Artificial Antigen Enhancer System for Immunolabeling Signal Amplification
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Solution Overview
Problem
Current immunolabeling methods face challenges with unspecific binding and limited signal amplification, especially in multi-immunolabeling processes, due to antibody cross-binding and the presence of endogenous biotin, which restricts the number of primary antibodies that can be amplified and introduces background signals.
Innovation Solution
A signal enhancer system using artificially formulated peptide sequences as unique antigens not present in biological samples, allowing for an unlimited number of amplification steps without antibody cross-binding, enabling the use of multiple primary antibodies from different species by employing a chain of enhancer antigens and antibodies, with the last antibody in the chain labeled with a reporter molecule for detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the indirect method with secondary antibodies is used for signal amplification, then the signal is amplified, but unspecific binding occurs due to secondary antibodies binding to endogenous antibodies in the sample
Solution Approach 1:
The patent introduces an intermediary system consisting of artificial enhancer antigens and their specific antibodies. Instead of using secondary antibodies that may cross-bind to endogenous antibodies, the system uses uniquely designed artificial antigens (e.g., peptide sequences not present in nature) as intermediaries. Primary antibodies are conjugated to these artificial antigens, and then specific anti-artificial-antigen antibodies bind to them, providing signal amplification without cross-reactivity with sample components.
Solution Approach 2:
The patent fundamentally changes the parameter of the antigen used in immunolabeling from natural biological antigens to artificially formulated peptide sequences. These artificial antigens have unique properties (not present in biological samples) that allow for specific antibody binding without cross-reactivity. This parameter change enables unlimited amplification steps while avoiding background signals from endogenous biotin or antibodies.
2Reliability
If biotinylated secondary antibodies and labeled streptavidin are used for further signal amplification, then amplification is achieved, but unspecific binding occurs due to endogenous biotin in biological samples
Solution Approach 1:
The patent replaces the biotin-streptavidin intermediary system with an artificial antigen-antibody intermediary system. Artificial enhancer antigens (such as unique peptide sequences) are conjugated to primary antibodies, and specific antibodies against these artificial antigens are used for detection. This intermediary system eliminates cross-reactivity with endogenous biotin while providing the necessary signal amplification capability.
Solution Approach 2:
The patent changes the detection parameter from biotin-based detection to artificial antigen-based detection. By using artificially formulated peptides that do not exist in biological samples as the basis for amplification, the system avoids all background signals from endogenous biotin while maintaining the ability to perform multiple amplification steps.
3Adaptability or versatility
If multiple primary antibodies from different species are used for multi-immunolabeling, then detection of multiple targets is enabled, but antibody cross-binding occurs due to secondary antibodies recognizing the same Ig class
Solution Approach 1:
The patent introduces artificial enhancer antigens as unique intermediaries for each primary antibody, regardless of the primary antibody's species origin. Each primary antibody is conjugated to a specific artificial antigen (e.g., different peptide sequences), and then species-specific anti-artificial-antigen antibodies are used for detection. This eliminates cross-binding issues because the detection antibodies recognize the artificial antigens, not the primary antibody's species-specific Ig class.
Solution Approach 2:
The patent creates a universal detection system based on artificial antigens that can be used with primary antibodies from any species. The artificial antigen-antibody system serves as a universal intermediary that works regardless of the primary antibody's origin, enabling multi-immunolabeling of multiple targets from different species without cross-reactivity problems.
4Productivity
If the direct method with labeled primary antibodies is used, then the process is simple and rapid, but the signal is too weak for applications like microscopy
Solution Approach 1:
The patent implements a nested structure where primary antibodies are first conjugated to artificial enhancer antigens, then multiple layers of anti-artificial-antigen antibodies with reporter molecules are added. This nested arrangement of multiple antibody-antigen binding events around each primary antibody creates signal amplification while maintaining the simplicity of the direct method workflow.
Solution Approach 2:
The patent performs preliminary conjugation of primary antibodies to artificial enhancer antigens before the immunolabeling process. This preliminary action prepares the primary antibodies for subsequent amplification steps, allowing the actual labeling process to remain simple and rapid while achieving strong signals through the pre-prepared conjugates.
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 allows for robust signal amplification without background noise, enabling the simultaneous detection of multiple target antigens with distinct signals, overcoming the limitations of existing methods by preventing unspecific binding and allowing for an unlimited number of amplification steps in both single and multi-immunolabeling processes.
Implementation Method 1
a first enhancer antibody that is selected to solely bind to the first enhancer antigen
Implementation Method 2
a second enhancer antibody that is selected to solely bind to the second enhancer antigen
Data Source
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AI summary
The present invention generally relates to method for preparing a biological sample for use in an immunolabeling process. The invention also relates to corresponding kits for use in the immunolabeling process.