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Updated: Aug 9, 2026
Intracellular Refolding Assay
Published on: January 24, 2012
Determination of intracellular heat shock protein 70 using a newly developed cell lysate immunometric assay
Rose Njemini1, Christian Demanet, Tony Mets
1Geriatric Unit, Academic Hospital, Free University of Brussels (VUB), Laarbeeklaan 101, B-1090, Brussels, Belgium.
Insights
A new cell lysate immunometric assay (CLIA) was developed to quantify intracellular heat shock protein 70 (Hsp 70). This cost-effective method offers a reliable alternative to existing techniques for Hsp 70 measurement.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Heat shock proteins (Hsp) are implicated in various clinical conditions.
- Existing methods for quantifying Hsp 70 have limitations.
Purpose of the Study:
- To develop and validate a novel cell lysate immunometric assay (CLIA) for intracellular Hsp 70 quantification.
- To establish a cost-effective and reliable alternative to current Hsp 70 detection methods.
Main Methods:
- Development of a CLIA utilizing two distinct monoclonal antibodies against Hsp 70.
- Quantification using recombinant human Hsp 70 as the standard.
- Validation through assessment of detection range, precision, recovery, linearity, and comparison with flow cytometry.
Main Results:
- The CLIA demonstrated a detection range of 4-4000 ng/ml.
- High precision with intra- and interassay coefficients of variation of approximately 5% and 12%.
- Excellent linearity (97-120%) and recovery (81-116%), with significant correlation (r=0.896) to flow cytometry.
Conclusions:
- The developed CLIA is a reliable and accurate method for quantifying intracellular Hsp 70.
- This assay provides a low-cost alternative to flow cytometry for Hsp 70 measurement.
- The findings support the utility of CLIA in clinical and research settings for Hsp 70 analysis.
Abstract:
Heat shock proteins (Hsp) have been associated to several clinical relevant conditions. Currently used methods to determine Hsp 70 possess certain drawbacks. Therefore, we developed a cell lysate immunometric assay (CLIA) for the quantification of intracellular Hsp 70. This CLIA uses a combination of two distinct monoclonal antibodies that recognize different epitopes on the Hsp 70 molecule. A recombinant human Hsp 70 was used as the standard material. The detection range of the CLIA was 4-4000 ng/ml. The intra- and interassay coefficients of variation were, on average, 5% and 12%, respectively. The recovery varied between 81% and 116%. The Hsp 70 levels assayed after serial dilution of cell lysates varied linearly with dilution (between 97% and 120%). The reliability of the CLIA was assessed by comparison with the values determined by flow cytometric procedure; these two sets of values showed a highly significant correlation (r=0.896, p<0.0001), indicating that the two methods are comparable. We conclude that this assay represents a low-cost alternative of the flow cytometric technique.
