Sequential Epitope Detection in FFPE Tissue Sections
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting multiple antigens in a single tissue section are limited by the need for specialized reagents and difficulty in removing antibodies without compromising antigenicity, which restricts the ability to study co-localization of antigens in the same cell.
Innovation Solution
A method involving labeling a formalin-fixed paraffin embedded (FFPE) tissue section with a first set of antibodies, treating with a protease to separate the antibodies from the tissue, washing, and then relabeling with a second set of antibodies, allowing for the detection of multiple antigens using distinguishable fluorescent labels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If multiple antigens are detected in the same tissue section using conventional methods, then more validating data regarding disease status can be obtained, but specialized reagents and complex procedures are required
Solution Approach 1:
The detection process is divided into separate cycles, with each cycle detecting one antigen. After detecting the first antigen, the fluorescent label is removed and the tissue section is re-labeled for the next antigen detection. This segmentation allows multiple antigens to be detected sequentially using the same tissue section without requiring complex multiplexed labeling systems.
Solution Approach 2:
The method employs periodic cycles of labeling, imaging, and label removal. Each cycle is repeated for different antigens, allowing the same tissue section to be analyzed multiple times. This periodic action enables the detection of multiple antigens through time-separated operations rather than simultaneous complex labeling.
2Adaptability or versatility
If antibodies are removed from the tissue section to enable detection of additional antigens, then more antigens can be studied, but the antigenicity may be compromised
Solution Approach 1:
The fluorescent label is extracted from the antibody complex using protease treatment, which cleaves the label from the antibody while leaving the antibody bound to the antigen. This extraction allows the label to be removed for subsequent detection cycles while maintaining the antibody-antigen complex integrity.
Solution Approach 2:
A protease enzyme acts as an intermediary to separate the fluorescent label from the antibody. The protease specifically cleaves the label-antibody linkage without affecting the antibody-antigen binding, enabling label removal while preserving antigenicity for subsequent detection cycles.
3Quantity of substance
If limited tissue samples are used for multiple antigen detections, then valuable biopsy material is conserved, but the tissue may not support multiple separate sections
Solution Approach 1:
The same tissue section is reused continuously across multiple detection cycles. After each antigen detection, the label is removed and the section is immediately re-labeled for the next antigen, maintaining continuous productive use of the limited tissue sample without requiring additional sections or biopsies.
Solution Approach 2:
The fluorescent label is discarded after each detection cycle and recovered for reuse in subsequent cycles. This allows the same tissue section to be analyzed repeatedly for different antigens, maximizing the information obtained from limited tissue samples without requiring new sections.
4Measurement precision
If conventional labeling methods are used for multiple antigens, then co-localization can be detected, but the presence of multiple labeled antibodies makes color differentiation difficult
Solution Approach 1:
The detection of multiple antigens is segmented into separate time-based cycles rather than simultaneous detection. Each antigen is detected individually with its own fluorescent label, eliminating the need to differentiate multiple colors in the same field of view. The temporal separation simplifies color differentiation while maintaining co-localization detection accuracy.
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 the detection of multiple antigens in the same tissue section without requiring specialized reagents, facilitating the analysis of co-localized antigens and increasing the number of markers that can be investigated by up to 4n-fold, where n is the number of digestive cycles performed.
Implementation Method 1
treating the labeled tissue with a protease, thereby digesting the first primary antibody and/or the first labeled secondary antibody and separating the label from the FFPE tissue section
Data Source
AI summary
A method for labelling a tissue section is provided. In certain embodiments, the method may comprise: (a) labeling a formalin-fixed paraffin embedded (FFPE) tissue section using a first set of labeling reagents that comprises a first primary antibody and a first labeled secondary antibody; (b) treating the labeled tissue with a protease, thereby digesting the first primary antibody and/or the first labeled secondary antibody and separating the label from the FFPE tissue section; (c) washing the tissue section to remove the separated label and the protease; and (c) labeling the FFPE tissue section using a second set of labeling reagents that comprises a second primary antibody and a second labeled secondary antibody. A kit for performing the method is also provided.