3 3 5 5 tetramethylbenzidine tmb solution

The binding activity of rTcCTL15 to PAMPs was examined using an enzyme-linked immunosorbent assay (ELISA) according to previous reports [8 (link)]. Briefly, 50 μL of LPS or PGN (80 μg/mL) was coated on the wells of a 96-well microplate at 37 °C overnight and then evaporated at 60 °C for 30 min. After blocking with BSA (100 μL/well of 1 mg/mL) in TBS for 2 h at 37 °C, 100 μL serial concentrations of rTcCTL15 (0–70 μg/mL) were added to each well and incubated at room temperature for 3 h. The plate was washed with TBS, and 100 μL/well of an anti-His mouse polyclonal antibody (TransGen, Beijing, China) diluted to 1:2000 with TBS was added and incubated at 37 °C for 1 h. The plate was rewashed with TBS, and 100 μL/well of a goat anti-mouse IgG horseradish peroxidase (HRP) conjugate (TransGen, Beijing, China) diluted to 1:5000 with TBS was added and incubated at 37 °C for 1 h. After washing with TBS, 100 μL/well of a soluble 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Solarbio, Beijing, China) was added and incubated at 28 °C for 10 min under darkness and then stopped with 100 μL/well of 2 mol L⁻¹ H₂SO₄. After the termination of the reaction, an automatic enzyme-linked immunosorbent assay reader (BioTek, Winosky, VT, USA) was utilized to measure the absorbance at 450 nm. Each binding assay was repeated three times.

EPS-HD270 (2.0 mg/mL, 100 μL) was loaded into 96-well ELISA plates (Nunc Maxisorb, Thermo) and immobilized at 4 °C overnight. The ELISA plates were washed three times with Tris-buffered saline (TBS) and blocked with TBST (TBS containing 0.1% Tween-20) containing 2.0% BSA (200 μL) at 37 °C for 2 h. The plates were washed with TBST three times and incubated with different concentrations of Vip3Aa11 (0, 0.5, 1, 2, 4, 8, 16, 32 nmol/L) (100 μL) for 1 h at 37 °C. After washing with TBST buffer 3 times, anti-Vip3A antibody (TBST 1:5000 dilution, 100 μL) was added. TBST (100 μL) containing 1/10,000 HRP-conjugated goat anti-mouse IgG (Solarbio Life Sciences, Beijing, China) was incubated at 37 °C for 1 h. For each treatment, three replicates were performed. After washing, the reaction was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Solarbio Life Sciences, Beijing, China) (100 μL) for 15 min in the dark at 37 °C. The reaction was terminated with HCl (2.0 mol/L, 100 μL), and the absorbance was immediately read at 450 nm using a microplate reader. The equilibrium dissociation constant (K_d) was analyzed using Sigma-plot Software (Version 12.0).

The blood was obtained from the mouse angular vein for the detection of the levels of antigen-specific antibody using ELISA as follows: the 96-well ELISA plates (Corning Laboratories, Corning, NY) were coated overnight at 4°C with 100 μL of 10 μg/mL CCF. 100 μL of 1 : 100 diluted serum was added into the plates and then incubated at 37°C for 1 h. Then 100 μL of diluted HRP-conjugated goat anti-mouse IgG1 and IgM (Santa Cruz, diluted 1 : 2000) was added as the second antibody and then incubated at 37°C for 1 h. The color reaction was performed through adding the substrate 3,3,5,5-tetramethylbenzidine (TMB) solution (Solarbio). The absorbance value was detected at 450 nm using a plate reader. Each sample was measured in triplicate.