Human madcam 1 duoset elisa kit

Two milliliter Blood was collected in SST II Advance BD Vacutainer (RWF 367956). Serum was aliquoted and stored at −80°C for batch analysis. Human MAdCAM-1 DuoSet ELISA kit (R&D Systems-DY6056-05) was used for estimation of soluble MAdCAM-1 in serum in accordance with manufacturer's protocol. Human/Mouse TGF beta 1 Uncoated ELISA kit (Invitrogen) was used for quantification of total TGF-β1 in serum. Serum was diluted 1:5 in Phosphate buffered saline and treated with 1N HCl to activate latent TGF-β1. Following 10 min incubation at room temperature, the reaction was neutralized using 1N NaOH and TGF-β1 was estimated in accordance with manufacturer's protocol.

Human MAdCAM-1 DuoSet ELISA kit (R&D Systems-DY6056-05, Minneapolis, USA) was used for estimation of soluble MAdCAM-1 in plasma in accordance with the manufacturer’s protocol. Briefly, wells coated with capture antibody and reagent diluent (1% BSA in PBS) were added for blocking. MAdCAM standard or 1:100 diluted plasma was added to wells and incubated overnight at 4°C. Incubation with detection antibody was followed by addition of streptavidin-HRP which was allowed to react with a substrate solution to give chromogenic readout. Reaction was stopped using 2N H2SO4, and absorbance was measured at 450 and 570 nm using Synergy H1 microplate reader (Biotek, Vermont, USA). Readings at 570 nm were subtracted from 450 nm and used to plot the standard curve. Plasma concentration of sMAdCAM was calculated by interpolating absorbance values and multiplying by dilution factor.