IL-33 Immunoassay Acid Dissociation Blocking
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Solution Overview
Problem
Commercially available immunoassays for detecting interleukin 33 (IL-33) suffer from interference by endogenous binding partners, leading to unreliable results due to interference from soluble ST2 receptor and other serum components, which can result in misinterpretation of patient samples and inappropriate treatment.
Innovation Solution
An IL-33 immunoassay that includes an acid pre-treatment step to dissociate soluble ligand:drug complexes, followed by neutralization with a Tris-base solution containing a ruthenium-labeled anti-human IL-33 antibody, and the addition of blocking agents to prevent complex reformation, using a streptavidin-coated plate with biotinylated anti-human IL-33 as the capture reagent, which reduces assay interference and allows for accurate quantification of total IL-33 in human serum.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If commercially available immunoassays are used to detect IL-33, then the assay can be performed with standard reagents, but the results are unreliable due to interference from endogenous binding partners such as soluble ST2 receptor
Solution Approach 1:
The patent applies preliminary action by performing acid pre-treatment on serum samples before the immunoassay to dissociate soluble IL-33:ST2 receptor complexes. This preliminary step separates the bound IL-33 from its endogenous binding partners, preventing interference during the subsequent detection steps and enabling reliable quantification of total IL-33
Solution Approach 2:
The patent utilizes parameter changes by altering the pH conditions through acid treatment to disrupt the binding between IL-33 and ST2 receptor. The acidification step changes the physical-chemical parameters of the sample, causing conformational changes or electrostatic repulsion that dissociates the complexes, thereby eliminating the harmful interference effect
2Measurement precision
If acid pre-treatment is applied to dissociate IL-33 complexes, then detection sensitivity improves, but the assay procedure becomes more complex
Solution Approach 1:
The patent merges multiple functions into the neutralization step: after acid pre-treatment, the same neutralization buffer simultaneously restores physiological pH and contains the detection reagents (anti-IL-33 antibodies). This consolidation reduces the number of separate steps and reagent additions, simplifying the overall procedure while maintaining high detection sensitivity
3Object-affected harmful factors
If blocking agents are added to prevent complex reformation, then assay interference is reduced, but the number of reagents and steps increases
Solution Approach 1:
The patent employs blocking agents as intermediary substances that compete with ST2 receptor for binding to IL-33. These blocking agents (such as excess soluble ST2 or IL-33 analogs) act as mediators to occupy the binding sites on IL-33, preventing endogenous ST2 from re-binding and causing interference, while being added in a single step with the detection reagents
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 method achieves a sensitivity of 12.5 pg/mL and tolerance to >50 ng/mL of ST2, providing precise and specific detection of IL-33 levels in human serum samples, improving the reliability of IL-33 measurement and reducing interference from endogenous binding partners.
Implementation Method 1
acid pre-treatment of serum samples to dissociate soluble ligand:drug complexes present in the samples
Implementation Method 2
electrochemiluminescence immunoassay... IL-33 captured on the plate is measured by a chemiluminescent signal generated by the ruthenium label when voltage is applied to the plate
Data Source
AI summary
Methods and compositions for detecting and quantifying cytokines are provided. The disclosed assays have reduced assay interference relative to commercially available assays and/or a control assay. The interference can be cytokine dependent, cytokine independent, or both. One embodiment provides an IL-33 immunoassay that reduces assay interference caused by endogenous soluble IL-33 binding molecules present in the sample. Exemplary soluble IL-33 binding molecules include, but are not limited to anti-IL-33 antibodies, soluble ST2 receptor, and serum components. In some embodiments a blocking agent is added to the sample to reduce, inhibit, or block IL-33 complexes in the sample from reforming after acid dissociation of the IL-33 complexes in the sample. In one embodiment, the blocking agent and the detection reagent do not compete for binding to IL-33.