Cell-Free Neutralizing Antibody Quantification Using Stabilized Prefusion Spike Proteins
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Solution Overview
Problem
Current methods for quantifying neutralizing antibodies against SARS-CoV-2 are technically demanding, time-consuming, and unreliable due to the need for biosafety level facilities and low infectivity of viral pseudotypes, leading to inaccurate estimations and challenges with emerging virus variants.
Innovation Solution
A cell-free method using prefusion viral proteins stabilized in a reactive conformation coupled to solid supports, combined with cell receptor proteins or competing antibodies, to detect and quantify neutralizing antibodies in biological samples, allowing for accurate measurement of neutralizing activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cell-based neutralization assays are used to detect SARS-CoV-2 neutralizing antibodies, then the ability to assess viral infectivity blocking is improved, but the technical complexity and biosafety requirements increase significantly
Solution Approach 1:
The invention extracts the essential functional component (prefusion spike protein) from the complete virus particle and presents it in a stabilized conformation on solid supports. This extraction eliminates the need for complex cell cultures and biosafety facilities while retaining the ability to detect neutralizing antibodies that block the critical virus-receptor interaction.
Solution Approach 2:
The invention creates a simplified copy of the viral entry mechanism by using stabilized prefusion spike proteins coupled to solid supports instead of live virus particles. This copy maintains the essential binding interface with ACE2 receptors while removing the complexities and biosafety hazards associated with live virus handling.
2Object-affected harmful factors
If viral pseudotypes are used instead of wild-type virus, then biosafety requirements are reduced, but the infectivity and reliability of neutralization assays decrease
Solution Approach 1:
The invention uses stable, non-infectious prefusion spike protein structures that can be produced and stored without the complex requirements of live virus or pseudotype systems. These stabilized proteins serve as reliable, repeatable assay targets that eliminate the infectivity and biosafety issues while maintaining assay functionality.
Solution Approach 2:
The invention changes the physical and structural parameters of the viral protein by stabilizing the prefusion conformation through specific mutations and chemical modifications. This stabilization preserves the ACE2 binding capability while eliminating the dynamic, infectious properties of live virus and pseudotype systems.
3Measurement precision
If live virus assays are used for neutralizing antibody quantification, then measurement accuracy is improved, but the time required for assay completion and result readout increases
Solution Approach 1:
The invention performs preliminary stabilization of the spike protein in its prefusion conformation and pre-coupling to solid supports before the actual neutralization assay. This preliminary preparation eliminates the need for time-consuming cell infection and incubation steps, enabling rapid readout while maintaining measurement accuracy.
Solution Approach 2:
The invention replaces the complex biological mechanical system of live virus infection and cell-based readout with a simplified protein-protein binding assay on solid supports. This substitution maintains the ability to measure neutralizing antibody activity while dramatically reducing assay time and complexity.
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 method provides a high-throughput, accurate, and reliable quantification of neutralizing antibodies, matching the performance of gold standard live virus assays and enabling assessment of neutralizing activities against multiple SARS-CoV-2 variants, with 96.7% sensitivity and 100% specificity.
Implementation Method 1
contacting the mixture with one or more cell receptor proteins that bind to the at least one prefusion viral protein(s) or one or more competing antibodies that bind to the at least one prefusion viral protein(s)
Data Source
AI summary
The invention provides an in vitro method for the cell-free quantification of virus neutralizing activity, such as the SARS-CoV-2 neutralizing activity, of biological samples and kit for carrying out the method. The invention also provides a method of screening biological samples or collections of molecules to identify neutralizing antibodies or antiviral drugs.