title: 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.
authors.affiliation: Department for Health, University of Bath, Claverton Down, Bath, BA2 7AY, UK. b.j.lee@bath.ac.uk.|Department of Biomolecular and Sport Sciences, Coventry University, Priory Street, Coventry, UK. b.j.lee@bath.ac.uk.
authors.forename: B J
authors.initials: BJ
authors.lastname: Lee
authors.affiliation: Department for Health, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
authors.forename: N M
authors.initials: NM
authors.lastname: Sukri
authors.affiliation: Department for Health, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
authors.forename: H
authors.initials: H
authors.lastname: Ogden
authors.affiliation: Department for Health, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
authors.forename: C
authors.initials: C
authors.lastname: Vine
authors.affiliation: Department of Biomolecular and Sport Sciences, Coventry University, Priory Street, Coventry, UK.
authors.forename: C D
authors.initials: CD
authors.lastname: Thake
authors.affiliation: Department for Health, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
authors.forename: J E
authors.initials: JE
authors.lastname: Turner
authors.affiliation: Department for Health, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
authors.forename: J L J
authors.initials: JL
authors.lastname: Bilzon
mesh_terms: D000293:Adolescent; D000328:Adult; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015444:Exercise / Q000502:physiology; D005080:Exercise Test; D018840:HSP70 Heat-Shock Proteins / Q000097:blood*; D006801:Humans; D008297:Male; D015203:Reproducibility of Results; D055815:Young Adult
chemical_list: D018840:HSP70 Heat-Shock Proteins
keywords: Acute heat stress; Acute hypoxia; Heat shock proteins (HSPs); Human