Figure 1: Validation of the lipHsp70 ELISA. (A) Representative eight point calibration curve obtained from the lipHsp70 ELISA using a four-parameter fit model (inset). The OD (mean corrected) is indicated on the Y-axis and the Hsp70 concentration (ng/ml) on the X-axis. (B) Linearity of the lipHsp70 ELISA was assessed by comparing first-, second- and third-order model fits of a relative concentration vs. system output plot. Linearity was shown within a concentration range of 0.36‑17.4 ng/ml. (C) Recovery of free Hsp70 in buffer: 2.5 ng/ml Hsp70 was spiked into dilution buffer. The lipHsp70 ELISA recovered 2.53 ± 0.09ng/ ml (left panel) and thus revealed a significantly better recovery (101 ± 3%, right panel) compared to the control ELISA (1.43 ± 0.07 ng/ml, left panel; 57 ± 3%,right panel). The data show the mean of n=20 tests. (D) Recovery of free Hsp70 in serum: 2.5 ng/ml and 5 ng/ml Hsp70 were spiked intoserum (diluted 1:5). The lipHsp70 ELISA recovered 1.98 ± 0.12 ng/ml and 3.82 ± 0.12 ng/ml for 2.5 and 5 ng/ml (left panel) and thus revealed a significantly better recovery (mean 78 ± 3%, right panel) compared to the control ELISA (1.41 ± 0.07 ng/ml and 2.18 ± 0.09 ng/ml, left panel; mean 50 ± 3%, right panel). Black bars: lipHsp70 ELISA, white bars: control ELISA, dashed line: expected amount of Hsp70. *p<0.05,***p<0.001 (t‑test).