HBsAg Assay Using Reducing Agent to Cleave Disulfide Bonds
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Solution Overview
Problem
Conventional methods for detecting hepatitis B virus surface antigen (HBsAg) face challenges such as false negativity due to surfactant treatment and the complexity of acid/alkali pretreatment, which can lead to decreased sensitivity and interference from autoantibodies.
Innovation Solution
A method involving sample pretreatment with a reducing agent to cleave S-S bonds of HBsAg, forming a linear structure, and using antibodies that react preferentially with the reduced peptide composed of amino acids 111 to 130 of HBsAg, eliminating the need for strong acid or alkali treatment and reducing autoantibody interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If acid/alkali pretreatment is used to treat immune complexes, then autoantibody interference is reduced, but measurement precision decreases due to decreased sensitivity
Solution Approach 1:
The invention changes the chemical parameter (redox state) of the HBsAg antigen by using reducing agents to break disulfide bonds. This structural modification allows the antigen to be recognized by antibodies against the linear peptide sequence (amino acids 111-130) without requiring acid/alkali treatment, thereby maintaining detection sensitivity while avoiding autoantibody interference
Solution Approach 2:
The invention extracts the specific linear peptide sequence (amino acids 111-130) from the complex three-dimensional structure of HBsAg by breaking disulfide bonds. This allows selective detection of the linear epitope using specific antibodies, avoiding interference from autoantibodies that target conformational epitopes
2Measurement precision
If surfactant treatment is used to expose inside antigens, then detection sensitivity is improved, but false negativity occurs due to complex higher-order structure disruption
Solution Approach 1:
Instead of using surfactants that disrupt membrane structures, the invention changes the redox parameter by using reducing agents to break disulfide bonds. This selectively linearizes the antigen structure while preserving the integrity of the lipid bilayer membrane and viral particle structure, avoiding false negativity
3Adaptability or versatility
If conventional antibodies against common antigenic determinant a are used, then broad HBsAg detection is achieved, but autoantibody interference increases due to complex higher-order structure recognition
Solution Approach 1:
The invention extracts the linear peptide sequence (amino acids 111-130) from the complex three-dimensional conformation of the common antigenic determinant a. By targeting this specific linear epitope that becomes accessible after disulfide bond cleavage, the method achieves HBsAg detection while avoiding recognition by autoantibodies that target conformational epitopes
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 provides a highly sensitive HBsAg assay that is less susceptible to autoantibodies and avoids the complications of acid/alkali treatment, enhancing detection accuracy and simplicity.
Implementation Method 1
a pretreatment step of mixing the sample with a pretreatment reagent containing a reducing agent, to reduce hepatitis B virus s antigen, thereby cleaving the S-S bonds of said antigen contained in the sample, causing dissociation of the spatial structure, to form a linear structure
Implementation Method 2
an immunoassay step of subjecting the pretreated sample to an immunoassay of hepatitis B virus s antigen using at least one antibody or antigen-binding fragment thereof capable of antigen-antibody reaction with a reduced, linear peptide composed of the amino acids at positions 111 to 130
Data Source
Figure 1
AI summary
Disclosed is a highly sensitive assay method and assay kit for HBsAg, which do not require treatment with a strong acid or alkali in the sample pretreatment, and which is less susceptible to influences by the autoantibodies. The assay method for hepatitis B virus s antigen in a sample separated from a living body includes: a pretreatment step of mixing a sample with a pretreatment reagent containing a reducing agent, to reduce hepatitis B virus s antigen; and an immunoassay step of subjecting the pretreated sample to an immunoassay of hepatitis B virus s antigen using at least one antibody or antigen-binding fragment thereof capable of antigen-antibody reaction with a reduced peptide composed of the amino acids at positions 98 to 179 of hepatitis B virus s antigen.