Pyy elisa kit

The mouse MPO ELISA kit was purchased from Abcam (Cat. No. ab275109), PYY ELISA kit from Aviva Systems Biology (Cat. No. OKCD05062, USA), mouse TNF-α and IL-6 ELISA kits from Biolegend (Cat. No. 431,301 and 430,901, USA). The protocols were performed as the manufacturers described. In brief, 96-well plates were coated with the corresponding capture antibody. After incubation with blocking buffer (PBS + 1% bovine serum albumin), a 100-µl aliquot of either standards, serum, or colonic protein extracts (5 mg tissue per mL PBS supplemented with protease inhibitors) was added into each well individually and the plates were incubated for 2 h at room temperature. The detector antibody was added to the wells, the plate was incubated, and unbound detector antibody was removed by washing. The avidin-peroxidase conjugate was added, the plate was incubated, and unbound conjugate was removed by washing. The enzymatic reaction catalyzed by horseradish peroxidase (HRP) was initiated by adding the substrate, 3,3’,5,5’ tetramethylbenzidine dihydrochloride (TMB). The absorbance was measured by using a microplate reader at a wavelength 450 nm after the reaction was stopped with 2 M H₂SO₄.

Millipore GLP-1 Total ELISA kit (Cat # EZGLP1T-36 K—sensitivity: 1.5 pM) and Aviva Systems Biology PYY ELISA kit (Cat # OKEH04432—sensitivity: 8.14 pg/mL) was used to analyze the portal plasma concentrations after fasting at the end of experiment 1. Samples where the absorbance did not fall within the ELISA standard curve range or where the coefficient of variability was above 10% were removed. ELISAs were run according to manufacturer guidelines and were read at 450 nm and 590 nm (GLP-1) and 450 nm (PYY) to determine pM gut peptide concentrations.