Pbs 2 bsa

Enzyme-Linked Immunosorbent Assay (ELISA) for sHLA-G and sHLA-E was performed as previously described [14 (link)]. Briefly, MaxiSorp Nunc-Immuno 96-microwell plates (Nunc A/S, Roskilde, Denmark) were coated overnight at 4°C with 1 µg/mL of MEM-G9, specific for HLA-G HC (Exbio, Prague), that recognizes sHLA-G1/G5, or 3D12 mAb, specific for HLA-E HC (eBioscience, Science Center Drive, San Diego, CA, USA). After three washes with PBS 0.05% Tween 20 (washing buffer), plates were saturated with 200 μL/w of PBS 2% BSA (Sigma, St. Louis, MO, USA) for 30 min at RT. One hundred μL of BM plasma samples and serial dilutions of 721.221.G1 cell line supernatant (for HLA-G) or total extract from normal peripheral blood mononuclear cells (standard) were added to each well and incubated at RT for 1 hour. After three washes, 100 μL of detection reagent (HRP-conjugated anti-β2 microglobulin mAb, Exbio, Vestec, CZ) was added, and plates were incubated for 1 hour at RT. After three washes, 100 μL of TMB (substrate for HRP, Sigma) was added, and reaction was stopped after approximately 30 minutes by adding H₂SO₄ 5 N. Absorbance at 450 nm was measured using Infinite® 200 PRO spectrometer (Tecan Group Ltd., Seestrasse, Männedorf, Switzerland). Results are expressed as ng/mL sHLA-G and arbitrary units/mL sHLA-E (1 unit = quantity of sHLA-E in 1 µg of total extract).