Mutant PlGF sFlt-1 Calibrator Assay Binding
Solution Overview
Problem
The challenge lies in preparing reference, calibrator, or control compositions comprising both soluble forms of sFlt-1 and PlGF together, as they form a stable association complex, making it difficult to detect them individually in analytical assays.
Innovation Solution
Modifying one or more amino acids or non-amino acid groups of sFlt-1 and PlGF to reduce or eliminate their mutual binding, allowing for the preparation of compositions where these proteins can be used together in known amounts for calibrating assays, involving steps like contacting the modified proteins with specific receptors, separating bound and free labeled receptors, and detecting signals to determine protein amounts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native sFlt-1 and PlGF are used together in calibrator compositions, then the physiological relevance is maintained, but the proteins form stable association complexes that prevent individual detection and quantification
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues in PlGF (specifically residues involved in sFlt-1 binding) to alter the binding affinity parameter. This creates mutant PlGF variants that no longer form stable complexes with sFlt-1, enabling individual detection while maintaining the ability to study their interaction in separate assays
Solution Approach 2:
The patent segments the PlGF protein by creating mutant variants with specific amino acid modifications that disrupt the binding interface with sFlt-1. This segmentation of the binding function allows the proteins to coexist in calibrator compositions without forming interfering complexes
2Difficulty of detecting and measuring
If amino acid modifications are made to PlGF to reduce binding to sFlt-1, then individual detection becomes possible, but the mutant protein may have altered epitopes that affect assay recognition
Solution Approach 1:
The patent applies local quality by making targeted amino acid modifications specifically at the binding interface between PlGF and sFlt-1, while leaving other regions of the protein unchanged. This localized modification approach preserves epitopes recognized by antibodies in assays while disrupting the specific binding interaction
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
Enables the calibration and confirmation of sFlt-1 and PlGF levels in samples by reducing mutual binding, allowing for accurate detection and quantification, thereby improving the precision and accuracy of analytical assays for pre-eclampsia diagnosis.
Implementation Method 1
contacting a reference, calibrator or control composition comprising PlGF and sFlt-1 for use in an assay for sFlt-1 and/or PlGF with a receptor specific for sFlt-1 or a receptor specific for PlGF
Implementation Method 2
contacting complexes formed in step 1) with a labeled receptor specific for sFlt-1 and/or a labeled receptor specific for PlGF; and (3) separating bound labeled receptor specific for sFlt-1 from free labeled receptor specific for sFlt-1
Data Source
Figure 1A~1B
Figure 2
AI summary
Disclosed herein are compositions and methods comprising two or more proteins in which at least one of the proteins has been altered to reduce their mutual recognition and binding. Such compositions are useful as reference, calibrators or controls in methods and assays for determining the amount of one or more of the proteins that may be present in a sample of interest or in confirming the presence of one or more of the proteins in the sample. More particularly, it relates to compositions and methods comprising altered placental growth factor-1 (PlGF-1) and soluble fms-like tyrosine kinase (sFlt-1) and methods for determining the amount or confirming the presence of sFlt-1 and/or PlGF-1 in a sample of interest.