Detection of heat-shock protein 70 in cow’s milk using ELISA

Abstract

Context Heat-shock protein 70 (HSP70) has been suggested as an effective biomarker to assess heat stress in livestock. Milk sampling being a non-invasive procedure in cows, the detection of HSP70 in milk would be beneficial for heat-stress studies in cows. Aims The objective of the present study was to optimise an ELISA system to detect HSP70 in cow’s milk and to assess its application in quantifying HSP70 in milk collected during two different weather conditions (hot and mild) and from two production groups (low and high yielding). Methods Foremilk samples were collected from 48 Holstein-Friesian cows under two different weather conditions (hot, n = 25 and mild, n = 23) and from two production groups (low yielding, n = 15 and high yielding, n = 33). Milk HSP70 was measured using a newly optimised, competitive in-house ELISA. The assay validation parameters (i.e. dilutional linearity, parallelism, recovery rate, intra-assay and inter-assay precision) were computed. Key results The minimum detection limit and the sensitivity range of the ELISA were 125 ng/mL and 125–4000 ng/mL respectively. The HSP70 concentrations in cow’s milk ranged from 536.88 to 1532.04 ng/mL. There were weak negative correlations (P < 0.05; n = 48) between HSP70 concentration and milk yield, calculated on either a per cow per day (r = −0.39), per cow per am or pm milking (r = −0.29), or per cow per quarter (r = −0.35) basis. Estimated marginal mean (EMM) HSP70 mass (HSP concentration × milk yield) in milk was higher during hot weather than during mild weather in all three of the above yield calculations (P < 0.0001). An increased (P < 0.05) EMM HSP70 mass was found in high-yielding group compared with low-yielding group when yield calculation was based on per cow per quarter. Conclusions A sensitive ELISA system was optimised to quantify HSP70 in cow’s milk. Higher milk HSP70 masses were found in milk samples collected during hot weather and from high-yielding cows. Implications The assay of HSP70 in milk is a useful tool for non-invasive detection of heat stress in cows. Further studies to measure HSP70 in milk samples collected from the same individual cows during different seasons would be needed to better explain and to confirm these findings.