RNase A Immunoassay With Switching Peptide for Wash-Free Quantification
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Solution Overview
Problem
Existing technologies lack effective inhibitors for RNase A and methods for quantitatively analyzing and capturing RNase A in samples.
Innovation Solution
Development of antibodies and peptides with specific amino acid sequences (SEQ ID NO: 5, 6, 7) that inhibit RNase A, and an immunoassay device using a substrate with a binding antibody, switching peptide, and quenching material for quantitative analysis, and a method involving beads with antibody-fixed surfaces for capturing RNase A.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional RNase A inhibitors (small molecules, nucleotide-based RIs) are used, then RNase activity can be inhibited, but the inhibition efficiency is insufficient and lacks specificity
Solution Approach 1:
The patent uses CDR3 regions from antibodies that naturally bind to RNase A as the basis for creating peptide inhibitors. By copying the critical binding interface (CDR3) from the antibody variable region, the invention creates simplified peptide sequences that retain high binding affinity and inhibition efficiency while being easier to manufacture and modify.
Solution Approach 2:
The patent extracts only the essential CDR3 region from the complete antibody structure to create the peptide inhibitor. This extraction isolates the most critical binding element, removing unnecessary complexity from the antibody molecule while preserving the core inhibitory function against RNase A.
2Measurement precision
If an immunoassay device with multiple components (substrate, binding antibody, switching peptide, fluorescent label, quenching material) is developed, then quantitative analysis capability is achieved, but device complexity increases
Solution Approach 1:
The patent combines multiple functional components into a single integrated immunoassay device structure. The substrate contains both the binding antibody and quenching material, while the switching peptide carries both the fluorescent label and the switching mechanism. This merging reduces the number of separate components and simplifies the overall device architecture while maintaining quantitative analysis capability.
Solution Approach 2:
The switching peptide serves multiple functions simultaneously: it acts as a fluorescent probe for detection, a competitor for RNase A binding, and a switching mechanism for signal modulation. This multi-functionality reduces the need for separate components and simplifies the device while achieving precise quantitative analysis.
3Measurement precision
If washing steps are included in the immunoassay protocol, then measurement accuracy is improved, but analysis time increases
Solution Approach 1:
The immunoassay system uses its own components to achieve separation and signal enhancement without external washing. The quenching material on the substrate automatically quenches fluorescence from unbound switching peptides, and the competitive binding mechanism naturally concentrates bound complexes. This self-service approach eliminates the need for time-consuming washing steps while maintaining measurement accuracy.
Solution Approach 2:
The patent replaces the mechanical washing process with a chemical/optical mechanism where quenching materials and competitive binding provide automatic separation and signal enhancement. This substitution eliminates the need for physical washing steps while achieving the same or better measurement precision.
4Productivity
If beads with antibody-fixed surfaces are used for RNase A capture, then capture efficiency is improved, but the complexity of capture methodology increases
Solution Approach 1:
The patent pre-fabricates beads with antibody-fixed surfaces, preparing the capture system in advance. This preliminary action allows the actual RNase A capture process to be simple and rapid, as the binding functionality is already established on the bead surfaces. The complex step of antibody immobilization is performed once during bead preparation, not during each capture operation.
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
The antibodies and peptides effectively inhibit RNase A, enabling quantitative analysis without washing and simple capture of RNase A from samples, applicable in RNA extraction, purification, PCR, cDNA synthesis, and DNA expression methods.
Implementation Method 1
an antibody or peptide that specifically binds to RNase A
Implementation Method 2
a quenching material arranged adjacent to the fluorescent label to quench the fluorescence from the fluorescent label
Implementation Method 3
a switching peptide comprising a peptide compound reversibly bound to the Fab region of the binding antibody
Data Source
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AI summary
The present invention relates to an antibody or peptide that specifically binds to RNase A. The invention can effectively inhibit RNase A and can therefore be usefully applied in various fields such as RNA extraction methods, RNA purification methods, PCR, cDNA synthesis methods, and DNA expression methods. An RNase detection immunoassay device is disclosed. The RNase detection immunoassay device includes a substrate; a binding antibody; a peptide compound and a switching peptide; and a quenching material, wherein the binding antibody specifically binds to RNase A and includes one selected from the group consisting of CDR3 comprising the amino acid sequence of SEQ ID NO: 5, CDR3 comprising the amino acid sequence of SEQ ID NO: 6, and CDR3 comprising the amino acid sequence of SEQ ID NO: 7.