Methods and systems for detecting and/or sorting targets

A target and sample detection technology, which is applied in the field of detection and/or sorting targets and systems, can solve problems such as sampling errors, and achieve the effects of improving sensitivity and minimizing protein denaturation

Inactive Publication Date: 2009-09-02
CALIFORNIA INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The heterogeneity of such biopsy samples can lead to significant sampling errors as multiple measurements of cells, mRNA, and protein are each made in different regions of the tissue

Method used

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  • Methods and systems for detecting and/or sorting targets
  • Methods and systems for detecting and/or sorting targets
  • Methods and systems for detecting and/or sorting targets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1: Production of Antibodies Encoded by Polynucleotides

[0155] according to figure 1 The two-step strategy shown produces antibodies encoded in DNA. Specifically, aldehyde functional groups were introduced into 5-aminated oligonucleotides via succinimide chemistry using commercially available reagents ( figure 1 Figure a). Similarly, the hydrazide moiety was introduced by reaction with the lysine side chain in the corresponding antibody ( figure 1 Figure a). DNA-antibody conjugate formation is then facilitated by stoichiometric hydrazone bond formation between the aldehyde and hydrazide functional groups. Verify conjugate formation by PAGE electrophoresis and control for DNA loading ( figure 1 Figure b).

[0156] For these experiments polyclonal goat anti-human IgG labeled with AlexaFluor 488, 594 and 647 were purchased from Invitrogen. Monoclonal rabbit anti-human interleukin-4 (clone 8D4-8), non-fluorescent and APC-labeled rabbit anti-human TNF-α (clon...

Embodiment 2

[0165] Example 2: Production of polynucleotide-encoded streptavidin

[0166] Production of DNA-encoded streptavidin was performed according to the same route described in Example 1 for the production of DNA-encoded antibodies. The only difference is that the SANH:streptavidin ratio was kept constant at 100:1.

Embodiment 3

[0167] Example 3: Optimizing the polynucleotide load of a polynucleotide-encoded antibody

[0168] Unfavorable steric effects when labeling antibodies with oligonucleotides are considerations when performing various assays, such as the immunoassays and cell sorting / capture experiments described herein. To this end, the ability of DNA-encoded antibodies to retain cell surface markers, visualized by fluorescence-activated cell sorting (FACS), was investigated. DNA loading was optimized using FACS for polynucleotide-encoded conjugates by using a fluorophore covalently labeled on the DNA instead of the antibody. For this analysis, 5′-aminated, 3′-FITC-labeled DNA was labeled with different stoichiometric ratios of SANH to antibody (5:1, 25:1, 50:1, 100:1, 300:1) in Anti-CD90.2 antibody on. This produced on average conjugates with 1, 2, 3, 4-5 and 6-7 FITC-DNA strands, respectively, as measured by gel mobility assay, see figure 1 Figure d. The ability of these conjugates to bin...

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Abstract

Provided herein are methods and systems for detecting and / or sorting targets in a sample based on the combined use of polynucleotide-encoded protein and- substrate polynucleotides. The polyhucleotide-encoded protein is comprised of a protein that specifically binds to a predetermined target and of an encoding polynucleotide that specifically binds to a substrate polynucleotide, wherein the substrate polynucleotide is attached to a substrate.

Description

[0001] Cross reference with related applications [0002] This application claims priority from U.S. Provisional Application Docket No. CIT-4707, Serial No. 60 / 834,823, filed August 2, 2006, entitled "A unified Platform for Multiplexed Cell Sorting and Detection of Genes and Proteins," and 2007 US Provisional Application, Docket No. CIT-4944, Serial No. 60 / 959,665, filed July 16, 1999, entitled "DigitalDEAL: A quantitative and digital Protein Detection Immunoassay," the disclosure of which is incorporated herein by reference in its entirety. [0003] Statement of Government Funding [0004] The United States Government has certain rights in this disclosure pursuant to Grant No. CA119347 awarded to the Frederick National Cancer Institute and Grant No. DAAD19-03-D-0004 / 0008 awarded to ARO-U.S. Army Robert Morris Acquisition Center and Grant No. 5U54CA 119347 . technical field [0005] The present disclosure relates to the detection of one or more targets, especially biomarkers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 盖布·克翁瑞安·贝利樊荣詹姆斯·R·希思
Owner CALIFORNIA INST OF TECH
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