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

摘要

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?% V?O2max. Participants in part B completed 60?min of cycling exercise at 50?% V?O2max 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) 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.