Fluorescent Cell Binding Assay Normalization
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Solution Overview
Problem
Current methods for measuring cell surface protein binding, such as apoptosis markers like Annexin V, face challenges in ensuring equal sample loading and accurate quantification due to manual cell counting and varying cell growth rates, leading to inconsistent results.
Innovation Solution
A fluorescent cell binding assay that involves pre-labelling cell interacting components, incubating them with cells, washing, lysing, and performing SDS PAGE followed by Western blotting, where signals from bound proteins are normalized to housekeeping proteins or total protein load, allowing for accurate quantification and multiplex detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If manual cell counting and pipetting are used to ensure equal sample loading, then cell binding measurement can be performed, but variation in cell number seeded in each well occurs leading to inaccurate quantification
Solution Approach 1:
The assay uses cells themselves as the internal standard by detecting constitutively expressed endogenous proteins (house-keeping proteins) that are present in all cells. This self-service approach eliminates the need for external normalization methods and manual cell counting, as the cells provide their own reference signal for quantification.
Solution Approach 2:
The invention changes the measurement parameter from total signal intensity to normalized signal intensity (bound protein signal divided by house-keeping protein signal). This parameter transformation allows accurate quantification regardless of variations in cell number, converting an unreliable measurement into a reliable one.
2Ease of manufacture
If cells are grown in multiwell plates with manual seeding, then cell binding assay can be performed, but cells grow at different rates in each well leading to unequal sample loading
Solution Approach 1:
The assay utilizes endogenous house-keeping proteins expressed by the cells themselves as normalization references. This eliminates the need for precise manual cell counting and seeding, as each well's cells provide their own internal standard for normalization.
Solution Approach 2:
The invention merges the detection of the cell binding protein signal with the detection of the house-keeping protein signal in the same assay system. By combining these two measurements and normalizing them, the method simultaneously achieves ease of setup and precision in cell number normalization.
3Measurement precision
If total protein quantification and pipetting are used for normalization, then sample loading can be adjusted, but accuracy relies on manual procedures involving pipetting errors
Solution Approach 1:
The assay uses endogenous house-keeping proteins as internal standards, eliminating the need for external total protein quantification methods. The cells themselves provide the normalization reference, removing the need for separate protein concentration measurements and complex pipetting procedures.
Solution Approach 2:
The house-keeping protein acts as an intermediary that links the cell binding measurement to the cell number. Instead of directly measuring and normalizing by cell number or total protein, the assay uses the house-keeping protein signal as an intermediate reference that correlates with cell number, simplifying the normalization process.
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 provides accurate and reliable quantification of cell surface protein binding by normalizing signals to housekeeping proteins, enabling precise measurement of protein interactions and confirming molecular weight, thus overcoming the limitations of existing techniques.
Implementation Method 1
pre-labelling said cell interacting component with a fluorescent dye
Implementation Method 2
electrophoretic separation of proteins in said cell lysate in a gel, preferably an SDS PAGE gel
Implementation Method 3
transferring proteins from the gel to a Western blotting membrane
Data Source
Figure 1
Figure 2A~2B
AI summary
The present invention relates to a fluorescent cell binding assay combining pre-labeling and Western blotting. Intact cells are incubated with pre-labelled binders preferably followed by SDS PAGE (sodium dodecylsulphate polyacrylamide gel) separation and Western blotting. More closely, the invention relates to a cell binding assay in which the degree or amount of binding of one or more cell interacting protein or protein component to the cell surface is measured with the ability to correlate the degree of cell binding to the sample load/total number of cells.