Ancillary reagent kit 1

The expression levels of EGFR, IGF-IR, and TGFβ1 were encountered in serum samples from NSCLC male patients. Healthy subjects were detected using ELISA, with the Human EGFR DuoSet ELISA (R&D System, Minneapolis, MN, USA, cat No. DY231), Ancillary Reagent Kit 2 (R&D System, cat No. DY008), IGFIR DuoSet ELISA (R&D System, cat No. DY 391), Ancillary Reagent Kit 3 (R&D System, cat No. DY240-05), TGF beta 1 DuoSet ELISA, and Ancillary Reagent Kit 1 (R&D System, cat No. DY 007).

For measurement of transforming growth factor beta-1 (TGF-β1), freshly thawed heparinized plasma (stored at -80°C and not previously thawed) was activated by adding 5 µL of 1.0 M HCl to 10 µL of plasma, and incubated for 10 minutes at room temperature. The reaction was then neutralized by addition of 5 µL of 1.2 M NaCl/0.5M HEPES and the resultant mixture was diluted to a final volume of 400 µL with diluent reagent. The concentration of TGF-ß1 in the diluted activated plasma (100 µL per well, in duplicate) was measured using a human TGF- ß1 DuoSet ELISA Kit and Ancillary Reagent Kit 1 (R&D Systems), with the optical density being read at 450 nm using a FLUOStar^® Omega microplate reader (BMG Labtech). IFN5 scores were determined by measuring the expression levels of five IFN-induced genes (EPSTI1, IFI44L, LY6E, OAS3, and RSAD2) in whole peripheral blood archived in Tempus tubes (Applied Biosystems), using a custom NanoString (NanoString Technologies) (12 (link), 17 (link)). Log₂ normalized expression levels of the 5 genes were summed to generate a composite IFN5 score. Serum IFN-α was measured using patient serum collected and archived at −80°C at the time of recruitment, as previously described (12 (link)).