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Enzyme activity measurements using bio-layer interferometry

A technology of enzyme activity and substrate, which is applied in the field of compositions for measuring enzyme activity, can solve the problems of increasing the time and cost of specific activity measurement

Inactive Publication Date: 2008-01-02
FORTEBIO
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Quantitative assays (eg, by enzyme-linked immunosorbent (ELISA)-based assays) also add time and expense to specific activity measurements and require additional samples

Method used

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  • Enzyme activity measurements using bio-layer interferometry
  • Enzyme activity measurements using bio-layer interferometry
  • Enzyme activity measurements using bio-layer interferometry

Examples

Experimental program
Comparison scheme
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example

[0047] The following are examples of specific embodiments used to practice the invention. These examples are given for illustrative purposes only and are not intended to limit the scope of the invention in any way. While every effort has been made to achieve accuracy with respect to numbers used (eg, amounts, temperature, etc.), some experimental errors and deviations should, of course, be allowed for. Procedures were performed at room temperature (typically 20 to 23 degrees Celsius) unless otherwise stated.

[0048] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained in detail in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers Company, recently added); Sambrook et al., ...

example 1

[0049] Example 1: BLI Molecular Weight Detection Features.

[0050] The lowest molecular weights of BLI-detectable binding molecules are shown in FIGS. 1 and 2 . In Figure 1 are presented data for a biotin-PEG conjugate with a molecular weight of 900 Daltons bound to a streptavidin-coated BLI sensor. BLI sensors and methods of coating them are described in detail in U.S. Nonprovisional Application Serial No. 10 / 981,901, filed November 4, 2004, and co-owned by Hong Tan et al., entitled "Fiber-Optic Assay Apparatus Based on Phase-Shift Interferometry," Attorney Docket No. 24377-09611US. Figure 2 demonstrates the binding of biotin (molecular weight about 230 Daltons) to a streptavidin-coated BLI sensor. These data show that the interferometry method readily detects the binding of molecules with a molecular weight of approximately 250 Daltons, and that molecules with molecular weights in the range of 500 to 1000 Daltons produce significant changes in optical layer thickness. ...

example 2

[0052] Example 2: Hydrolase activity measurement.

[0053] Hydrolases are enzymes that catalyze the cleavage of C-O, C-N, C-C or phosphate anhydride bonds.

[0054] Subgroup 1: Proteases (enzymes that act on peptide bonds)

[0055] fixed substrate format

[0056] This format features a protease substrate immobilized on the surface of a BLI fiberglass sensor using U.S. Nonprovisional Application No. 10 / 981,901, filed November 4, 2004 and co-owned by Hong Tan et al. (titled " Fiber-Optic Assay Apparatus Based on Phase-Shift Interferometry", Docket No. 24377-09611US (incorporated herein by reference)) and the methods set forth below. The fiber was immersed in an enzyme-containing sample and the change in optical layer thickness was monitored.

[0057] The basic assay protocol is to grow a biolayer interferometer (BLI) sensor on which an enzyme substrate has been immobilized in an enzyme-containing solution. Substrate depletion is quantified by, for example, optical phas...

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Abstract

Disclosed are enzyme assays using biolayer interferometry. Assays may be carried out using immobilized substrate or with a substrate capture format. In certain embodiments, the assays are carried out using unlabeled substrates.The methods are broadly applicable to enzyme assay measurements, can be carried out in vivo or in vitro, and are easily multiplexed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the rights of U.S. Provisional Application No. 60 / 645,153, filed January 19, 2005, and U.S. Provisional Application No. 60 / 642,454, filed January 7, 2005, both of which, for various purposes, Both applications are incorporated herein by reference in their entirety. [0003] Statement Regarding Federally Sponsored Research or Development [0004] Not applicable. technical field [0005] The present invention relates to methods based on interferometry and compositions for measuring enzyme activity. Background technique [0006] Enzymes are a large class of proteins that catalyze biochemical reactions and have many therapeutic and industrial applications. Enzyme activity is often measured during the development and manufacture of enzyme products. Simple methods of enzymatic activity (preferably unlabeled to avoid interference with enzyme / substrate interactions) will be widely used. During th...

Claims

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Application Information

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IPC IPC(8): C12Q1/00C12Q1/34C12Q1/42C12Q1/48H01L21/20
Inventor 罗伯特·朱克朱赛马维磊克丽斯塔·威特
Owner FORTEBIO
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