3 3 5 5 tetramethylbenzidine (tmb)

To screen for positive hybridoma cell clones and linear B cell epitopes, indirect ELISA was performed as previously described (Qiu et al., 2012 (link)), with some modifications. Briefly, immuno-microtiter plates were coated with 5 μg/mL of peptides or 3 μg/mL of proteins in carbonate-buffered saline (pH = 9.6) at 4°C overnight. Plates were washed three times with PBST (0.01 M PBS, pH 7.2; 0.05% Tween 20) and blocked with 4% bovine serum albumin at 37°C for 2 h. Hybridoma cell supernatant (1:1) and serum (1:50 to 1:102,400) were diluted and added to the plate in a volume of 100 μL/well and incubated at 37°C for 1 h. After washing five times with PBST, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:10,000 diluted) was added as the secondary antibody and plates were incubated at 37°C for 45 min. After washing five times with PBST, 100 μL chromogenic solution (TMB, Sangon Biotech) was added and incubated at 37°C for 20 min. The absorbance was measured using a microplate reader at an absorption of 450 nm. An mAb to the P30 protein of African swine fever virus (ASFV) was used as a negative control, and test results were calculated as the sample to cut-off (S/CO) ratio: S/CO value = sample detection value/cutoff (CO) value, where the CO value = 2.1 × negative-control detection value. An S/CO ratio ≥ 1.0 was considered as positive.

The affinity of the purified VHH antibody with CTGF was evaluated by ELISA. The VHH or mouse anti-CTGF-C mAb was immobilized onto a 96-well microtiter plate by adding 200 μg/ml × 100 μl protein to each well followed by an overnight incubation at 4 °C. The uncoupled protein was washed away and 100 μl 1 % BSA was added to block the uncoupled sites. The plate was incubated at 37 °C for 2 h after 50 μl of CTGF ranging from 256 to 0.125 μg/ml was added to each well. Afterwards, a volume of 50 μl s antibody (1:4000 dilution, horseradish peroxidase-conjugated mouse anti-human CTGF polyclonal antibody) was added. The plate was washed with 2.0 % Tween-20/TBS before each round of addition. A volume of 50 μl 3,3′,5,5′-tetramethylbenzidine (TMB, Sangon, China) of 100 μg/ml was added to each well and incubated for 10 min. H₂SO₄ (1 M, 50 μl/well) was added and the plate was sent for colorimetry to measure the absorbance at 450 nm (Alisei, Italy).