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Quantitative Analysis of Liposomal Heat Shock Protein 70 (Hsp70) in the Blood of Tumor Patients Using a Novel LipHsp70 ELISA

Stephanie Breuninger, Janina Erl, Clemens Knape, Sophie Gunther, Ivonne Regel, Franz Rödel, Udo S Gaipl, Jun Thorsteinsdottir, Lydia Giannitrapani, Anne M Dickinson and Gabriele Multhoff

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.