Indirect ELISA for pB602L Protein

Indirect ELISA was performed as described previously [12 (link)]. In brief, purified recombinant pB602L protein diluted in Tris-HCl buffer (pH 8.5) to a final concentration of 2.5 μg/mL was coated onto 96-well ELISA plates (100 μl/well) and incubated overnight at 4 ℃. The supernatant was discarded, and the plate was washed three times with phosphate-buffered saline (PBS) (pH 7.2-7.4) containing 0.5% Tween-20 (PBST). After blocking with 5% Difco skim milk for 1 h at room temperature, the monoclonal antibodies purified from ascites were added to the ELISA plate (100 μl/well), which was incubated at 37 ℃ for 30 min. After six washes with PBST, a rabbit anti-mouse IgG horseradish peroxidase (HRP) conjugate (whole molecule) (Sigma-Aldrich, USA) was added as a secondary antibody at a dilution of 1:2000 and incubated at 37 ℃ for 30 min, followed by six washes with PBST. TMB substrate solution (TianGen Biotech, China) was added to the plate (100 μl/well), which was incubated in the dark at room temperature for 20 min. The reaction was terminated by addition of 2M H₂SO₄ (50 μl/well), and the optical density was measured at 450 nm using a microplate reader (ELx808, BioTek, USA).