Interleukin 6 (il 6)

ELISA was performed on purified phage preparations of different concentrations (10⁹, 10⁷, and 10⁵ PFU/well) using commercially available kits IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α (Mabtech AB, Stockholm, Sweden) according to the manufacturer’s instructions. Optical density (OD) was determined at 405 nm on a micro-plate reader (Molecular Devices Corp, Sunnyvale, CA, USA). Data were analyzed in the SoftMax Pro 5.2 rev C software package (Molecular Devices Corp, Sunnyvale, CA, USA).

Cytokine release was quantified using human interleukin (IL)-6 (Mabtech, Stockholm, Sweden), IL-8, and IL-1α (R&D Systems, Minneapolis, MN, USA) enzyme-linked immunosorbent assay kits according to the manufacturers’ instructions. The optical density was measured at 450 nm with reference measurement at 620 nm using a plate photometer (SPECTROstar Omega, BMG Labtech GmbH, Ortenberg, Germany). Interleukin concentrations were calculated according to a standard curve using a 4-parameter fit with lin-log coordinates for optical density. The concentration was expressed as fold secretion compared to the control.

Secreted levels of the cytokines IL-1ra (R&D Systems-BioTechne), IL-1β, IL-6, IL-10, IL-17A and IFN-γ (MabTech AB) were measured in cell culture supernatants using sandwich ELISA kits according to the manufacturer’s instructions. Absorbance was measured at a wavelength of 405 nm using a micro-plate reader (Molecular Devices Corp.) and results analysed using SoftMax Pro 5.2 rev C (Molecular Devices Corp.).

Primary human monocyte-derived macrophages were plated in 96 well plates at concentration of 3x10⁴ cell/well. Next day cells were pretreated with 1 μM of the TLR4 inhibitor TAK-242 (Invivogen, San Diego, CA) for 2 h followed by treatment with 20 ng/ml ProTα variants or controls: ultra-pure LPS (10 ng/ml) or TLR3 ligand PolyI:C (50 μg/ml) (Invivogen, San Diego, CA). Cells were then either infected with VSV envelope-pseudotyped HIV-1 (MOI: 0.01) expressing the luciferase reporter gene or left uninfected. Supernatants collected at 6 h and 48 h post-infection were analyzed by ELISA for the presence of IFN-β (VeriKine-HS^TM kit from PBL,Piscataway, NJ; sensitivity of the assay is 1.2 pg/ml), and TNF-α and IL-6 (Mabtech, Cincinnati, OH; assay sensitivity for both IL-6 and TNF-α is 13 pg/ml). The anti-HIV-1 activity was assessed by measuring in parallel the firefly luciferase expression in the cell lysates. Luciferase values were normalized relative to the corresponding sample’s protein concentration.

Enzyme-linked immunosorbent assays (ELISAs) for quantification of IP-10 (CXCL10), RANTES (CCL5), eotaxin-1 (CCL11), VEGF-A, IL-8 (CXCL8) (all Peprotech), TNF and IL-6 (both Mabtech), were performed according to the manufacturer’s protocols.
The production of nitric oxide (NO) was measured indirectly in cell culture supernatants by determination of the level of nitrite using the Griess assay as previously described [20 (link)].