Acidic-pH Staining for Unassociated Non-Enveloped Virus Flow Cytometry
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Solution Overview
Problem
Existing flow cytometry techniques face challenges in accurately and reliably quantifying unassociated non-enveloped virus particles due to nonspecific staining and reduced accessibility of fluorogenic dyes and fluorescent antibody stains, leading to inconsistent and less accurate results.
Innovation Solution
Preparation of a fluorescently-stained fluid sample at an acidic pH range (3.0 to 6.5) for flow cytometry evaluation, using fluorogenic dyes or fluorescent antibody stains, enhances stain accessibility and particle integrity, resulting in improved accuracy and repeatability of quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional flow cytometry techniques using fluorogenic dyes or fluorescent antibody stains are used to detect unassociated non-enveloped virus particles, then particle detection is achieved, but staining specificity and quantification accuracy deteriorate due to nonspecific binding and reduced accessibility
Solution Approach 1:
The patent applies parameter changes by modifying the pH condition from neutral to acidic (pH 3.0-6.5), which fundamentally alters the binding characteristics of fluorogenic dyes to non-enveloped virus particles. This pH modification increases stain accessibility while maintaining particle integrity, thereby improving both quantification accuracy and staining consistency simultaneously
Solution Approach 2:
The patent implements preliminary action by pre-adjusting the sample pH to the acidic range (3.0-6.5) before adding the fluorescent stain. This preparatory step ensures optimal binding conditions are established in advance, allowing the stain to access particle components more effectively and produce consistent, accurate results
2Reliability
If fluorogenic dyes are used to stain non-enveloped virus particles at neutral pH, then staining is achieved, but particle integrity and stain accessibility worsen leading to inconsistent results
Solution Approach 1:
The patent modifies the pH parameter to an acidic range (3.0-6.5), which simultaneously improves stain accessibility and maintains particle integrity. The acidic environment appears to create favorable binding conditions without compromising the structural stability of non-enveloped virus particles
3Measurement precision
If conventional staining protocols are used for non-enveloped virus particles, then general virus detection is achieved, but detection accuracy for non-enveloped particles specifically deteriorates
Solution Approach 1:
The patent applies local quality by creating a specific acidic environment (pH 3.0-6.5) tailored to the unique requirements of non-enveloped virus particles. This localized pH modification optimizes staining conditions specifically for this particle type without affecting the general applicability of flow cytometry for other virus types
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
The method significantly improves the accuracy and repeatability of flow cytometry results for unassociated non-enveloped virus particles by optimizing stain accessibility and particle integrity at acidic pH, overcoming previous performance deficiencies.
Implementation Method 1
detecting for the fluorescent emission response from the investigation zone and counting identified occurrences of the unassociated labeled particles
Implementation Method 2
fluorescent staining the biological material in a fluid sample composition with a said fluorescent stain to prepare a stained fluid sample composition
Data Source
AI summary
A method for flow cytometry evaluation of unassociated non-enveloped viral particles having a non-enveloped viral capsid includes preparing a fluorescently-stained fluid sample in which at least one fluorescent staining step is performed at an acidic pH in an acidic pH range and then subjecting the fluorescently-stained fluid sample to flow cytometry evaluation. A kit includes a plurality of sealed container with a first sealed container containing a fluorescent stain composition and a second sealed container including an aqueous dilution liquid at an acidic pH.