.

[0477] 1) Spot-blot serial dilutions of the capture antibody from 1 mg / ml to 0.004 mg / ml on a 16-well FAST glass slide (Watchman). Alternatively a 2-fold serial dilution of each capture antibody (0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml) can be used, and double or quadruple spots can be used for each antibody dilution. For detection of BCR-ABL fusion proteins, exemplary capture antibodies include anti-BCR monoclonal or polyclonal antibodies.

[0478] 2) Dry overnight and block ...

Embodiment 2

[0486] Example 2. Generation of activation profiles for drug selection.

[0487] Drugs for cancer therapy can be selected using the compositions and methods of the invention. As a non-limiting example, the present invention is used to identify the presence and / or activity of one or more oncogenic fusion proteins associated with hematological malignancies in order to provide appropriate treatment for patients with such cancers. A typical protocol entails generating two distributions, a reference activation profile and a test activation profile, and then comparing the two distributions to determine the effect of a particular drug regimen (see, figure 2 ).

[0488] Reference activation distribution

[0489] To generate a reference activation profile, a blood sample is obtained from a patient with a particular type of cancer (eg, leukemia such as CML) prior to treatment with an anticancer drug. Tumor cells in a blood sample can be isolated using, for example, any of the tech...

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Abstract

The present invention provides antibody-based arrays for detecting the activation state and / or total amount of one or a plurality of oncogenic fusion proteins in a biological sample such as whole blood or tumor tissue and methods of use thereof. In certain instances, the activation state and / or total amount of oncogenic fusion protein(s) present in a sample can be measured in combination with one or a plurality of signal transduction molecules. The compositions and methods of the present invention have the advantages of specificity associated with enzyme-linked immunosorbent assays, sensitivity associated with signal amplification, and high-throughput multiplexing associated with microarrays.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 61 / 253,393, filed October 20, 2009, U.S. Provisional Application No. 61 / 305,084, filed February 16, 2010, U.S. Provisional Application No. 61 / 327,487, filed April 23, 2010 and priority to U.S. Provisional Application No. 61 / 383,037, filed September 15, 2010, which is hereby incorporated by reference in its entirety for all purposes. Background of the invention [0003] Fusion proteins, also known as chimeric proteins, are proteins produced by linking two or more genes that originally coded for separate proteins. Translation of the fusion gene produces a single polypeptide with functional properties derived from the respective original proteins. A chimeric mutant protein occurs when a large-scale mutation (usually a chromosomal translocation) produces a new coding sequence that contains parts of the coding sequence of two different genes. Naturally occurring fusio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50
CPCG01N33/5011G01N33/574G01N33/57426G01N33/5748G01N2800/52G01N2800/56A61P35/02G01N33/68
Inventor S·辛格X·刘
Owner SOC DES PROD NESTLE SA