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Improved methods for conducting multiplexed assays

A technology for detection reagents and detection labels, applied in laboratory containers, chemical instruments and methods, biochemical equipment and methods, etc., to achieve the effect of improving productivity and flexibility

Active Publication Date: 2016-03-02
MESO SCALE TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]Commercially available assays are generally supplied in pre-set configurations, offering little or no flexibility for the user to evaluate targets that may be of unique value to him or her flexibility

Method used

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  • Improved methods for conducting multiplexed assays

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0268] Example 1 - Direct Assay Format

[0269] The procedure for the preparation and use of multiwell plates for direct assays is shown in Figure 2. The experimental layout, i.e. which oligonucleotide sequence is located in which binding domain of the multiwell assay plate, is annotated. Multiwell assay plates were obtained from MesoScale Discovery, a division of MesoScale Diagnostics, LLC (Rockville, MD). A working solution (550 uL) of each individual oligonucleotide sequence complement was prepared by diluting the stock solution of the sequence complement approximately 50-fold in Diluent100 (the stock solution of the oligonucleotide sequence complement and Diluent100 are available from MesoScaleDiscovery).

[0270] Using a bifunctional coupling reagent (4-( N -maleimidomethyl)-1-sulfosuccinimidyl cyclohexanecarboxylate ("SMCC")) and the conventional coupling scheme shown in Figure 5(d), using an oligo with a terminal thiol group Nucleotide labeling of each capture ant...

Embodiment 2

[0273] Example 2—Indirect Assay Format Using Oligonucleotide-SA Conjugates

[0274]The procedure for the preparation and use of multiwell plates for indirect assays using oligonucleotide-SA conjugates is shown in Figures 9(a)-(c). As in Example 1, annotate the experimental layout for the multi-well assay plate. A working solution (550 uL) of each individual oligonucleotide sequence complement conjugated to streptavidin (SA) was prepared by diluting the stock solution of the sequence complement approximately 50-fold in Diluent 100 (oligonucleotide Stock solutions of sequence complements and Diluent100 are available from MesoScale Discovery). A solution of biotinylated antibody was added to the working solution of the complement of the desired oligonucleotide sequence to prepare a set of each biotinylated capture antibody / oligonucleotide-SA mixture. The concentration of biotinylated antibody in the mixture ranged from about 5-30 ug / mL. The mixture was mixed gently for 30-45...

Embodiment 3

[0277] Example 3 - Indirect assay format using neat SA / biotinylated oligonucleotides

[0278] The procedure for the preparation and use of multiwell plates for indirect assays using biotinylated capture antibodies, neat streptavidin, and biotinylated oligonucleotides is shown in Figure 10(a)-(c). As in Example 1, annotate the experimental layout of the multiwell assay plate. A working excess (550 uL) of each individual oligonucleotide sequence complement conjugated to streptavidin (SA) was prepared by diluting the stock solution of the sequence complement approximately 50-fold in Diluent 100 (oligonucleotide Stock solutions of nucleotide sequence complements and Diluent100 are available from MesoScale Discovery). A solution of biotinylated antibody was added to the working solution of the complement of the desired oligonucleotide sequence to prepare a set of each biotinylated capture antibody / oligonucleotide-SA mixture. The concentration of biotinylated antibody in the mix...

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Abstract

The invention relates to methods for conducting solid-phase binding assays. One example is an assay method having improved analyte specificity where specificity is limited by the presence of non-specific binding interactions.

Description

[0001] Cross References to Related Applications [0002] Reference is made to US Provisional Application Serial Nos. 61 / 775,860, filed March 11, 2013, and 61 / 778,727, filed March 13, 2013, respectively, the disclosures of each are hereby incorporated by reference in their entirety. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with Federal Government support under 5RCA130391-04 awarded by the National Cancer Institute. The US Government has certain rights in this invention. field of invention [0005] Improved methods and products for performing binding assays are provided. These methods include the use of linker complexes to enable the user to configure the assay to suit his / her needs with a standard set of assay materials. The products and methods of the invention greatly increase productivity and flexibility in assay development. Background of the invention [0006] A large body of literature develops techniques for sensitive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/483C12N15/11C12Q1/68
CPCG01N33/54353C12Q1/6816G01N33/54306C12Q2563/131G01N33/532G01N2470/04C12Q1/6876B01L3/50853B01L2300/021B01L2300/043B01L2300/0609B01L2300/0829C12Q1/6804G01N33/6845G01N2458/10
Inventor E.N.格勒策尔S.库马P.奥贝罗瓦G.西加尔M.齐安斯基
Owner MESO SCALE TECH LLC