Human tgf beta 1 duoset

Human milk levels of TGF-β1, TGF-β2, and IgA levels were measured via commercial enzyme-linked immunosorbent assay (ELISA) kits. TGF-β1 and TGF-β2 levels were measured with a Human TGF-beta 1 DuoSet and a Human TGF-beta 2 DuoSet, respectively (R&D Systems, Minneapolis, MN, USA). IgA levels were measured with a Human IgA ELISA Quantitation Set (Bethyl Laboratories Inc., Montgomery, AL, USA). All ELISAs were conducted according to the respective manufacturers' instructions. The detection limits were 31.2 pg/mL for TGF-β1 and TGF-β2, and 7.8 ng/mL for IgA.

Protein levels of TGF-β1, TNF-α and IL-6 were measured in the supernatants using sandwich ELISA kits purchased from R&D systems (Human TGF-beta 1 DuoSet, Human TNF-alpha DuoSet, Human IL-6 DuoSet ELISA). Each well of a 96-well plate was coated overnight at room temperature with mouse anti-human capture antibody. The plates were washed with TBS, 0.05% Tween 20, blocked for a minimum of 1 hour with 5% BSA (Sigma-Aldrich) in TBS and washed once more with TBST. Then, 100 μl of each sample diluted with 1% BSA in TBS was incubated for 2 hours at room temperature. Recombinant human TNF-α, TGF-β1 and IL-6 were used as standards. The plates were washed with TBST and then incubated with detection antibody for 2 hours at room temperature. The plates were washed again with TBST and incubated for 20 minutes with HRP-streptavidin at room temperature. The plates were washed once again with TBST and incubated with the substrate solution for 20 minutes. Reactions were stopped with 2N H₂SO₄. The optical density of the colorimetric reaction in each well was determined at 450 nm.