Levels of serum anti-Hsp70 IgG autoantibodies in patients with EBA and healthy controls

This dataset contains the levels of anti-Hsp70 IgG autoantibodies measured in serum of epidermolysis bullosa acquisita (EBA) (n=20) patients and healthy donors (n=40) by home-made ELISA test and expressed as optical density (OD). 

Method:

Levels of IgG against human Hsp70 were evaluated in the serum samples by a home-made enzyme-linked immunosorbent assay (ELISA), as described previously (Mantej et al. 2019). Medium-binding 96-well plates were coated with commercially available full-length native human Hsp70 protein at a concentration of 0.5 μg/ml in 0.1 M bicarbonate buffer at 4 °C overnight. The wells were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature (RT) for 1.5 h. After a washing step (three times with PBS containing 0.05% Tween 20), the sera were diluted (1:100) in PBS containing 0.1% BSA, added to the wells and were incubated at RT for 1.5 h. Plates were then incubated with horseradish peroxidase (HRP)–conjugated anti-human IgG (Sigma) secondary antibodies diluted in PBS containing 0.1% BSA at RT for 1 h. The TMB substrate solution (Sigma) was used to visualize HRP enzymatic reaction, and the reaction was stopped by adding 0.5 M H2SO4. Optical density measurements were performed at 450 nm with an ELISA plate reader (VICTOR Multilabel Plate Reader, PerkinElmer).

The assay was performed at the Department of Molecular Biology, University of Gdańsk, Poland.

Sera collection of EBA patients and healthy donors belong to Department of Dermatology, Venerology and Allergology, University of Lubeck, Germany and Department of Molecular Biology, University of Gdańsk, Poland, respectively.