Human il 17 duoset elisa kit

Levels of SDF-1α, VEGF, MMP-9, IL-8 and IL-17 in serum samples were measured by commercial quantitative colorimetric sandwich enzyme-linked immunosorbent assay (Human CXCL12/SDF-1α Quantikine ELISA Kit, Human VEGF Quantikine ELISA Kit and Human IL-17 DuoSet ELISA Kit, all from R&D Systems, Minneapolis, MN, USA; Human IL-8/NAP-1 INSTANT ELISA Kit and Human MMP-9 Platinum ELISA Kit, both from eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. The detection range was 0.156–10.0 ng/ml for SDF-1α, 31.2–2,000 pg/ml for VEGF, 0.23–15.0 ng/ml for MMP-9, 15.6–1,000 pg/ml for IL-8 and 15.6–1,000 pg/ml for IL-17. Serum samples were diluted 1:250 for the MMP-9 assay. Concentrations were calculated using a standard curve generated with specific standards provided by the manufacturer. Each sample was measured in duplicate.

A modified ELISA was used for measuring interferon-γ (IFN-γ) secretion in cell-culture
supernatants. Enhanced binding plates (Thermo Scientific) were coated with human IFN-γ
capture antibody (Thermo Fisher Scientific) in a binding buffer
(0·1 m-Na₂HPO₄) and incubated overnight at +4°C. Blocking
was performed using 1 % bovine serum albumin in PBS. The plates were washed with 0·05 %
Tween in PBS. IFN-γ in undiluted culture supernatant samples was detected using
biotinylated secondary IFN-γ antibody (Thermo Fisher Scientific) and biotin-specific
streptavidin–alkaline phosphatase (Invitrogen) with
p-nitrophenylphosphate (Sigma-Aldrich) for colour formation and intensity
readings at 405 nm. Recombinant human IFN-γ (R&D Systems) at different dilutions
was used for constructing a standard curve for calculation of the concentration of
secreted IFN-γ in the samples. Secreted IL-17A in cell-culture supernatants was detected
using the Human IL-17 DuoSet ELISA Kit (catalogue no. DY317) according to the
manufacturer's instructions (R&D Systems). To prevent inter-assay variation, the
supernatant samples from one experiment including different treatments were always
analysed in the same assay, i.e. on the same ELISA plate. The detection limit was
determined as the lowest standard dilution in the analysis (0·78 ng/ml for IFN-γ and
15·6 pg/ml for IL-17A).