Cyclic Antibody Staining for Fluorescence Signal Amplification
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Solution Overview
Problem
Current bioimaging technologies face limitations in fluorescence signal amplification, including low signal-to-noise ratio, limited multicolor imaging, low spatial resolution, and complexity, which hinder high-throughput imaging and require long scan times for 3-D large-volume imaging.
Innovation Solution
The method involves cyclic staining of target molecules using fluorophore-conjugated complementary antibodies and the removal of cross-reactions between orthogonal antibodies using an agarose gel, allowing for amplified fluorescence signals and multicolor imaging without additional equipment, enabling high image processing throughput.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If conventional fluorescence imaging methods are used, then imaging can be performed with simple procedures, but the fluorescence signal intensity is insufficient requiring long scan times
Solution Approach 1:
The patent implements nested antibody structures where secondary antibodies bind to primary antibodies, and tertiary antibodies bind to secondary antibodies, creating a nested arrangement that amplifies the fluorescence signal. This nested doll approach allows multiple antibody layers to be stacked around the target antigen, significantly increasing signal intensity and reducing scan time.
Solution Approach 2:
The patent employs cyclic staining procedures where antibody-antigen binding and fluorescence detection are performed in repeated cycles. Each cycle involves incubating with fluorophore-conjugated antibodies, washing, and imaging, then repeating with additional antibody layers. This periodic action progressively builds signal amplification while managing background noise through intermediate washing steps.
2Adaptability or versatility
If multiple biomarkers are imaged simultaneously, then comprehensive spatiotemporal distribution can be obtained, but cross-reactions between antibodies occur reducing measurement precision
Solution Approach 1:
The patent introduces species-specific antibodies as intermediaries to prevent cross-reactions. By using antibodies from different host species (e.g., mouse, rabbit, goat) in a hierarchical binding sequence, the system ensures that each antibody layer binds specifically to its target without cross-reacting with other antibody layers. This intermediary approach enables multiplexed imaging while maintaining measurement precision.
Solution Approach 2:
The patent segments the multicolor imaging process into distinct sequential steps, where different fluorophore-conjugated antibody pairs are applied in a specific order. Each imaging cycle focuses on one or two biomarkers before proceeding to the next, allowing cross-reactivity to be managed through temporal and procedural segmentation rather than attempting simultaneous detection of all markers.
3Area of stationary object
If 3-D large-volume imaging is performed, then comprehensive tissue mapping is achieved, but the high number of image acquisitions requires excessively long total imaging time
Solution Approach 1:
The patent implements periodic signal amplification through cyclic application of fluorophore-conjugated antibodies before each imaging session. By repeating the staining process multiple times with increasing signal intensity, the system accumulates sufficient fluorescence signal to reduce the number of image acquisitions needed for 3-D reconstruction, thereby reducing total imaging time while maintaining comprehensive tissue mapping.
4Illumination intensity
If fluorescence signal is amplified through multiple antibody layers, then signal intensity increases, but background signals and cross-reactions increase reducing signal-to-noise ratio
Solution Approach 1:
The patent extracts and removes background signals through intermediate washing steps between each antibody incubation cycle. By thoroughly washing away unbound and non-specifically bound antibodies before adding the next layer, the system eliminates background noise that would otherwise accumulate with each amplification cycle. This extraction approach maintains high signal-to-noise ratio despite multiple staining cycles.
Solution Approach 2:
The patent uses species-specific antibodies as intermediaries to prevent cross-reactions between different antibody layers. By ensuring that each antibody layer recognizes a unique epitope or is from a different host species, the system prevents non-specific binding and cross-reactivity that would generate background signals, thereby maintaining clean fluorescence imaging even with multiple amplification layers.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly reduces imaging time by enhancing fluorescence signal intensity, allowing for high-resolution, high-throughput 3-D imaging and simultaneous multicolor fluorescence imaging, while minimizing background signals in tissue samples.
Implementation Method 1
fluorophore-conjugated complementary antibodies
Implementation Method 2
binding the primary antibody to the target protein, and binding the antibody pairs to the primary antibody
Implementation Method 3
removal of a cross reaction between antibodies using an agarose gel
Data Source
AI summary
Various embodiments can provide a method of amplifying a fluorescence signal through the cyclic staining of complementary antibodies conjugated with fluorophores. According to various embodiments, the method of amplifying a fluorescence signal may be configured to prepare a primary antibody and antibody pairs with respect to each target protein, bind the primary antibody to the target protein, and bind the antibody pairs to the primary antibody. The multicolor fluorescence signals may be amplified and fabricated by forming fluorescence signals in a way to prepare different antibodies for which cross reactions with different types of target proteins, respectively, have been removed using an agarose gel and to bind different antibody pairs according to combinations of different antibodies to different primary antibodies bound to target proteins, respectively.