For detection of active GLP-2, a Luminex based assay compatible with the Luminex platform was developed by NMI, University of Tübingen, Germany, in collaboration with Boehringer Ingelheim GmbH & Co. KG, Biberach/Riss, Germany. The Peptide sequence (HADGSFSD) in the N-terminus was synthesized as Cys-Doa-Doa (8-amino-3,6-dioxaoctanoic acid) to increase accessibility of the immunogenic peptide containing variants of the primary sequence of interest. Cystein was included to allow immobilization via maleimide groups on the activated carrier proteins (BSA and Ovalbumin). Monoclonal and polyclonal antibodies to the above sequence were generated in rats and rabbits, respectively. Cross reactivity to GLP-1 was analyzed using the multiplex Luminex assay platform on a Luminex 100 system in accordance to classical Luminex based assays. Briefly, 20 µl of sample were incubated in 100 µl ELISA blocking buffer (#1112589, Roche Diagnostics GmbH, Mannheim, Germany) in the presence of 20 µl suspension of microparticles (1000 beads linked to Mab to active GLP-2) and incubated overnight at 10°C. After washing 3 times with 100 µl 0.05% Tween 20 in PBS, 40 µl biotinylated detection polyclonal antibody solution (active GLP-2, stock solution 0.1 mg/ml; diluted 1:50 in ELISA blocking buffer) was added and incubated on a shaker for 2h in the dark (22°C). After washing, 40 µl of streptavidin-PE (2 µg/ml diluted 1:500 in ELISA blocking buffer) was added and incubated on a shaker for 30 min. in the dark (22°C). After washing, plate was run on a Luminex 100. Detection limit of active GLP-2 was 20 pg/ml with minimal cross reactivity to GLP-1.

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