Multiplexed immunosignal amplification using hybridization chain reaction-based method

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...

AI Technical Summary

Benefits of technology

[0015]The present method may also comprise using grapheme oxide (GO) to absorb unassembled HCR amplifiers. If the amplifiers are terminally modified and / or internally modified with fluorescent dye, grapheme oxide (GO) may also quench the fluorescence. Therefore, the kit of the present invention may also comprise grapheme oxide. Graphene Oxide in the present invention has a particle size of <500 nm. Crucially, in addition to abolishing the fluorescence of HCR amplifiers, the addition of HCR initiators along with HCR amplifiers and GO resulted in substantial recovery of fluorescence, likely because the initiators triggered the formation of double-strand-nicked polymers of HCR amplifiers, thereby protecting them from the adsorption activity of GO. That is, GO can be used to suppress background levels, further enhancing the performance of isHCR.

[0016]The addition of GO reduced the background but did not diminish the signal intensity, resulting in an improved signal-to-noise ratio as compared to isHCR amplification without GO. Surprisingly, further analysis using antibody serial dilution experiments showed that isHCR with GO significantly increased signal intensity as compared to a standard IHC staining method, achieving a greater than 80× amplification factor when the primary antibody was highly diluted.

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.

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