Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Cell Stress and Chaperones Aims and scope

A Letter to the Editor to this article was published on 04 August 2015

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Amorim FT, Yamada PM, Robergs RA et al (2008) The effect of the rate of heat storage on serum heatshock protein 72 in humans. Eur J Appl Physiol 104:965–972

  • Armstrong LE, Maresh CM, Castellani JW, Bergeron MF, Kenefick RW, LaGasse KE, Riebe D (1994) Urinary indices of hydration status. Int J Sport Nutr 4(3):265–279

  • Asea A (2006) Initiation of the immune response by extracellular Hsp72: chaperokine activity of Hsp72. Curr Immunol Rev 2:209

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Borg G (1976) Simple rating methods for estimation of perceived exertion. Phys Work Effort 39–46

  • Campisi J, Leem TH, Fleshner M (2003) Stress-induced extracellular Hsp72 is a functionally significant danger signal to the immune system. Cell Stress Chaperones 8:272

  • Cheuvront SN, Chinevere TD, Ely BR et al. (2008) Serum S-100 beta response to exercise-heat strain before and after acclimation. In:DTIC Document

  • Chung J, Nguyen A-K, Henstridge DC et al (2008) HSP72 protects against obesity-induced insulin resistance. Proc Natl Acad Sci 105:1739–1744

  • Dulin E, García-Barreno P, Guisasola MC (2010) Extracellular heat shock protein 70 (HSPA1A) and classical vascular risk factors in a general population. Cell Stress Chaperones 15:929–937

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Febbraio MA, Steensberg A, Walsh R et al (2002) Reduced glycogen availability is associated with an elevation in HSP72 in contracting human skeletal muscle. J Physiol 538:911–917

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Febbraio MA, Mesa JL, Chung J et al (2004) Glucose ingestion attenuates the exercise-induced increase in circulating heat shock protein 72 and heat shock protein 60 in humans. Cell Stress Chaperones 9:390

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Fehrenbach E, Niess A, Voelker K et al (2005) Exercise intensity and duration affect blood soluble HSP72. Int J Sports Med 26:552–557

    Article  CAS  PubMed  Google Scholar 

  • Fink AL (1999) Chaperone-mediated protein folding. Physiol Rev 79:425–449

    CAS  PubMed  Google Scholar 

  • Garrido C, Gurbuxani S, Ravagnan L et al (2001) Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun 286:433–442

    Article  CAS  PubMed  Google Scholar 

  • Gibson OR, Dennis A, Parfitt T et al (2014) Extracellular Hsp72 concentration relates to a minimum endogenous criteria during acute exercise-heat exposure. Cell Stress Chaperones 19:389–400

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Girard O, Racinais S (2014) Combining heat stress and moderate hypoxia reduces cycling time to exhaustion without modifying neuromuscular fatigue characteristics. Eur J Appl Physiol 114:1521–1532

    Article  PubMed Central  PubMed  Google Scholar 

  • Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381:571–579

    Article  CAS  PubMed  Google Scholar 

  • Horowitz M (2007) Heat acclimation and cross-tolerance against novel stressors: genomic–physiological linkage. Prog Brain Res 162:373–392

    Article  CAS  PubMed  Google Scholar 

  • Hubbard R, Bowers W, Matthew W et al (1977) Rat model of acute heatstroke mortality. J Appl Physiol 42:809–816

    CAS  PubMed  Google Scholar 

  • Ianaro A, Ialenti A, Maffia P et al (2001) HSF1/hsp72 pathway as an endogenous anti-inflammatory system. FEBS Lett 499:239–244

    Article  CAS  PubMed  Google Scholar 

  • Jones AM, Doust JH (1996) A 1% treadmill grade most accurately reflects the energetic cost of outdoor running. J Sports Sci 14:321–327

    Article  CAS  PubMed  Google Scholar 

  • Kampinga HH, Hageman J, Vos MJ et al (2009) Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones 14:105–111

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Krause M, Heck TG, Bittencourt A et al. (2015) The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediat Inflamm 2015

  • Kregel KC (2002) Invited review: heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol 92:2177–2186

    Article  CAS  PubMed  Google Scholar 

  • Lancaster G, Møller K, Nielsen B et al (2004) Exercise induces the release of heat shock protein 72 from the human brain in vivo. Cell Stress Chaperones 9:276

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lee BJ, Emery-Sinclair EL, Mackenzie RW et al (2014a) The impact of submaximal exercise during heat and/or hypoxia on the cardiovascular and monocyte HSP72 responses to subsequent (post 24 h) exercise in hypoxia. Extreme Physiol Med 3:15

    Article  Google Scholar 

  • Lee BJ, Mackenzie RW, Cox V et al. (2014b) Human monocyte heat shock protein 72 responses to acute hypoxic exercise after 3 days of exercise heat acclimation. BioMed Research International, Article ID 849809

  • Luo X, Zuo X, Zhou Y et al (2008) Extracellular heat shock protein 70 inhibits tumour necrosis factor-alpha induced proinflammatory mediator production in fibroblast-like synoviocytes. Arthritis Res Ther 10:R41

    Article  PubMed Central  PubMed  Google Scholar 

  • Marfell-Jones M, Olds T, Stewart A et al. (2006) ISAK: Potchefstroom: International Standards for Anthropometric Assessment

  • Moran DS, Shitzer A, Pandolf KB (1998) A physiological strain index to evaluate heat stress. Am J Physiol-Regul Integr Comp Physiol 275:R129–R134

    CAS  Google Scholar 

  • Moseley PL (1997) Heat shock proteins and heat adaptation of the whole organism. J Appl Physiol 83:1413–1417

    CAS  PubMed  Google Scholar 

  • Najafizadeh SR, Ghazizadeh Z, Nargesi AA et al. (2015) Analysis of serum heat shock protein 70 (HSPA1A) concentrations for diagnosis and disease activity monitoring in patients with rheumatoid arthritis. Cell Stress Chaperones:1–7

  • Ogawa K, Kim H-K, Shimizu T et al (2012) Plasma heat shock protein 72 as a biomarker of sarcopenia in elderly people. Cell Stress Chaperones 17:349–359

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ortega E, Giraldo E, Hinchado M et al (2006) Role of Hsp72 and norepinephrine in the moderate exercise-induced stimulation of neutrophils’ microbicide capacity. Eur J Appl Physiol 98:250–255

  • Ortega E, Hinchado M, Martin-Cordero L et al (2009) The effect of stress-inducible extracellular Hsp72 on human neutrophil chemotaxis: a role during acute intense exercise. Stress 12:240–249

  • Périard J, Patricia R, Caillaud C et al. (2012a) Plasma Hsp72 and Hsp27 during moderate and intense exercise to exhaustion in the heat. In: Proceedings of The Physiological Society. The Physiological Society

  • Périard JD, Ruell P, Caillaud C et al (2012b) Plasma Hsp72 (HSPA1A) and Hsp27 (HSPB1) expression under heat stress: influence of exercise intensity. Cell Stress Chaperones 17:375–383

    Article  PubMed Central  PubMed  Google Scholar 

  • Pockley A, Shepherd J, Corton J (1998) Detection of heat shock protein 70 (Hsp70) and anti-Hsp70 antibodies in the serum of normal individuals. Immunol Investig 27:367–377

    Article  CAS  Google Scholar 

  • Rodrigues-Krause J, Krause M, O’hagan C et al (2012) Divergence of intracellular and extracellular HSP72 in type 2 diabetes: does fat matter? Cell Stress Chaperones 17:293–302

    Article  PubMed Central  PubMed  Google Scholar 

  • Ruell PA, Simar D, Périard JD et al (2014) Plasma and lymphocyte Hsp72 responses to exercise in athletes with prior exertional heat illness. Amino Acids 46:1491–1499

    Article  CAS  PubMed  Google Scholar 

  • Ruell PA, Thompson MW, Hoffman KM et al (2006) Plasma Hsp72 is higher in runners with more serious symptoms of exertional heat illness. Eur J Appl Physiol 97:732–736

  • Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ, Stachenfeld NSl (2007) American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sports 39(2):377–390

  • Schick C, Arbogast M, Lowka K et al (2004) Continuous enhanced expression of Hsc70 but not Hsp70 in rheumatoid arthritis synovial tissue. Arthritis Rheum 50:88–93

    Article  CAS  PubMed  Google Scholar 

  • Selkirk GA, Mclellan TM, Wright HE et al (2009) Expression of intracellular cytokines, HSP72, and apoptosis in monocyte subsets during exertional heat stress in trained and untrained individuals. Am J Physiol-Regul Integr Comp Physiol 296:R575–R586

    Article  CAS  PubMed  Google Scholar 

  • Taylor HL, Buskirk E, and Henschel A (1955) Maximal oxygen intake as an objective measure of cardio-respiratory performance. J Appl Physiol 8:73–80

  • Whitham M, Fortes M (2006) Effect of blood handling on extracellular Hsp72 concentration after high-intensity exercise in humans. Cell Stress Chaperones 11:304

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Whitham M, Fortes MB (2008) Heat shock protein 72: release and biological significance during exercise. Front Biosci 13:1328–1339

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Morgan Mathieu, Ph.D., for his technical advice during the optimization steps of the ELISA assays. We would also like to thank all the volunteers for their time during the study.

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Department for Health, University of Bath, Claverton Down, Bath, BA2 7AY, UK

    B. J. Lee, N. M. Sukri, H. Ogden, C. Vine, J. E. Turner & J. L. J. Bilzon

  2. Department of Biomolecular and Sport Sciences, Coventry University, Priory Street, Coventry, UK

    B. J. Lee & C. D. Thake

Authors
  1. B. J. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. N. M. Sukri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. Ogden
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. Vine
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. D. Thake
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J. E. Turner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J. L. J. Bilzon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B. J. Lee.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee, B.J., Sukri, N.M., Ogden, H. et al. A comparison of two commercially available ELISA methods for the quantification of human plasma heat shock protein 70 during rest and exercise stress. Cell Stress and Chaperones 20, 917–926 (2015). https://doi.org/10.1007/s12192-015-0610-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12192-015-0610-3

Keywords

Search

Navigation