ELlSA was performed using the method described by Hirai et al.32. For preparation of antigens, TGC obtained from 10-week-old normal mice (n = 3) was homogenized in carbonate–bicarbonate buffer (Sigma-Aldrich, MO, USA). Antigen concentrations were determined using the Bradford method with bovine serum albumin as the standard. Antigen was then added to each well of a microtiter plate (Nunc 96-well plate; Thermo Fisher Scientific, Kanagawa, Japan) and incubated at 37 °C for 30 min. After removing the coating solution, the wells were washed three times with PBS-Tween 20; Then, 200 μl of a rabbit anti-mouse monoclonal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (housekeeping gene; Bethyl Laboratories, Inc., TX, USA; 1/200 and 1/1000 dilution) or experimental mouse serum samples serially diluted with 1% goat serum in PBS (each sample run in duplicate) were added to microELISA wells and incubated for 2-h at room temperature. The wells were then washed five times with PBS-Tween 20 and incubated for 2-h at RT with 50 μl of HRP-conjugated goat anti-rabbit IgG (Cappel, PA, USA; 1:1,000), anti-GAPDH antibody, or HRP-conjugated goat anti-mouse IgG (Cappel; 1:1,000). A solution of one Alkaline Phosphatase Yellow (pNPP) tablet and one Tris Buffer tablet (Sigma-Aldrich) dissolved in 5 ml of water was used as a soluble substrate to detect alkaline phosphatase activity. After the wells were again washed five times with PBS-Tween 20, 200 μl of freshly prepared pNPP solution was added to each well. Thirty minutes later, the absorbance at 492 nm was determined using a microtiter plate reader (MPR-A4, Toyo Soda, Tokyo, Japan).