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Kits for multiparametric phospho analysis

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Kits for multiparametric phospho analysis

As disclosed herein, the present invention provides for kits and a composition for diagnosis, prognosis, drug discovery, drug development, and patient stratification. The kits can comprise a plurality of binding elements for cell surface markers, and a plurality of binding elements for state-specific intracellular markers. The kits can further comprise a plurality of modulators directed for the particular cell function or signaling pathways. The kits can further include fixatives, permeabilizing agent, buffers, containers, instructions, and software for data analysis/compilation.

Browse recent Nodality, Inc. patents - South San Francisco, CA, US
Inventors: David Soper, David Rosen, Todd Covey, Ying-Wen Huang, Wenday Fantl, Ralph Lin
USPTO Applicaton #: #20120276558 - Class: 435 724 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay >Involving A Micro-organism Or Cell Membrane Bound Antigen Or Cell Membrane Bound Receptor Or Cell Membrane Bound Antibody Or Microbial Lysate >Animal Cell >Leukocyte (e.g., Lymphocyte, Granulocyte, Monocyte, Etc.)

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The Patent Description & Claims data below is from USPTO Patent Application 20120276558, Kits for multiparametric phospho analysis.

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This application is a continuation application which claims the benefit of U.S. application Ser. No. 12/730,170, filed Mar. 23, 2010; which claims the priority of U.S. Provisional App. Ser. No. 61/162,673, filed on Mar. 23, 2009 and U.S. Provisional App. Ser. 61/245,000, filed on Sep. 23, 2009 the disclosures of which are hereby incorporated by reference their entirety for all purposes.


Multiparametric analyses of cells provide an approach for the simultaneous determination of the activation states of a plurality of cellular components. The activation status of the plurality of cellular components can be measured after exposure of cells to extracellular modulators and in so doing allows the signaling capacity of signaling networks to be determined when compared to the activation status of those networks in the absence of such modulators. The induced activation status of a protein rather than the frequently measured basal phosphorylation state of a protein has been shown in several studies to be more informative, as it takes into account (and reveals) signaling deregulation that is the consequence of numerous cytogenetic, epigenetic and molecular changes characteristic of transformed cells. For example, multiparameter flow cytometry at the single cell level can measure the activation status of multiple intracellular signaling proteins and can assign activation states of these molecules to the varied cell sub-sets within complex primary cell populations.

However, usually multiparametric analyses of cells, e.g., multiparametric flow cytometry, require the use of multiple reagents at precise concentrations to produce robust and reproducible results. Since these data can be used as tools to inform clinical decisions, as well as therapeutic development, it would be beneficial to provide kits comprised of components relevant to a particular application with accompanying relevant usage information.

Protein phosphorylation is a critical post translational process in controlling many cell functions such as migration, apoptosis, proliferation and differentiation. Site specific phosphorylation of proteins can be detected, for example, by incubating cells with fluorochrome-conjugated phospho-specific antibodies using flow cytometry. However, only reagents whose parameters (including but not limited to, concentration, kinetics, fluorochrome to protein ratio) have been optimized can be used to generate robust and reproducible data that can be applied to a specific purpose. Kits comprising two or more reagents recognizing intracellular markers and/or extracellular markers along with an appropriate modulator or modulators to evoke a signaling response appropriate for the signal transduction pathway, specific cell type, disease state, or cellular function can save the end user from the tedious and often costly process of selecting, optimizing and standardizing reagents thereby providing the user with a more streamlined and cost-saving approach for profiling cellular networks in single cells.

It is therefore an objective of the present invention to provide kits that meet such demands.



The present invention involves the preparation of kits to be utilized in multi-parametric analyses (e.g. flow cytometry) on cell populations for the identification of the activation states of cellular signaling molecules (called nodes) in cells. Profiles of node states in cell populations are useful for diagnosis, prognosis, drug discovery, drug development, patient stratification (for example, who will and who will not respond to a drug) and other applications. Methods for determining cell populations and activation states have been disclosed in U.S. Pat. Nos. 7,381,535, 7,393,656, 7,563,584 and U.S. Ser. No. 61/120,320, which are hereby incorporated by reference in their entirety.

One embodiment of the present invention is a kit comprising a combination of binding element cell surface markers and state-specific intracellular markers. The kit can also comprise one or more modulators, therapeutic agents, fixatives, buffers, physical devices and software as described below.

In some embodiments, kits can be directed toward applications such as prediction of a response to a therapeutic agent, diagnosis and prognosis of various diseases or conditions, profiling signaling in specific cell types, analyzing the functional effects of genetic mutations, etc.

In some embodiments, kits can be prepared based on cell types of interest. For example, a kit can have a panel of antibodies that recognize extracellular markers specific to T cells, B cells, myeloid cells, stromal cells, neuronal cells or epithelial cells.

In some embodiments, the cell-type-specific kits can be supplemented with modulators for different signaling pathways. For example, the kit can include one or more cytokines and or growth factors that activate pathways including, but not limited to, JAK/STAT, PI3K/Akt, Ras/Raf/Erk, phosphatase signaling, metabolism, apoptosis, DNA damage response or transcriptional activation pathways. The kits can further comprise control cells, compounds and/or protocols.

In some embodiments, the invention involves kits for analyzing the effect of a compound on a cancer cell, comprising one or more binding elements that recognize particular surface markers expressed by cells in certain disease states. The kits can also comprise a compound used for treating a condition such as cancer. The kits can also comprise binding elements recognizing the activated state of signaling elements that can be activated in response to a compound, including but not limited to phosphorylated, acetylated, methylated or cleaved proteins.

In some other embodiments of the present invention, kits can additionally comprise consumable hardware, such as plates for holding the reagents or performing reactions, pipette tips, and software or files required to carry out the experiment. In some embodiments, the kit can further comprise a software package for data analysis of cell signaling profiles, which can include reference profiles for comparison with a test profile. The kit can also include software to manage or perform the experiment, including the use of the reagents and protocols for conducting appropriate reactions.

In some other embodiments, kits of the present invention enable the detection of activatable elements by sensitive cellular assay methods, such as immunohistochemistry and flow cytometry, which are suitable for clinical applications in detection, prognosis, and screening of cells and tissues from patients who have a disease involving aberrant signaling networks, for example leukemia.


The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 illustrates some embodiments of apoptosis pathway kits, comprising various intracellular markers involved in intrinsic and extrinsic apoptosis pathways.

FIG. 2 illustrates some embodiments of apoptosis pathway kits, comprising various intracellular markers involved in intrinsic and extrinsic apoptosis pathways.

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