CD31 Isoform Detection via Cytometric Bead Array

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Solution Overview

Problem

Current methods for analyzing signaling pathways are limited in their ability to discriminate between different soluble forms of proteins, quantify specific isoforms, and determine the activation state of signaling cascades, leading to incomplete understanding of CD31's role in immune responses and its association with diseases like atherothrombosis.

Innovation Solution

A method using cytometric beads and fluorescently labeled antibodies to differentiate between shed and soluble splice variants of CD31, allowing for precise quantification and detection of these forms in biological samples, and adapted for analyzing other transmembrane proteins and signaling pathways.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional ELISA methods are used to detect soluble CD31, then total soluble CD31 levels can be measured, but the method cannot discriminate between different soluble forms (shed ectodomain vs. splice variant)

Engineering Contradiction:

Improvediscrimination between soluble CD31 formsVSAvoidassay complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The invention segments the detection process by using multiple antibodies, each specific to different regions of the CD31 protein. The first antibody binds to a region present in all soluble forms, while the second antibody binds to a region present only in the shed ectodomain, enabling discrimination between soluble forms through segmented epitope recognition

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces an intermediary calculation step where the amount of shed ectodomain is determined by subtracting the amount of splice variant from the total soluble CD31. This mathematical intermediary allows indirect measurement of the shed form without requiring direct specific antibodies for all forms

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If previous studies measured plasma CD31 levels to assess atherothrombosis risk, then broad ranges of values were obtained, but the results were contradictory and inconclusive

Engineering Contradiction:

Improvediagnostic reliability for atherothrombosisVSAvoidinformation loss due to inability to distinguish isoforms

Core Design Contradiction:

ReliabilityVSLoss of information

Solution Approach 1:

The invention extracts and isolates the measurement of the shed ectodomain form from the total soluble CD31 measurement. By specifically quantifying only the shed form through the two-site ELISA approach, the method removes the confounding information from splice variants, providing reliable diagnostic data for atherothrombosis risk assessment

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention applies local quality by making the measurement specific to a particular region/form of CD31 (the shed ectodomain) rather than measuring all soluble forms equally. This localized measurement approach provides clinically relevant information that was previously lost in bulk measurements

Inventive Principle:

Principle #3Local quality

3Measurement precision

If the invention uses a two-site ELISA method with multiple antibodies and calculations, then precise quantification of shed CD31 is achieved, but the assay procedure becomes more complex

Engineering Contradiction:

Improvequantification accuracy of shed CD31VSAvoidassay procedure simplicity

Core Design Contradiction:

Measurement precisionVSEase of operation

Solution Approach 1:

The invention performs preliminary actions by pre-coating plates with the first antibody and pre-incubating samples with blocking reagents. These preparatory steps simplify the subsequent measurement steps and reduce the complexity of the overall procedure by organizing the multi-step process into logical sequences

Inventive Principle:

Principle #10Preliminary action

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

Enables rapid, sensitive, and specific analysis of CD31 isoforms, providing a diagnostic tool for thrombotic and autoimmune disorders by accurately measuring shed CD31 levels, improving upon existing assays by allowing multiple replicates, low sample requirements, and simultaneous detection of multiple parameters.

Implementation Method 1

A method using cytometric beads and fluorescently labeled antibodies to differentiate between shed and soluble splice variants of CD31

Methodology Applied

Scientific EffectAntibody-antigen binding:

Implementation Method 2

providing a diagnostic tool for thrombotic and autoimmune disorders by accurately measuring shed CD31 levels

Methodology Applied

Scientific EffectFlow cytometry:

Data Source

PatentUS9977030B2Detection of shed CD31, diagnosis of atherothrombosis and autoimmune disorders, and methods for analyzing signaling pathways

Publication Date: 2018.05.22 INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)

AI summary

The present invention stems from the finding that the extracellular domain of CD31 proteins present on blood leukocytes is shed and released in the circulation as a soluble form of CD31. A method for detecting shed CD31 is further disclosed. The invention therefore relates to a method for detecting a shed ectodomain of a transmembrane protein such as CD31 and to the use of such a method as a diagnostic tool. The invention further provides methods for determining whether a candidate protein is part of a molecular complex.