Two-Step Antigen Retrieval for Multiplex Protein Detection
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Solution Overview
Problem
Current tissue processing methods for diagnostic cell imaging, such as immunohistochemistry, often mask antigens due to formaldehyde fixation, making it difficult to detect multiple proteins simultaneously, and existing antigen retrieval techniques are not uniformly effective across different samples.
Innovation Solution
A two-step antigen retrieval method involving incubation of formaldehyde-fixed tissue samples in a first solution at greater than 90°C, followed by transfer to a second solution at the same temperature, using buffer solutions with specific pH ranges to hydrolyze cross-link bonds and retrieve unretrieved antigens, enhancing protein detection for multiplex analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If formaldehyde fixation is used to preserve tissue samples, then tissue preservation is improved, but antigen detection capability deteriorates
Solution Approach 1:
The patent applies preliminary action by performing antigen retrieval treatment before the actual antigen detection step. The method involves heating the fixed tissue sample in a buffer solution to reverse formaldehyde cross-linking and mask effects, thereby restoring antigen accessibility prior to antibody application. This preliminary restoration step enables subsequent successful detection of multiple antigens that would otherwise be masked by fixation.
2Ease of operation
If single-step antigen retrieval is used, then process simplicity is improved, but retrieval effectiveness for multiple antigens deteriorates
Solution Approach 1:
The patent applies segmentation by dividing the antigen retrieval process into two distinct sequential steps: first heating in a low pH buffer solution (pH 6.0) to retrieve certain antigens, then heating in a high pH buffer solution (pH 8.0) to retrieve additional different antigens. This segmented approach allows each buffer condition to target specific antigen types, thereby achieving comprehensive multi-antigen retrieval that a single-step method cannot accomplish.
3Productivity
If automated antigen retrieval processes are used, then processing speed is improved, but staining quality and epitope stability deteriorate
Solution Approach 1:
The patent applies parameter changes by optimizing specific parameters including buffer pH values (6.0 and 8.0), heating temperature (95-100°C), and heating duration (20 minutes each step). These precisely controlled parameter changes enable effective antigen retrieval while preserving epitope stability and staining quality, overcoming the limitations of automated processes that use fixed, non-optimizable parameters.
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 method significantly improves antigen retrieval, allowing for enhanced staining intensity and reduced background noise, enabling more effective detection of multiple proteins in a single sample, as demonstrated by increased pixel intensity and preserved epitope stability compared to single-step or automated processes.
Implementation Method 1
incubating the formaldehyde-fixed tissue sample in a first antigen retrieval solution at a temperature of greater than 90°C, transferring the tissue sample to a second antigen retrieval solution, and incubating the tissue sample in the second antigen retrieval solution at a temperature of greater than 90°C
Data Source
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AI summary
The invention provides a method for antigen retrieval of a formaldehyde-fixed tissue sample comprising incubating a formaldehyde-fixed tissue sample in a first antigen retrieval solution at a temperature of greater than 90oC, transferring the tissue sample to a second antigen retrieval solution, and incubating the tissue sample in the second antigen retrieval solution at a temperature of greater than 90oC. The invention also provides a kit and sample delivery device for carrying out the method.