Anti gal 3 antibody

Experiments was performed as described previously, with slight modifications^{36 (link)}. The 96-well ELISA plates (Costar High binding 3590, Fisher Scientific, Japan) were coated with Aggregatibacter actinomycetemcomitans-LPS (A.a.-LPS) kindly provided by Dr. Tatsuji Nishihara of Kyushu Dental College, Japan), P.g.-LPS, Escherichia coli-LPS (E. coli-LPS; Wako Pure Chemical Industries, Ltd., Ohsaka, Japan) (50 μg/ml each), or 3% bovine serum albumin (BSA; as a control) and incubated overnight at 4 °C. Plates were washed twice with ELISA buffer (Tris-buffered saline with 0.05% Tween 20, 1 mM CaCl₂, and 0.1% BSA, pH 7.4), and 3% Gelatin was used for blocking at 37 °C for 1 h. rhGal-3 was added to each well at different final concentrations (0, 0.05, 0.15, 0.6, or 2 μg/ml) and incubated overnight at 4 °C. An anti-Gal-3 antibody (BioLegend, Inc.) was also added to each well, and the plates were incubated overnight at 4 °C. For detection, a horseradish peroxidase-conjugated anti-rat IgG (GE Healthcare Life Sciences) was added, and tetramethyl benzidine substrate (colour reagent A & B, 1: 1, R&D Systems) was used for visualization. After 20 min, stop solution (R&D Systems) was added. O.D.s were measured at 450 nm.