Automated Virus Titration via Metabolic Activity Detection
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Solution Overview
Problem
Current methods for determining virus titration, such as plaque assays and endpoint dilution assays, are labor-intensive, time-consuming, and difficult to automate, limiting their throughput and sensitivity, especially for viruses with small lytic activity and non-adherent cell lines.
Innovation Solution
A cell viability-based method that measures the intracellular metabolic activity of infected cells using markers like ATP or LDH, comparing the results to a standard curve to determine virus titers, which can be applied to both adherent and non-adherent cell lines and is suitable for a wide range of cytopathogenic viruses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If plaque assays or endpoint dilution assays are used to determine virus titre, then measurement precision is improved, but productivity deteriorates due to labor-intensive procedures and long time requirements
Solution Approach 1:
The patent replaces manual mechanical procedures (plaque formation, agarose overlay, manual counting) with an automated metabolic activity detection system using ATP or LDH markers and plate readers, enabling high-throughput automated virus titre determination while maintaining measurement precision
Solution Approach 2:
The patent changes the measurement parameter from visual plaque counting or endpoint dilution to quantitative metabolic activity markers (ATP concentration or LDH release), allowing for automated detection and significantly increased throughput while maintaining accuracy
2Measurement precision
If plaque assays or endpoint dilution assays are used to determine virus titre, then measurement precision is improved, but loss of time increases due to 7-day assay duration
Solution Approach 1:
The patent performs preliminary action by pre-measuring metabolic activity markers in infected cells at multiple time points to establish a standard curve, which then allows rapid determination of unknown virus titres without requiring the full 7-day incubation period of traditional methods
Solution Approach 2:
The patent substitutes time-consuming manual plaque formation and counting with rapid automated metabolic activity detection using ATP or LDH markers, reducing assay duration from 7 days to significantly shorter periods while maintaining precision
3Productivity
If immunodetection methods are used to determine virus titre, then productivity is improved through faster reading, but device complexity increases due to need for specialized equipment and trained technicians
Solution Approach 1:
The patent uses disposable cell culture plates with metabolically active infected cells that are read by standard plate readers, eliminating the need for expensive flow cytometers or specialized equipment, while maintaining high productivity through automated metabolic activity detection
Solution Approach 2:
The patent replaces complex immunodetection systems requiring flow cytometers and specialized antibodies with a simpler metabolic activity detection system using ATP or LDH markers that can be measured by standard plate readers, reducing device complexity while improving productivity
4Productivity
If reporter genes are incorporated in viruses to determine titre, then productivity is improved through rapid detection, but reliability deteriorates due to potential effects on viral characteristics
Solution Approach 1:
The patent uses an intermediary approach by measuring metabolic activity markers (ATP or LDH) in infected cells rather than directly detecting viral components or reporter proteins, thereby maintaining the integrity of native viral characteristics while achieving rapid and reliable titre determination
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 rapid, sensitive, and universal virus titer determination across a broad range (1E+02 to 1E+06 TCID 50/ml), is easily automated, and can be used for high-throughput assays, overcoming the limitations of existing methods.
Implementation Method 1
determining the level of the viability of the host cells in the samples by measuring a significant cell viability parameter, preferably by determining the intracellular concentration, quantity or activity of a cell viability marker
Implementation Method 2
more preferably by determining the intracellular concentration, quantity or activity of a cell viability marker
Data Source
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AI summary
The present invention is directed to a titration method for cytopathogenic agent, particularly infectious viruses. In particular, the present invention is based on a new method for in vitro determining the titre of infectious agent, such as viruses in solution by comparing the viability of the infected cells (in monolayer conformation or in suspension) against a standard curve obtained by measuring the cell viability of infected host cells cultured in monolayer or in suspension with a known cytopathogenic agent stock. The invention is of special utility in the pharmaceutical industry for cytopathogenic agent clearance studies and for the identification and optimization of antiviral compounds involved in drug development and/or in pharmaceutical compositions.