Opd substrate

BD Heparin Binding Plates (BD Biosciences) were used in all assays. HS, DS, and heparin (Iduron) were dissolved in PBS at concentrations of 0.05–12.8 μm based on approximate “average” disaccharide MWs. Plates were coated with GAG overnight at room temperature then washed with Wash Buffer (100 mm NaCl, 50 mm NaAc, 0.2% Tween20, pH 6.0), blocked with Wash Buffer +1% BSA (Sigma) for 90 min at 37 °C. Following incubation with 200 ng/ml CXCL12 in PBS for 90 min at 37 °C, wells were blocked with Wash Buffer/5% goat serum (Vector Labs) before adding 1:1000 polyclonal rabbit αhuman CXCL12 antibody (AbCam) for 1 h at room temperature. Wells were stained with biotinylated goat αrabbit IgG (Vector Labs,1:2000), amplified with Vectastain Elite ABC reagent (Vector Labs) and developed with OPD substrate (Sigma) as per manufacturer's instructions. Absorbance at 490 nm was measured using a Synergy HT Microplate Reader (BioTek).

Synthetic peptides (Chinese Peptide Company) were immobilized on 96-well microtiter plate (50 μg/mL) in 50 mM carbonate buffer, pH 9.0 (OV at 4°C). Wells were washed with PBS supplemented with Tween-20 (0.05%) (PBST), blocked for 2 h at RT with PBS/BSA and incubated with patient sera in PBS (dilutions 1/100 up to 1/1,600). After 2 h at RT, wells were washed with PBST, incubated with goat anti-human IgG conjugated to horseradish peroxidase (Sigma Aldrich), washed with PBST and developed with OPD substrate (Sigma).

Antibody titers present in sera of immunized pigs were determined by iELISA with the chimeric protein and horseradish peroxidase-conjugated anti-pig IgG (H + L) (Bethyl, Montgomery, Texas, USA). ELISA plates were coated overnight at 4°C with 4 µg/mL recombinant protein (100 µL per well) in bicarbonate buffer, and the plates were washed 3 times with PBS-T. The plates were blocked with PBS-T containing 5% skimmed milk (Svelty-Nestlé) for 1 h at 37°C and 200 rpm and washed 3 times. A total of 100 µL of each serum was added to the plates from days 0 and 31 after immunization at serial dilutions from 1:2,000 to 1:4,096,000. The plates were incubated at 37°C for 1 h and 200 rpm and washed 3 times. A 1:200,000 diluted anti-pig IgG (100 µL per well) was added and incubated for 45 min at 37°C and 200 rpm, and the plates were washed three more times. OPD substrate (Sigma Aldrich, Germany) was added and incubated for 25 min, and the optical density (O. D.) was measured at 450 nm. Statistical significance was tested by Tukey’s multivariable comparison test, α=0.05.

Antigen-capture ELISA was performed as described [12 (link)] to determine the antibody titers of anti-VLP sera. In brief, 50 μL of diluted fish sera (1:100) were coated in a well of 96-well microtiter plate at 4 °C overnight. The plate was conducted to room temperature and washed three times with 200 μL PBS. After blocking with 5% BSA in PBS for 1 h and washing three times with 1% PBST (all wash steps below indicated three times of 200 μL 1% PBST), 1 ng VLP was added to each well and incubated for 1 h. After wash, the plate was incubated with in-house produced mouse anti-VLP antiserum at a dilution of 1:1000 in PBST for 1 h. The plate was washed and peroxidase-conjugated goat anti-mouse IgG (Sigma) at a dilution of 1:3000 was added and incubated for 1 h. Following thorough washes (five times), 100 μL of OPD substrate (Sigma) was added and the color development was conducted at room temperature. The reaction was stopped by the addition of 4 M sulfuric acid and the absorbance at 492 nm was determined. Statistical differences of titers from different groups were assessed by paired Student’s t tests. Numerical results are presented as mean ± standard deviation with 95% confidence intervals and p < 0.05 was considered statistically significant.

Recombinant SAV3 E2 protein was used for coating ELISA plates. The preparation of the E2 protein is described elsewhere (35 (link)). Briefly, 96 well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with recombinant E2 protein (200 ng/well) and subsequently blocked with a protein free blocking buffer (Thermo Fisher Scientific) before incubation with 1:80 diluted individual sera samples in duplicate. Following overnight incubation at 4°C, wells were washed four times (PBS containing 0.05% Tween-20). Mouse anti-trout IgM mAb (IgF1-18 (6-1-18), 1:500) and HRP conjugated goat anti-mouse (Bio-Rad, 1:1500) were added sequentially and incubated at RT each for 1 h. Plates were developed for 30 min in the dark with OPD substrate (Sigma). Optical densities were measured immediately at 450 nm on a VersaMax microplate reader (Molecular devices).

The indexed tests were unit-based ELISA protocol for IgG; Strongyloides ratti as antigen, described previously, was used with slight modifications [11 (link), 12 (link)]. The 96-well plates were coated with 2.5 ug/ml S. ratti antigen, respectively, at 4 °C overnight. The plates were washed twice using phosphate-buffered saline (PBS) containing 0.05% Tween-20 (pH 7.2) before blocking with 3% skimmed milk in PBS containing 0.5% Tween-20 at ambient temperature for 2 h. One hundred microliters of undiluted urine or diluted serum samples (diluted at 1:8000) was added and incubated at 37 °C for 1 h. After triplicate washing of the plates, 100 µl of horseradish peroxidase conjugate goat antihuman IgG (Abcam, USA) was added and incubated at 37 °C for 1 h. After being washed three times, the OPD substrate (Sigma-Aldrich, USA) in citrate–phosphate buffer pH 5.0 (100 µl/well) was added and the plates incubated at room temperature for 1 h in a humidified dark container. Finally, the reaction was stopped by adding 4 N sulfuric acid (50 µl/well), and optical density (OD) was measured at 492 nm using an ELISA reader (TECAN Sunrise, Austria). The OD was transformed to arbitrary antibody units (per ml urine or serum) based on a standard curve generated from serial dilutions of pooled positive serum samples [11 (link), 12 (link)].