Indirect ELISA
The iELISA was performed according to the previous procedure (Gimenez-Lirola et al., 2016). In brief, purified recombinant pB602L protein was coated on 96-well ELISA plates(2.5 μg/ml, 100 μl/well) in Tris-HCl buffer (pH 8.5) and incubated overnight at 4℃. The coating solution was discarded and the plate was rinsed with phosphate buffered saline (PBS) (pH 7.2-7.4) add 0.5% Tween-20 (PBST) for three times. The hybridoma cell or monoclonal antibodies purified from ascites was added to the ELISA plate (100 μl/well) and incubated for 30 min at 37℃. After six washes with PBST for 1 min each, the rabbit anti-mouse IgG (whole molecule) (Sigma-Aldrich, USA) as secondary antibody was added at a dilution of 1:2000 and incubated for 30 min at 37℃, followed by six washes with PBST. Antibody binding was analyzed by adding TMB Substrate Solution (100 μl/well) (TianGen Biotech, China) for 20 min. The reaction was stopped by 2M H2SO4 (50 μl/cell), and the optical density value was measured at 450 nm using a microplate reader (ELx808, BioTek, U.S.A).
The antibody subtypes were identified using the SBA Clonotyping System-HRP Kit (SouthernBiotech, USA). The capture antibody was diluted to a concentration of 5-10 μg/mL in PBS and added to the ELISA plates (100 μl/well) incubating at 4℃ overnight, then the HRP-labeled second antibodies (IgA-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, Kappa-HRP, and Lambda-HRP) were added (1:500 dilution in 5% Difco Skim Milk) and incubated for 1 h at 37℃. The other steps are the same as above.