Enzyme-Linked Immunoassay Mass Spectrometry Detection

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Solution Overview

Problem

Current methods for measuring proteins at low concentrations, such as ELISA and RIA, face limitations in sensitivity, particularly in detecting molecules present in femto mol to atto mol ranges, and LC-MS/MS methods struggle with sensitivity in complex biological samples.

Innovation Solution

A method involving immobilizing a target substance on a solid phase, forming complexes with a reporter enzyme detection probe, and using mass spectrometry to detect ionizable products generated from a substrate reaction, allowing for sensitive detection and quantification of proteins in low concentrations through ionizable products.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If ELISA or RIA methods are used to detect proteins, then the detection can be performed with standard equipment and procedures, but the sensitivity is limited and cannot detect proteins in femto mol to atto mol ranges

Engineering Contradiction:

Improvedetection sensitivityVSAvoidmethod complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent uses an enzyme reporter as an intermediary between the target protein and the detection system. The enzyme catalyzes conversion of a substrate to an ionizable product, which is then detected by mass spectrometry. This intermediary amplifies the signal while maintaining specificity, enabling detection at femto mol to atto mol levels without requiring complex radioactive labeling or specialized equipment

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces the optical detection system (photodetectors, light sources) used in traditional ELISA with a mass spectrometry-based detection system. This substitution enables direct measurement of ionizable products with higher sensitivity and eliminates the limitations of optical detection, allowing detection at concentrations below the detection limit of conventional ELISA methods

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Measurement precision

If LC-MS/MS is used to detect proteins in complex biological samples, then direct measurement of ionized peptides or small molecules can be achieved, but the sensitivity is insufficient for low abundance proteins

Engineering Contradiction:

Improvedetection sensitivityVSAvoiddetection difficulty in complex samples

Core Design Contradiction:

Measurement precisionVSDifficulty of detecting and measuring

Solution Approach 1:

The patent performs preliminary enrichment of the target protein using capture antibodies before the enzymatic reaction. This preliminary action concentrates the low-abundance target from complex biological samples, removing it from the complex matrix and placing it in a simplified environment where the subsequent enzymatic amplification and mass spectrometry detection can proceed with high sensitivity

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent employs an enzyme reporter that continuously catalyzes the conversion of substrate to ionizable product as long as substrate is available. This continuous amplification process maintains a steady-state signal that is proportional to the amount of target protein, enabling reliable detection and quantification even at very low concentrations where discrete signaling methods would fail

Inventive Principle:

Principle #20Continuity of useful 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 detection and quantification of proteins in the femto mol to atto mol range with high sensitivity, surpassing the limitations of existing methods by utilizing mass spectrometry to analyze ionizable products from enzyme-substrate reactions, facilitating precise measurement of proteins in complex biological samples.

Implementation Method 1

incubating the target: enzyme detection probe complex with a reporter enzyme detection probe substrate in substrate reaction solution to generate one or more ionizable products

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 2

detecting one or more of the one or more ionizable products using mass spectrometry (MS)

Methodology Applied

Scientific EffectIonization: Ionisation

Data Source

PatentUS9964538B2Methods and compositions for enzyme linked immuno and hybridization mass spectrometric assay

Publication Date: 2018.05.08 YYZ PHARMATECH INC

AI summary

Disclosed are sensitive methods for detecting a target substance in low concentrations. The method can include immobilizing a target substance to a solid phase; incubating the immobilized target substance with a reporter enzyme detection probe in solution under conditions for forming target: enzyme detection probe complexes; washing the solid phase to remove any unbound reporter enzyme detection probe; incubating the target: enzyme detection probe complex with a reporter enzyme detection probe substrate in substrate reaction solution to generate one or more ionizable products; and detecting one or more of the one or more ionizable products using mass spectrometry (MS).