*2.7. ELISA*

The protein expression of KRT19 and HSPD1 in lesional and healthy skin was evaluated using ELISA kits (MyBiosource, Inc., San Diego, CA, USA, MBS2703060 and MBS450548, respectively) according to the manufacturer's protocol. Briefly, tissue samples were prepared in lysis buffer (25 mg per 1 mL), homogenized, centrifuged (10,000 g; 5 min; 4 ◦C), aliquoted, and stored at −80 ◦C. Before the experiment, samples, blanks, and standards were loaded on 96-well plates and incubated for 1 h at 37 ◦C. Then, the solutions were replaced by detection reagent A and incubation continued for the same period of time. After washing with wash solution (3 × 2 min) detection reagent B was added and incubation continued for another 20 min. Then, the wells were washed again (5 × 2 min). The presence of antigen was visualized with the chromogenic substrate 3,3′ ,5,5′ -tetramethylbenzidine (TMB) and assayed using a microplate reader (Bio-Rad, Hercules, USA) at the wavelength 450 nm. The antigen was quantified with a standard curve generated with standards of known concentrations.

The blood levels of E2, PG, and TS were analyzed using ELISA kits (Diagnostics Biochem Canada, Inc., London, ON, Canada CAN-F-430, CAN-PRE-4500, and CAN-TE-250) according to the instructions provided by the manufacturer. To perform an assay, the calibrator, control, and specimen samples were loaded on 96-well plates and mixed with aliquots of horse radish peroxidase (HRP) conjugated to a tested hormone. The plate was incubated for 1 h at room temperature on a shaker (200 rpm). After washing 3 times with provided washing buffer, TMB was added and the incubation continued for another 10–15 min. Then, the presence of a tested hormone was revealed by measuring the absorbance using a microplate reader (Bio-Rad, Hercules, CA, USA) at the wavelength 450 nm. The antigen was quantified with a standard curve generated with standards of known concentrations.
