A comparison of two commercially available ELISA methods for the quantification of human plasma heat shock protein 70 during rest and exercise stress

Abstract

This study compared resting and exercise heat/hypoxic stress-induced levels of plasma extracellular heat shock protein 70 (eHSP70) in humans using two commercially available enzyme-linked immunosorbent assay (ELIS)A kits. EDTA plasma samples were collected from 21 males during two separate investigations. Participants in part A completed a 60-min treadmill run in the heat (HOT70; 33.0 ± 0.1 °C, 28.7 ± 0.8 %, n = 6) at 70 % V̇O2max. Participants in part B completed 60 min of cycling exercise at 50 % V̇O2max in either hot (HOT50; 40.5 °C, 25.4 relative humidity (RH)%, n = 7) or hypoxic (HYP50; fraction of inspired oxygen (FIO2) = 0.14, 21 °C, 35 % RH, n = 8) conditions. Samples were collected prior to and immediately upon termination of exercise and analysed for eHSP70 using EKS-715 high-sensitivity HSP70 ELISA and new ENZ-KIT-101 Amp'd(™) HSP70 high-sensitivity ELISA. ENZ-KIT was superior in detecting resting eHSP70 (1.54 ± 3.27 ng · mL(-1); range 0.08 to 14.01 ng · mL(-1)), with concentrations obtained from 100 % of samples compared to 19 % with EKS-715 assay. The ENZ-KIT requires optimisation prior to running samples in order to ensure participants fall within the standard curve, a step not required with EKS-715. Using ENZ-KIT, a 1:4 dilution allowed for quantification of resting HSP70 in 26/32 samples, with a 1:8 (n = 3) and 1:16 (n = 3) dilution required to determine the remaining samples. After exercise, eHSP70 was detected in 6/21 and 21/21 samples using EKS-715 and ENZ-KIT, respectively. eHSP70 was increased from rest after HOT70 (p < 0.05), but not HOT50 (p > 0.05) or HYP50 (p > 0.05) when analysed using ENZ-KIT. It is recommended that future studies requiring the precise determination of resting plasma eHSP70 use the ENZ-KIT (i.e. HSP70 Amp'd(®) ELISA) instead of the EKS-715 assay, despite additional assay development time and cost required.

Keywords: Acute heat stress; Acute hypoxia; Heat shock proteins (HSPs); Human.

Fig. 1

Standard curves (mean ± SD; n = 4) generated in duplicate from the same pre-prepared standards analysed on the same 96-well plate. The amplification steps produce a clear increase in the assay sensitivity when compared to the EKS-715 kit reagents allowing for determination of low levels of eHSP70 in plasma samples

Fig. 2

a, b The optical density and eHSP70 values obtained from each assay. EKS-715 was able to detect eHSP70 in 6 of the 32 resting observations (3.57 ± 2.68 ng·mL⁻¹), whereas ENZ-KIT measured eHSP70 in all 32 resting observations (1.54 ± 3.19 ng·mL⁻¹). When data for an individual was available from both assays (n = 12, c, d) the ENZ-KIT tended to indicate lower values, though this was not statistically significant (p = 0.501; R ² = 0.73). Between test reliability for samples assayed on two different occasions was high (e), with the between test CV 7.86 % and a correlation coefficient of 0.99 (f)

Fig. 3

eHSP70 was detected at all time points in each trial using the ENZ-KIT and was only elevated postexercise in HOT70 (a, n = 6), with no postexercise change in eHSP70 expression observed after HOT50 (b, n = 7) and HYP50 (c, n = 8). Individual data are shown with bars representing mean eHSP70