Bispecific Antibody SPR Assay Multimer Interference
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Solution Overview
Problem
Existing assays for determining the biological activity of multivalent, multispecific antibodies, such as bispecific antibodies, are sensitive to functional multimers due to avidity-based binding, leading to inaccurate results.
Innovation Solution
The use of a binding site with a smaller dissociation constant for immobilization on a solid surface and determining the bridging binding signal with a site having a higher dissociation constant, employing techniques like surface plasmon resonance or ELISA, to reduce interference from multimeric forms and accurately assess biological activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a general assay setup is used for determining biological activity of multivalent multispecific antibodies, then the assay can detect binding signals, but it becomes sensitive to functional multimers due to avidity-based binding, leading to inaccurate results
Solution Approach 1:
The patent applies local quality by differentiating between the two binding sites of the multispecific antibody and assigning them different functional roles based on their specific properties. The site with higher affinity (lower Kd) is designated for immobilization while the site with lower affinity (higher Kd) is used for detection, optimizing each site's contribution to the assay outcome and eliminating multimer interference.
Solution Approach 2:
The patent changes the parameter of affinity matching between capture and detection reagents. By selecting a capture antigen with affinity that matches the higher-affinity binding site and a detection antigen with affinity matching the lower-affinity binding site, the assay parameters are optimized to distinguish monomers from multimers, thereby improving measurement accuracy.
2Reliability
If the binding site with smaller dissociation constant is used for immobilization, then the assay robustness is improved, but the assay setup becomes more specific and complex
Solution Approach 1:
The patent applies preliminary action by pre-characterizing the affinity parameters (Kd values) of both binding sites before setting up the assay. This preliminary information is then used to rationally select which binding site should be used for immobilization and which for detection, eliminating the need for empirical optimization and reducing overall assay complexity.
Solution Approach 2:
The patent introduces affinity matching as an intermediary principle that mediates between the antibody's dual binding sites and the assay components. By selecting capture and detection antigens with affinities that match the respective binding sites, the system achieves robustness without excessive complexity, as the affinity matching rule provides a clear selection criterion.
3Quantity of substance
If functional multimers are present in the sample, then the quantity of antibody is increased, but the measurement precision deteriorates due to avidity-based binding
Solution Approach 1:
The patent converts the harmful effect of multimer presence into a beneficial discrimination mechanism. By using asymmetric affinity matching, the assay design causes multimers to bind preferentially to capture antigens while monomers can bind both capture and detection antigens. This transforms the multimer interference problem into a useful discrimination tool that enhances measurement precision.
Solution Approach 2:
The patent inverts the conventional approach by not trying to eliminate multimers but rather designing the assay to exploit their different binding behavior. Instead of assuming all bound antibody is functional monomer, the assay recognizes that multimers will show asymmetric binding patterns and uses this to distinguish them from functional monomers, thereby improving precision.
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 enhances the specificity and robustness of the assay by minimizing the impact of functional multimers, providing a more accurate measurement of biological activity by focusing on the interaction with the antigen having a higher dissociation constant.
Implementation Method 1
Surface plasmon resonance (SPR) is a well know technology for the determination of antibody-antigen interactions of monospecific antibodies
Data Source
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AI summary
Herein is reported the use of a binding assay of a bivalent, bispecific antibody that has the smaller kD value (dissociation constant) for the interaction with its antigen for the immobilization of the bivalent, bispecific antibody to a solid surface for the determination of the biological activity of the bivalent, bispecific antibody.