Alkaline Protein Extraction from Fixed Cells
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Solution Overview
Problem
Conventional cytological tests for detecting oncogenic HPV strains are prone to errors due to contaminants like mucous and blood cells, leading to false results, and existing methods struggle to reliably detect proteins from fixed cells using conventional means, such as ELISA, due to protein precipitation or aggregation caused by certain fixatives.
Innovation Solution
A method involving increasing the pH of fixed cells to at least pH 10.0 and then neutralizing it in the presence of a non-ionic detergent to produce a protein extract suitable for molecular detection assays, allowing for the extraction of proteins that are otherwise insoluble or difficult to detect.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional cytological tests are used to detect oncogenic HPV strains, then the detection process is simple, but the reliability is poor due to contaminants causing false results
Solution Approach 1:
The patent extracts and removes contaminants (mucous, blood cells, inflammatory cells) from the cellular sample before performing the detection assay. This extraction step isolates the target HPV proteins from interfering substances, thereby eliminating false positive and false negative results while maintaining a streamlined detection process.
Solution Approach 2:
The patent performs preliminary actions including cell lysis, protein extraction, and contamination removal before the actual detection step. These preliminary steps prepare the sample by removing interfering substances and concentrating target proteins, ensuring accurate detection without requiring complex subsequent processing.
2Stability of the object's composition
If fixatives are used to preserve cells for cytological examination, then the cells remain stable for analysis, but the proteins precipitate or aggregate making them insoluble and difficult to detect
Solution Approach 1:
The patent changes the chemical parameters of the fixed cell sample by adjusting pH to alkaline conditions (pH 10-11) and using specific buffers during protein extraction. This parameter change dissolves aggregated proteins and restores their solubility, enabling detection while maintaining the fixed state of cells for cytological analysis.
Solution Approach 2:
The patent employs intermediary reagents including alkaline buffers (Tris, glycine), detergents (Triton X-100, NP-40), and chelating agents (EDTA) that mediate between the fixed cell state and the soluble protein state. These intermediaries facilitate protein solubilization without compromising the fixed morphology of cells, enabling both cytological and molecular analysis.
3Ease of operation
If conventional ELISA methods are used to detect proteins from fixed cells, then the detection process is straightforward, but the results are unreliable due to protein precipitation and aggregation
Solution Approach 1:
The patent performs preliminary protein solubilization and deaggregation steps using alkaline buffers and detergents before the ELISA detection. This preliminary action ensures proteins are in a soluble, detectable state, making the subsequent ELISA procedure straightforward and reliable without requiring complex sample preparation during the assay itself.
4Productivity
If contaminants are present in cytological samples, then the sample processing remains simple, but false results occur due to interference with detection
Solution Approach 1:
The patent extracts and removes contaminants (mucous, blood cells, inflammatory cells) through lysis and filtration steps before protein detection. This extraction maintains processing efficiency by using straightforward removal techniques while significantly improving detection accuracy by eliminating interfering substances that cause false results.
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 effectively produces a protein extract that is readily detectable using capture agents, reducing errors and improving the reliability of HPV protein detection, especially for oncogenic strains like HPV 16 and 18, by solubilizing proteins and preventing re-aggregation during neutralization.
Implementation Method 1
contacting the fixed cells with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0
Implementation Method 2
contacting the intermediate composition with a neutralizing reagent to neutralize the pH of the intermediate composition
Implementation Method 3
in the presence of a non-ionic detergent, neutralizing the pH of the intermediate composition to produce the protein extract
Data Source
AI summary
Methods for producing a protein extract from cells, such as cells containing viral proteins, are provided. In general terms, the methods involve: increasing the pH of the cells to a pH of at least about pH 10.0 to produce an intermediate composition, and then, in the presence of a non-ionic detergent, neutralizing the pH of the intermediate composition to produce the protein extract. Kits and compositions for practicing the subject methods are also provided.