Fig 2: Representative standard dose-response curve and precision profile for HSP27 measurement. HSP27 was measured by quantifying signals from bound analyte on beads in units of average enzymes per bead (AEB). The average AEB (n = 2), standard deviation (SD) and coefficient of variation (CV) of each calibration point are portrayed in the embedded table. The error bars are indicated.

Fig 3: Quantification of HSP27 and 70 by Western blotting (A) and the newly developed SIMOA technique (B). Peripheral blood mononuclear cells from samples from 7 independent volunteers were lysed unheated or heat shocked at 42 °C for 1h before being lysed. S1 = sample 1 unheated; S1HS = sample 1 heat shocked; HSP = heat shock protein. For samples 1, 5 and 6, the water in the water bath—where samples were heat shocked—penetrated the Petri dishes. However, we decided not to discard them, but rather to use them—for comparison purpose—in both the SIMOA and Western blotting techniques. The signal intensity of HSP—relative to actin—obtained using Western blotting correlated significantly with HSP values using the present SIMOA technique: Spearman correlation test (r = 0.815, p < 0.001 and r = 0.895, p < 0.001 for HSP70 and HSP27, respectively).

Fig 4: HSP25 content increased in proportion to the number of LC. (A) Western blot analysis of HSP25 and GAPDH content in each condition. (B) The 15P, 0P60, 5P60, and 15P60 all significantly elevated HSP25 content relative to CON * p < 0.05. Additionally, 5P60 and 15P60 significantly elevated HSP25 content compared to 5P ** p < 0.01 and 15P60 HSP25 content was significantly higher than that in 15P # p < 0.05. Values are expressed as mean ± SEM.