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Multiplexed immunosignal amplification using hybridization chain reaction-based method

a multi-assay, hybridization chain technology, applied in the field of antibody-based immunoassays, can solve the problems of reducing spatial resolution, difficult to use for simultaneous detection of multiple amplified signals, unsuitable for large-volume samples in several powerful new tissue expansion and clearing techniques, etc., to achieve substantial fluorescence recovery, suppress background levels, and eliminate the fluorescence of hcr amplifiers

Inactive Publication Date: 2021-07-01
GENANS BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a method for detecting biomolecules in cells using a technique called HCR (homing endonuclease-assisted recombination). The method involves using two types of initiators that can be directly conjugated to target biomolecules, allowing for simultaneous amplification of multiple targets. The initiators can be linked to the biomolecules using chemical linkers like amine-reactive or thiol-reactive linkers. The method also involves using grapheme oxide to absorb unassembled HCR amplifiers and enhance the sensitivity of the amplification process. The technical effects of the method include improved sensitivity and signal-to-noise ratio, as well as increased amplification factor compared to standard staining methods.

Problems solved by technology

A major limitation in the use of immunoassays is that the low abundance of a given target molecule in a sample often necessitates signal amplification before detection is possible.
Although very useful and widely-employed, current amplification methods have several drawbacks: they often generate high background, they can reduce spatial resolution due to dye diffusion, they are difficult to use for the simultaneous detection of multiple amplified signals (Carvajal-Hausdorf, D. E., Schalper, K. A., Neumeister, V. M. & Rimm, D. L. Lab. Invest. 95, 385-396 (2015)), and they are unsuitable for use with large-volume samples in several powerful new tissue expansion and clearing techniques.

Method used

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  • Multiplexed immunosignal amplification using hybridization chain reaction-based method
  • Multiplexed immunosignal amplification using hybridization chain reaction-based method

Examples

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Effect test

example 1

[0051]Multiplexed Labeling Using isHCR.

[0052]FIG. 1. (a) shows images of the dorsal striatum in mouse brain sections double immunostained for TH (green) and choline acetyltransferase (ChAT, red). The signals of each antigen were visualized sequentially using corresponding biotinylated secondary antibodies and isHCR. (b) HCR initiators were conjugated to GFP-nanobodies (LaG-16-2) using SMCC as the linker. mGFP proteins were expressed in the orbitofrontal cortex neurons in CaMKII-Cre transgenic mice using adeno-associated virus (AAV) vectors. The GFP signals in the superior colliculus were amplified using HCR initiator-conjugated GFP-nanobody and isHCR-546. (c) Schematic of rapid labeling using genetically encoded protein tags. Functionalized HCR initiators bind directly to protein tags and initiate the amplification process. AAV vectors that bear the Cre-dependent double-floxed inverse (DIO) open reading frame cassette containing genes encoding the SNAPf tag were constructed and pack...

example 2

[0053]Simultaneous Detection of Multiple Targets Using isHCR.

[0054]FIG. 2. (a) shows that two orthogonal HCR initiators can be conjugated directly onto secondary antibodies using either SMCC or Click Chemistry groups as linkers. (b) Western blot of a protein mixture containing purified hGBP1 and purified GFP-hGBP1 proteins. An anti-GFP primary antibody and an anti-hGBP1 primary antibody were applied. The two primary antibodies were detected using two secondary antibodies that were conjugated with different HCR initiators. (c) Images of HEK 293T cells immunostained against the nuclear protein Ki67 (red) and a mitochondria protein Tom20 (green) using two HCR initiator-conjugated secondary antibodies. The signals were then simultaneously amplified using isHCR-546 for Ki67 and isHCR-488 for Tom20. (d) Images of the dorsal striatum in mouse brain sections double immunostained against dopamine transporter (DAT, red) and neuronal nitric oxide synthase (nNOS, green) using two HCR initiator-...

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Abstract

The invention provides a method for optimizing isHCR for multiplexed labeling, which combines binder-biomolecule interactions with hybridization Chain Reaction (HCR).

Description

BACKGROUND[0001]Owing to their ease of use, speed, and cost effectiveness, antibody-based immunoassays remain the most popular methods for detecting and identifying the location of proteins and other biomolecules in biological samples. These methods use a primary antibody that binds selectively to a target molecule (antigen), and this antibody-antigen interaction can be visualized via a conjugated reporter or a labeled secondary antibody that can recognize and react with the primary antibody-epitope complex (Han, K. N., Li, C. A. & Seong, G. H. Annu. Rev. Anal. Chem. 6, 119-141 (2013)). A major limitation in the use of immunoassays is that the low abundance of a given target molecule in a sample often necessitates signal amplification before detection is possible. Amplification can be achieved using conjugated enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase, which catalyze the deposition of chromogenic substrates on target complexes (Bobrow, M. N., Harris, T. D...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/6804C12Q1/6832
CPCC12Q1/6804C12Q1/6832C12Q2563/131C12Q2565/601C12Q2600/16G01N33/582G01N33/6875
Inventor LIN, RUILUO, MINMIN
Owner GENANS BIOTECHNOLOGY CO LTD
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