Simplified ELISA Method Reducing Washing Steps
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Solution Overview
Problem
Traditional ELISA operations are cumbersome, time-consuming, and prone to human errors due to multiple steps and extensive washing procedures, which hampers efficiency and repeatability.
Innovation Solution
A simplified ELISA method that integrates pre-treatment steps before adding TMB chromogenic substrate into a single step, using pre-prepared homogeneous microspheres of detecting antibodies and HRP enzyme in an ELISA plate, reducing the number of washing steps and overall operation time to under 90 minutes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional ELISA method with multiple washing steps is used, then detection accuracy is maintained, but operation time increases and operation complexity increases
Solution Approach 1:
The patent combines multiple washing steps into a single washing operation performed after the antibody incubation step. The microsphere structure allows all unbound reagents to be removed in one washing step rather than requiring separate washes after each addition, thereby reducing operation time while maintaining detection accuracy through the inherent specificity of the antibody-antigen binding.
Solution Approach 2:
The patent performs preliminary coating of capturing antibodies on the ELISA plate before the actual assay. This pre-coating step ensures that the plate is ready for immediate use, and the capturing antibodies are already in position to bind target antigens, eliminating the need for time-consuming setup steps during the actual detection process.
2Measurement precision
If traditional ELISA method with multiple steps is used, then detection sensitivity is maintained, but operation complexity increases
Solution Approach 1:
The patent merges the functions of detecting antibodies and HRP enzyme into a single biotinylated antibody-HRP conjugate. This eliminates the need for separate addition steps for detecting antibodies and enzyme conjugates, reducing operation complexity while maintaining detection sensitivity through the combined functionality of the conjugate.
Solution Approach 2:
The biotinylated detecting antibody serves multiple functions: it binds to the captured antigen, provides a binding site for HRP enzyme through biotin-streptavidin interaction, and enables signal amplification. This multi-functional design reduces the number of reagents and steps required while maintaining high detection sensitivity.
3Reliability
If multiple washing steps are performed, then human errors are reduced, but operation time increases
Solution Approach 1:
The patent consolidates multiple washing operations into a single washing step performed after antibody incubation. The microsphere-based assay design allows all unbound reagents to be removed in one step, reducing the number of repetitive operations that can lead to human errors while improving overall operation efficiency.
Solution Approach 2:
The biotinylated antibody-HRP conjugate system provides self-service through the high-affinity biotin-streptavidin interaction, which ensures stable binding without requiring multiple washing steps to ensure proper binding. This self-stabilizing mechanism reduces the need for repetitive washing operations.
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 significantly reduces the overall experiment time, minimizes the chances of operational errors, and enhances detection repeatability by streamlining the ELISA process while maintaining high sensitivity and specificity.
Implementation Method 1
adding horseradish peroxidase (HRP) enzyme, adding TMB (3,3′,5,5′-tetramethylbenzidine) chromogenic substrate
Implementation Method 2
adding TMB (3,3′,5,5′-tetramethylbenzidine) chromogenic substrate, incubating at room temperature, adding 100 μl of stop solution into each well to terminate the reaction
Data Source
AI summary
The present invention discloses a method for performing enzyme linked immunosorbent assay (ELISA) with simplified operation, including: preparing standards of a target protein with gradient dilutions and pre-diluting test samples; setting up wells for the standards and wells for the test samples on an ELISA plate, adding 100 μl of the diluted standards and 100 μl of the diluted samples into their respective wells, incubating, discarding the liquid, drying, washing the residue; adding 100 μl of TMB chromogenic substrate into the wells of the standards and the wells of the test samples respectively, incubating at room temperature, adding 100 μl of a stop solution to terminate the reaction in each well; placing the ELISA plate into an ELISA reader for dual-wavelength detection, calculating the concentration of a target protein in the samples. The overall operation time can be controlled within 90 minutes at minimum.