Human hsp70

Serum autoantibodies against human HSP60 (Abcam, Waltham, Boston, MA, USA) and human HSP70 (Abcam, Waltham, Boston, US) were evaluated using in-house developed ELISA. High-binding 96-well plates (Nunc maxisorp) were coated with the respective HSP at a concentration of 1 μg/mL in ELISA Coating Buffer (Bio-Rad, Hercules, CA, USA) at 4 °C overnight. The wells were blocked using an alternative combined blocking buffer (0.5% polyvinyl alcohol solution combined with bovine gelatin solution, at a ratio of 2:1) at room temperature (RT) for 2 h. After being washed with PBS + 0.05% Tween 20 (washing buffer; WB), sera were diluted (1:200 in WB) and incubated at RT for 1 h at 37 °C. The secondary antibodies were incubated at 37 °C for 40 min (horseradish peroxidase-conjugated antihuman IgG and IgM, polyclonal rabbit antihuman, (Agilent-Dako Santa Clara, CA, USA). TMB substrate solution (Sigma-Merck, Munich, Germany) was used to visualize the HRP enzymatic reaction, and the reaction was stopped by 1 M H₂SO₄. Reading was performed at λ = 450/620 nm using the BEP2000 Advanced automated system. Results are expressed in absorbance (OD) and in quantitative (standard curve-based) results. For data comparison, results were handled as continuous, non-normally distributed integers and the alterations of titers were considered.

An in-house ELISA was used to determine anti-Hsp60 and anti-Hsp70 IgM and IgG autoantibodies according to the protocol described previously [21 (link)]. Briefly, ninety-six-well polystyrene plates (Nunc, Roskilde, Denmark) were coated with human Hsp60 (Abcam, Waltham, Boston, MA, USA) and human Hsp70 (Abcam, Waltham, Boston, MA, USA) at a concentration of 1 μg/mL in ELISA coating buffer (Bio-Rad, Hercules, CA, USA) at 4 °C overnight. After blocking the wells with a 2:1 solution of 0.5% polyvinyl alcohol and bovine gelatin, serum samples were incubated in duplicate at 1:200 dilution in washing buffer (PBS, 0.05% Tween 20) for 1 h at 37 °C. After incubation with HRP-conjugated anti-human IgG- and IgM-specific secondary antibodies (Agilent-Dako Santa Clara, CA, USA) for 40 min at 37 °C, the reaction was visualized using TMB solution, and the OD was read at λ = 450/620 nm using the BEP 2000 Advance automated system (Siemens, Marburg, Germany).