Flow Proxy Assay for CITE-Seq Reagent Optimization
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Solution Overview
Problem
Current methods for measuring protein expression in cells, such as single-cell CITE-Seq, are costly and inefficient when detecting a large number of AbSeq markers, as they require estimating marker expression levels and can result in wastage of reagents due to low or high expression markers.
Innovation Solution
The method involves contacting cells with cellular component-binding reagents and detectable conjugates, where each reagent has a unique identifier oligonucleotide and each conjugate has a detectable moiety specific to the identifier, allowing for the measurement of emissions to indicate the amount of reagent bound to cellular targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If single-cell CITE-Seq is used to detect a large number of AbSeq markers, then protein expression analysis capability is improved, but cost increases and reagent waste increases
Solution Approach 1:
The patent applies preliminary action by performing flow proxy assays before single-cell CITE-Seq experiments to estimate marker expression levels. This allows researchers to determine the appropriate AbSeq reagent amounts in advance, preventing reagent waste from detecting markers with low or universally high expression that would not provide useful information.
Solution Approach 2:
The patent implements feedback by using flow cytometry data from flow proxy assays to guide the design of AbSeq panels. The expression level information obtained from flow proxy assays feeds back into the experimental design, allowing optimization of reagent quantities and panel composition before committing to costly single-cell CITE-Seq experiments.
2Adaptability or versatility
If single-cell CITE-Seq is used to detect a large number of AbSeq markers, then protein expression analysis capability is improved, but cost increases
Solution Approach 1:
The patent applies preliminary action by performing flow proxy assays before single-cell CITE-Seq experiments to estimate marker expression levels. This allows researchers to determine the appropriate AbSeq reagent amounts in advance, preventing reagent waste from detecting markers with low or universally high expression that would not provide useful information.
Solution Approach 2:
The patent implements feedback by using flow cytometry data from flow proxy assays to guide the design of AbSeq panels. The expression level information obtained from flow proxy assays feeds back into the experimental design, allowing optimization of reagent quantities and panel composition before committing to costly single-cell CITE-Seq experiments.
3Productivity
If flow proxy assay is used to estimate marker expression levels, then reagent usage optimization is improved, but the number of detectable markers is limited by available dye-oligonucleotide conjugates
Solution Approach 1:
The patent applies segmentation by separating the flow proxy assay function from the single-cell CITE-Seq function. Flow proxy assays use fluorescent antibodies against AbSeq reagents to estimate expression levels, while single-cell CITE-Seq uses sequencing to detect protein markers. This segmentation allows optimization of reagent usage in flow proxy assays without limiting the number of markers that can be detected in the subsequent sequencing experiment.
Solution Approach 2:
The patent uses flow cytometry as an intermediary tool between reagent optimization and marker detection. Flow proxy assays provide expression level estimates that guide AbSeq panel design, while the actual marker detection is performed by sequencing. This intermediary approach allows flexible optimization of reagent quantities without constraining the diversity of detectable markers.
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 enables cost-effective measurement of cellular component target expression, optimizing reagent usage and improving the efficiency of protein expression analysis, while also allowing for the simultaneous measurement of gene expression.
Implementation Method 1
each of the plurality of first detectable conjugates comprises a detectable moiety, or precursor thereof, and a unique identifier specific oligonucleotide comprising a sequence configured to bind a unique identifier sequence
Implementation Method 2
measuring emissions of the detectable moiety of each first detectable conjugate with an instrument as an indication of the amount each of first cellular component-binding reagent bound to a cellular component target
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for detecting cellular component-binding reagents comprising a cellular component-binding reagent specific oligonucleotide having a unique identifier sequence for the cellular component-binding reagent. Provided herein include first detectable conjugates comprising a detectable moiety, or precursor thereof, and a unique identifier specific oligonucleotide having a sequence configured to bind a unique identifier sequence. Also provided herein are second detectable conjugates comprising a detectable moiety, or precursor thereof, and a shared oligonucleotide having a sequence configured to bind a shared sequence of the cellular component-binding reagent specific oligonucleotides.