Grp94-IgG Complex Detection via Sandwich ELISA
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Solution Overview
Problem
Current diagnostic tools are inadequate for measuring Glucose-regulated protein 94 (Grp94) in blood and other biological fluids, which is essential for its consideration as a tumor biomarker and for predicting anti-tumor therapy efficacy, due to Grp94's stable non-immune binding with Immunoglobulin G (IgG), making it difficult to detect using existing ELISA methods.
Innovation Solution
A sandwich ELISA method using primary anti-Grp94 antibodies for coating and secondary anti-human polyclonal antibodies conjugated with detection systems to measure Grp94-IgG complexes, allowing for reliable quantitative detection of the protein in biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional ELISA methods are used to detect Grp94, then the detection process is simple, but the detection sensitivity and specificity are insufficient due to stable non-immune binding with IgG
Solution Approach 1:
The detection method is segmented into two independent antibody binding steps: first capturing Grp94 with anti-Grp94 antibodies, then detecting the IgG component with anti-human polyclonal antibodies. This segmentation allows specific detection of the Grp94-IgG complex despite the stable non-immune binding, resolving the contradiction between detection precision and method complexity.
Solution Approach 2:
The patent uses anti-Grp94 antibodies as an intermediary to capture the Grp94 component of the complex, and anti-human polyclonal antibodies as a second intermediary to detect the IgG component. These intermediary antibodies enable specific detection of the complex without being affected by the stable non-immune binding between Grp94 and IgG.
2Reliability
If Grp94 is measured as a circulating antigen, then it can be used as a tumor biomarker, but the stable binding with IgG makes it difficult to detect
Solution Approach 1:
Instead of attempting to detect Grp94 as a free circulating antigen, the method segments the detection into two parts: capturing Grp94 and detecting IgG separately within the complex. This segmentation overcomes the detection difficulty caused by stable binding while maintaining reliability as a biomarker.
Solution Approach 2:
The patent changes the detection parameter from measuring free Grp94 antigen to measuring the Grp94-IgG complex through dual antibody recognition. This parameter change transforms the detection challenge into an opportunity, as the complex formation becomes the basis for specific detection rather than an obstacle.
3Ease of operation
If existing ELISA kits are used for Grp94 measurement, then the measurement process is straightforward, but they cannot reliably measure Grp94 in complexes with IgG
Solution Approach 1:
The ELISA protocol is segmented into distinct steps: coating with anti-Grp94 antibodies, blocking, adding sample, washing, adding anti-human IgG antibodies, and detecting with HRP-conjugated secondary antibodies. This segmented approach maintains operational ease while achieving precise complex detection through systematic separation of capture and detection functions.
Solution Approach 2:
The detection system uses universal ELISA components (microplates, blocking buffers, HRP-conjugated antibodies) adapted for complex detection. The multi-functionality of standard ELISA reagents is leveraged to detect Grp94-IgG complexes, maintaining ease of operation while improving measurement precision for complexes.
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 new method provides a sensitive and specific measurement of Grp94-IgG complexes, demonstrating high sensitivity and specificity as a tumor marker, effectively distinguishing between healthy and diseased subjects, and showing potential for use in both diagnostic and prognostic purposes across various tumor types.
Implementation Method 1
A sandwich ELISA method using primary anti-Grp94 antibodies for coating and secondary anti-human polyclonal antibodies conjugated with detection systems to measure Grp94-IgG complexes
Data Source
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AI summary
Matter of the invention is a new method of immune-detection, a sandwich ELISA, for the measurement in plasma of complexes formed by Glucose-regulated protein94 (Grp94) with Immunoglobulins G (IgG) identified as diagnostic and prognostic tumor marker. The method for measuring Grp94-IgG complexes was proved to be effective in a sample of patients with primary solid tumors of the gastro-enteric tract and in a group of healthy control subjects. The measurement of complexes at a fixed dilution of plasma showed that the complex concentration was significantly higher in patients than in control subjects. The cut-off between patients and control subjects permitted to determine the sensitivity and specificity of the method that turned out to be 100% in the sample examined. The new ELISA method for measuring the tumor marker gave positive immune reactions in all patients regardless of the type and site of origin of the tumor of the gastro-enteric tract.