Thioether Cross-linked Antibodies for Reducing Condition Stability

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Solution Overview

Problem

Antibodies dissociate under reducing conditions, which limits their use in analyzing proteins, particularly in detecting splice variants and phosphorylation sites hidden by protein folding, and in studying urinary micelles and serum exosomes, where harsh conditions are necessary to reveal masked variations.

Innovation Solution

The development of an immunoassay kit with antibodies or fragments that incorporate non-disulphide thioether cross-links between heavy and light chains, allowing for stable binding under reducing conditions, using cross-linkers like bismaleimidoethane to form thioether bonds that maintain antibody stability and activity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If antibodies are used under reducing conditions to detect splice variants and phosphorylation sites, then the ability to detect masked variations is improved, but the antibody dissociates and binding activity is reduced

Engineering Contradiction:

Improvedetection of splice variants and phosphorylation sitesVSAvoidantibody binding activity

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent applies preliminary anti-action by pre-forming thioether cross-links between heavy and light chains before the reducing conditions are applied during assay. This cross-linking creates a protective structural constraint that prevents dissociation even when reducing agents are present, allowing the antibody to maintain binding activity while exposing hidden epitopes through denaturation

Inventive Principle:

Principle #9Preliminary anti-action

Solution Approach 2:

The patent creates a composite antibody structure by introducing thioether cross-links that combine the natural disulphide bond framework with additional covalent connections. This composite structural approach provides enhanced stability under reducing conditions while preserving antigen-binding capability, resolving the contradiction between maintaining reliability and enabling harsh condition analysis

Inventive Principle:

Principle #40Composite materials

2Adaptability or versatility

If harsh reducing conditions are applied to denature proteins and reveal hidden variations, then the detection capability for masked epitopes is improved, but the antibody structure destabilizes and dissociates

Engineering Contradiction:

Improveanalysis of denatured proteins and masked variationsVSAvoidantibody structure stability

Core Design Contradiction:

Adaptability or versatilityVSStability of the object's composition

Solution Approach 1:

The thioether cross-links are formed in advance to counteract the destabilizing effect of reducing conditions. This preliminary structural reinforcement allows the antibody to withstand harsh denaturing conditions that reveal hidden splice variants and phosphorylation sites without losing its own structural integrity

Inventive Principle:

Principle #9Preliminary anti-action

Solution Approach 2:

The patent changes the chemical parameter of the antibody structure by replacing or supplementing disulphide bonds with thioether cross-links. This parameter change increases the stability of the antibody composition under reducing conditions, enabling versatile analysis of denatured proteins while maintaining structural stability

Inventive Principle:

Principle #35Parameter changes

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 cross-linked antibodies maintain specific antigen binding activity and stability under conditions that would normally cause dissociation, enabling effective detection of epitopes and analysis of proteins in denatured states, enhancing the detection of clinically relevant variants and changes.

Implementation Method 1

the antibody or fragment thereof comprises one or more non-disulphide cross-links between at least one heavy chain or fragment thereof and at least one light chain or fragment thereof

Methodology Applied

Scientific EffectThioether bond formation: Chemical Bonding

Implementation Method 2

using cross-linkers like bismaleimidoethane to form thioether bonds that maintain antibody stability and activity

Methodology Applied

Scientific EffectCross-linking: Chemical Bonding

Data Source

PatentUS20230391851A1antibodies

Publication Date: 2023.12.07 THE BINDING SITE GROUP

AI summary

A method for analyzing protein(s) in a sample using an immunoassay kit includes creating protein-reducing and/or protein-denaturing conditions by contacting the sample with a reducing and/or denaturing agent provided in the immunoassay kit, to provide a partially or fully denatured protein population. One or both of a presence and an amount of one or more protein-associated analytes are determined under the created protein-reducing and/or protein-denaturing conditions by contacting the partially or fully denatured protein population with one or more specific antibodies or binding fragments thereof provided in the immunoassay kit. The one or more specific antibodies or binding fragments thereof include one or more chemically-introduced non-disulfide cross-links between at least one heavy chain or binding fragment thereof and at least one light chain or binding fragment thereof.