P9135

Fourier Transform Infrared (FTIR) Spectroscopy was used to verify the chemical nature of the extracted material.
Each sample was incorporated with KBr (weight ratio KBr:pectin 10:1) and pressed into pellets. The FTIR spectra were collected at the absorbance mode in the region of 500–4000 cm⁻¹ with an FTIR Vertex 70v Bruker instrument (Billerica, MA, USA). Every measure is the averaged result of 128 consecutive acquisitions.
Commercial pectin (Sigma-Aldrich P9135, St. Loius, MO, USA) was analyzed for comparison.

The ELISA binding assay was performed according to Decreux and Messiaen (2005 (link)) with modifications. Nunc Maxisorp flat-bottom plate wells (Invitrogen, CA, USA) were coated with commercial pectin from citrus peel (P9135; Sigma-Aldrich, USA; https://www.sigmaaldrich.com) or pectin extracted from C. plantagineum leaves (250 µg ml⁻¹, 100 µl well^–1) at 4 °C overnight and then wells were incubated with the following solutions for the indicated time: 100 µl of 3% (w/v) low fat dried milk in wash buffer (20 mM Tris–HCl, 150 mM NaCl, pH 8.0) for 2 h; 50 µl of purified His-tagged CpGLP1 protein in binding buffer (1% low fat dried milk, 20 mM Tris–HCl, 150 mM NaCl, 2 mM CaCl₂, pH 8.0) for 2 h; wash buffer (four times, briefly); 50 µl of anti-His tag antibody (1:10,000) (Jung et al. 2019 (link)) in incubation buffer (1% low fat dried milk, 20 mM Tris–HCl, 150 mM NaCl, pH 8.0) for 1 h; wash buffer (four times, briefly); 50 µl of goat anti-rabbit IgG peroxidase antibody (1:10,000) (Sigma, A9169) in incubation buffer for 1 h; wash buffer (six times, briefly). The bound recombinant CpGLP1 protein was visualized in the presence of the TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Sigma, T2885). The absorbance was measured at 450 nm after color development in the dark and the reaction was stopped by adding 50 µl of 10% (v/v) phosphoric acid.