Abstract
Elevated levels of the stress-inducible heat shock protein 70 (Hsp70) in the peripheral circulation have been reported for many tumor entities. In line with these findings we have shown that Hsp70 membrane-positive tumor cells actively release Hsp70 in exosome-like lipid vesicles. Since most commercial Hsp70 Enzyme-Linked Immunosorbent Assays (ELISAs) are not validated for the detection of liposomal Hsp70 in serum, the lipHsp70 ELISA was established using the monoclonal antibody cmHsp70.1 as a detection reagent. This antibody has been reported to recognize free and membrane-bound Hsp70 on living tumor cells. Validation of the ELISA showed a high assay precision and linearity in a concentration range of 0.36-17.4 ng/ml. A comparison of the recovery of spiked Hsp70 in buffer and serum samples revealed a significantly better recovery using the lipHsp70 ELISA compared to a commercial ELISA. With respect to lipid-associated Hsp70 a tenfold higher recovery was found with the lipHsp70 ELISA compared to the commercial ELISA. The analysis of blood samples of healthy human volunteers (n=114) revealed a mean serum Hsp70 concentration of 6.4 ± 2.7 and 2.8 ± 1.3 ng/ml, respectively, using the lipHsp70 and the control ELISA. No significant age-related differences in Hsp70 serum levels were detected. The lipHsp70 ELISA is equally suitable for serum and plasma and the measured Hsp70 concentrations were not impacted by food intake, repeated freezing and thawing of the sample or moderate hemolysis. A comparison of the Hsp70 levels in patients with head and neck, lung, colorectal, pancreatic cancer, glioblastoma or hematological malignancies and healthy human volunteers revealed significantly higher levels in tumor patients. In summary, the lipHsp70 ELISA provides a highly sensitive and robust method for measuring liposomal and free Hsp70 in serum and plasma and thus could provide a useful tool for tumor detection and for monitoring the clinical outcome of patients.
Highlights
Heat Shock Proteins (Hsp) are molecular chaperones that play a key role in maintaining protein homeostasis and transport
In line with these findings we have shown that heat shock protein 70 (Hsp70) membrane-positive tumor cells actively release Hsp70 in exosome-like lipid vesicles
To quantify free as well as lipid-bound Hsp70 derived from exosomes in the serum of tumor patients, we developed the novel lipHsp70 sandwich Enzyme-Linked Immunosorbent Assays (ELISAs)
Summary
Heat Shock Proteins (Hsp) are molecular chaperones that play a key role in maintaining protein homeostasis and transport. Heat shock proteins with a molecular weight of approximately 70 kDa (Hsp70) are involved in assisting protein folding, preventing protein aggregation and transporting proteins across membranes [1,2]. The heat shock cognate protein 70 (Hsc70) and the major stress-inducible heat shock protein 70 (Hsp70), which are present in all nucleated eukaryotic cells, show a high sequence homology of 86% [3]. Following a variety of different stress stimuli, the synthesis of Hsc is moderately [4], while that of Hsp is highly upregulated in normal cells [1]. In contrast to normal cells, tumor cells frequently overexpress inducible Hsp already under physiological conditions [5] and present it on their plasma membrane [6]. Since Hsp on the membrane of tumor cells could not be removed with high salt, acid or basic washes [7,8], experimental evidence is Received September 10, 2014; Accepted October 09, 2014; Published October 16, 2014
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