2.2.2. Calumenin Assay

Serum calumenin was determined using commercial sandwich ELISA kit (ELH-CALU-1 Raybiotech, Peachtree Corners, GA, USA) according to the manufacturer's instructions. The intra-assay and inter-assay coefficient of variation were 10% and 12%. The minimum detectable concentration of calumenin was 0.55 ng/mL.

#### 2.2.3. Cytohesin 2 Assay

Serum cytohesin 2 levels were assayed using commercial competitive ELISA kit (ABIN857811, MyBiosource, San Diego, CA, USA). The intra-assay and inter-assay coefficient of variation were 10% and 10%. The minimum detectable concentration of cytohesin 2 was 0.1 ng/mL.

All samples were analyzed in duplicate, with optical densities evaluated at 450 nm using a microplate reader (Dynex, Buštˇehrad, Czech Republic). In order to evaluate the standard curve and estimate the concentration of the samples, a four-parameter logistic regression curve was used (Aladyn software, Prague, Czech Republic). Results were presented as nanograms per milliliter (ng/mL).
