Pbst buffer

Polystyrene microtiter 96-well plates (Costar; USA) were coated
The underlined portions of the primer sequences were the restriction enzyme cutting sites (ClyA-F (forward): SacI; ClyA-R (reverse): XbaI; galT-F: XbaI; galT-R: HindIII).
with purified recombinant GalT (2.5 μg/ml in 0.02 M carbonatebicarbonate buffer, pH 9.6), and each well was coated with 100 μl of diluted protein and incubated overnight at 4 o C. The plates were blocked with 3% skim milk at 37 o C for 1.5 h then washed three times using PBST buffer (250 μl/well; Solarbio, China). Serum samples were serially diluted 2-fold in blocking buffer in a range of 10 -2 -10 -5 , and added to the wells (100 ul) and incubated 1 h. The plates were then washed three times. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was diluted (1:5000) with PBST buffer and added (100 μl) onto plates for incubation at 37 o C for 30 min. After washing five times, soluble tetramethylbenzidine (TMB) substrate solution (Tiangen, China) was added (100 μl), and the plates were incubated in the dark at room temperature for 30 min. The reaction was terminated with 2 M H 2 SO 4 and absorbance of each well was read at a wavelength of 450 nm in a microplate absorbance reader (Bio-Rad, USA).