96 well polystyrene microplate

The protein (50 ng) was diluted with 50 µL PBS and then coated onto 96-well polystyrene microplates (Corning Inc., Corning, NY, USA), followed by incubation overnight at 4 °C. The plates were washed six times with 100 µL of PBS-T (0.1% Tween-20) and blocked with 100 µL of 1% ovalbumin in PBS for 2 h at room temperature. After six rounds of additional washing, equine plasma samples (antivenom) were diluted (1:20,000) in PBS and added to each pre-coated well for another 2 h incubation at room temperature, and excess antibodies were removed by washing 6 times with PBS-T. Afterwards, rabbit anti-horse IgG conjugated with horseradish peroxidase (HRP) (Bethyl Laboratories, Montgomery, TX, USA) was added to each well and incubated at room temperature for 1 h. After final washing six times, 50 µL of tetramethylbenzidine (TMB) substrate (Clinical Science Products Inc., Mansfield, MA, USA) was added to each well and reacted for 10 min. The reaction was terminated by adding 25 µL of 2N H₂SO₄ (J.T Baker, Radnor, PA, USA), and absorbance was measured using a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA) with excitation and emission wavelengths of 450 and 540 nm, respectively. Each assay and sample extraction was performed in triplicate.

The procedure was carried out as described previously, with minor modifications [53 (link)]. 1 µg/mL rFL HA in carbonate–bicarbonate buffer pH 9.6 was immobilized on the surface of 96-well polystyrene microplates (Corning Inc., Corning, NY, USA) overnight at 4 °C. The plates were washed three times with TPBS and incubated for 1 h with blocking buffer at 37 °C and 350 rpm. Afterward, plates were rinsed three times with TPBS. TPCK-treated trypsin (25 ng/mL) was added after incubation for 1 h at 37 °C, and the plates were washed again. Citrate buffer 0.1 M with the addition of 150 mM NaCl with varying pH (7.4, 6 and 5) was added to the wells, and immunoplates were incubated for 1 h at room temperature without shaking. Plates were washed three times and VHH-Fcs diluted in blocking buffer were added. The conventional ELISA protocol was followed as described above.

Venom proteins (10 ng per well) were coated onto 96-well polystyrene microplates (Corning Inc., Corning, NY, USA) by incubating at 4 °C overnight and then blocked with 1% BSA at room temperature (RT) for 1 h. After blocking, the plate was washed with PBST six times, and dilution of anti-CTX antibodies (1:5000) were added to each well and incubated at RT for 1 h. Afterward, the plate was washed with PBST six times and incubated with related secondary antibodies, respectively, at RT for 1 h. After washing with PBST six times, 50 μL of TMB buffer (Clinical Science Products Inc., Mansfield, MA, USA) was added into each well and incubated at RT for 10 min. The reaction was stopped with 25 μL H₂SO₄, and absorbance of each well measured with a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA) at excitation and emission wavelengths of 450 and 540 nm, respectively.

For evaluation of mouse serum antibodies specific for PEDV-N protein, recombinant N proteins of different PEDV strains (400 ng/well) were used to coat wells of 96-well polystyrene microplates (Corning Inc.) in a volume of 100 μL PBS (pH 8.0) per well overnight at 4°C. Plates were blocked with 5% skim milk in PBS containing 0.5% Tween20 (Sigma-Aldrich). Diluted mice serum was added to wells then plates were incubated for one hour at 37°C followed by washing with PBS containing 0.5% Triton X-100 (Sigma-Aldrich). Binding of an antibody to its corresponding antigen was detected using HRP-conjugated goat anti-mouse IgG antibodies (GenScript) followed by visualization of results using a TMB substrate kit (Tiangen Biotech, Beijing, China). The absorbance of each well was measured using a Victor ™ X5 Multilabel Plate Reader (Perkin Elmer) at 450 nm.

The semi-quantitative adhesion test was performed for assessing biofilm formation, using the method described by Stepanovic et al., with some modifications (Stepanovic et al., 2000 (link)). In brief, cultures of all 69 V. parahaemolyticus strains were adjusted to 3 × 10⁵ CFU/ml, and incubated in 96-well polystyrene microplates (Corning, NY, USA), at 35°C for 30 h. Suspended cultures were transferred to a fresh microplate to measure the CFU. The original microplate was first rinsed three times with phosphate buffered saline (PBS; Leagene, Beijing, China), followed by fixing with Bouin’s fluid (Leagene, Beijing, China) for 20 min. Then the microplate was rinsed three more times with PBS, and subsequently stained with crystal violet (Leagene, Beijing, China) for 30 min, followed by a final rinsing with PBS three times. Stained biofilm on each air-dried well was washed with crystal violet using ethanol (95%, v/v solution; Sinopharm, Beijing, China) until it was completely rinsed off. An iMark microplate reader (Bio-Rad, CA, USA) was used for measuring the absorbance at 570 nm (OD_{570 nm}). The normalized BF was calculated using the following formula: normalized BF_sample = (original BF_sample − BF_control)/log_(CFU/ml).

A sandwich ELISA-based PRRSV virion capture assay was conducted as previously described [26 (link)]. Briefly, the wells of 96-well polystyrene microplates (Corning, Corning, NY, USA) were coated with 400 ng of mAb-PN9cx3 in 100 μL of PBS (pH 8.0) per well. The plates were incubated overnight at 4 °C, and each well was then blocked with 2.5% gelatine in PBS supplemented with 0.5% Tween 20 (Sigma-Aldrich). Subsequently, HP-PRRSV-SD16 (100 μL, 10^6.5 TCID₅₀/mL) was added to the wells, and the plates were incubated for 1 h at 37 °C. For the isotype control, all the steps were performed using mAb-2G8 at the same amount as mAb-PN9cx3. Anti-PRRSV convalescent pig serum (1:200 dilution in PBS) was used to detect the captured PRRSV virions. The binding of pig antibodies was revealed using HRP-conjugated goat anti-pig IgG (Jackson ImmunoResearch, West Grove, PA, USA) and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (TianGen Biotech, Beijing, China). The absorbances of the individual wells were measured using a Victor^TMX5 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA) at a wavelength of 450 nm.

Each fraction of N. atra venom (50 ng) was diluted in 100 μl phosphate-buffered saline (PBS) and coated onto 96-well polystyrene microplates (Corning Inc., Corning, New York, USA) by incubating at 4°C overnight. The plates were washed six times with 200 μl of PBST (contain 0.1% Tween-20) and blocked with 200 μl of 1% ovalbumin in PBS at room temperature for 2 hours. After repeating the washing step for six times, FNAV (8 mg/ml) was diluted (1:5000) in PBS, and added to each well. After incubation at room temperature for 2 hours, the excess antibodies were removed by washing 6 times with PBST. Then, the alkaline phosphatase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, Texas, USA) was diluted (1:5000) in PBS and added to each well and incubated at room temperature for 1 hour. After finally washing each well 6 times with PBST, the substrate 4-methyl umbelliferyl phosphate (100 μM, 100 μl/well) was added to each well and incubated for 10 minutes. The fluorescence was measured with a SpectraMax M5 microplate reader (Molecular Devices, Silicon Valley, CA, USA) at excitation and emission wavelengths of 355 and 460 nm, respectively.