Thermostable Affinity Polypeptides for Interference-Resistant Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing affinity pairs in diagnostic tests, such as biotin-streptavidin and digoxigenin-anti-digoxigenin, are prone to interference due to increased biotin content in medical samples and the presence of dig-binding proteins, limiting their reliability.
Innovation Solution
Utilizing a novel affinity pair comprising polypeptides with specific amino acid sequences (SEQ ID NO:1 and SEQ ID NO:2 or sequences at least 85% identical) for robust analyte detection, avoiding interference by incorporating Colicin E9-derived polypeptides with modified residues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If biotin-streptavidin affinity pair is used for analyte detection, then the detection method is well-established and easy to implement, but interference occurs due to increased biotin content in medical samples
Solution Approach 1:
The patent changes the fundamental parameter of the affinity pair from biotin-streptavidin to a novel Colicin E9-derived polypeptide system. This parameter change eliminates interference from endogenous biotin while maintaining high affinity binding capabilities, thus resolving the contradiction between ease of implementation and detection reliability
Solution Approach 2:
The invention creates a copy of the successful biotin-streptavidin system using a different molecular basis (Colicin E9 polypeptides). This copying approach replicates the functional advantages of the original system while avoiding its specific vulnerability to biotin interference in clinical samples
2Reliability
If digoxigenin-anti-digoxigenin affinity pair is used for analyte detection, then the detection system is robust and widely used, but interference occurs due to presence of dig-binding proteins in samples
Solution Approach 1:
The patent changes the affinity pair parameter from digoxigenin-anti-digoxigenin to a novel Colicin E9 polypeptide system. This change eliminates interference from endogenous dig-binding proteins while maintaining robust detection capabilities, thus resolving the contradiction between detection robustness and sample interference
Solution Approach 2:
The invention introduces an intermediary affinity system (Colicin E9 polypeptides) that mediates between the detection requirements and sample complexity. This intermediary system provides high-specificity binding without cross-reactivity to endogenous interfering proteins
3Strength
If E. coli-derived Colicin/Immunity Protein affinity pair is used, then the affinity binding is strong with femtomolar binding, but the complexes are unstable with melting points below 45°C limiting biotechnological application
Solution Approach 1:
The patent changes the physical parameter of thermal stability by engineering Colicin E9-derived polypeptides with enhanced thermostability. The modified polypeptides maintain femtomolar binding affinity while achieving melting points above 60°C, thus resolving the contradiction between binding strength and complex stability
Solution Approach 2:
The invention creates a more stable, reusable affinity system that replaces the thermally unstable E. coli-derived complexes. The engineered polypeptides can withstand higher temperatures and more rigorous processing conditions, making them suitable for various biotechnological applications
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
Enhances the reliability and specificity of analyte detection by minimizing interference from biotin and dig-binding proteins, ensuring accurate diagnostic results.
Implementation Method 1
The affinity pair is based on protein-protein interactions and comprises a first partner and a second partner
Data Source
Figure 1~2
Figure 3~4
Figure 5~6
AI summary
The present invention relates to a method of determining an analyte in a sample, said method comprising (a) contacting said sample with (i) a binding compound binding to said analyte, said binding compound comprising a binding agent and a first partner of an affinity pair (first affinity partner); and (ii) a second partner of the affinity pair (second affinity partner) coupled to a solid surface, to an indicator, and/or to a second binding agent; and (b) determining said analyte based on complexes formed in step (a); wherein one of said first affinity partner and said second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO:l or a sequence at least 50% identical thereto, and wherein the other of said first affinity partner and said second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a sequence at least 50% identical thereto, the present invention also relates to a polypeptide comprising an amino acid sequence as specified in SEQ ID NO:l or a sequence at least 50% identical thereto, wherein the amino acid at the position corresponding to position 77 in SEQ ID NO: 1 is not a histidine; and to a polypeptide comprising an amino acid sequence as specified in SEQ ID NO:2 or a sequence at least 50% identical thereto, wherein (i) the amino acid at the position corresponding to position 17 in SEQ ID NO:2 is not a cysteine, in an embodiment is alanine, serine, leucine, isoleucine, or glycine and/or (ii) said polypeptide further comprises at least one functional peptide; and to fusion polypeptides, polypeptide complexes, polynucleotides and kit related to the aforesaid.