T0440

A 96-well microplate was coated with human Fc-CAP1 and was incubated with 160 µM of 6× His-PCSK9 (rhPCSK9-His, human Fc-CAP1 were kindly provided by Y-Biologics, Seoul, Republic of Korea). Additionally, either 10 or 50 µM of IgG, evolocumab, or hFc-CAP1 was added and incubated for 2 h at room temperature. Following incubation with anti-6× His secondary antibody conjugated with HRP (Invitrogen, PA1-23024), 3,3′,5,5′-tetramethylbenzidine substrate at 100 µl/well (Sigma-Aldrich; T0440) was added to generate a detectable signal using ELISA. The reaction was stopped by the addition of acidic stop solution (1 N H₂SO₄), and the plate was read on the microplate reader (GloMax Discover Microplate Reader, Promega) at 450 nm.

Antibody response post challenge was measured by whole-cell ELISA using sera from experimental fish [27 (link)]. Briefly, high-binding 96-well ELISA plates (3590, Costar, Washington, DC, USA) were coated overnight at 4 °C with inactivated P3SAB cells (100 uL/well) resuspended in carbonate-bicarbonate coating buffer (pH 9.6) to an OD600 = 1.0. The next day, plates were washed with PBS containing 0.05% Tween 20 (PBST) before blocking with conjugate buffer (1% Bovine serum albumin in PBST) at 22 °C for 2 h. Diluted sera (1:10) from surviving fish were applied to the wells and incubated at 22 °C for 2 h before washing with PBST. The secondary antibody was an anti-Asian seabass IgM monoclonal antibody (AquaMab F-02, Aquatic Diagnostics, Scotland, UK). Plates were incubated with secondary antibody for 2 h at 22 °C. After washing, Horseradish peroxidase-conjugated tertiary antibody (Goat anti-mouse IgG; A16066, Invitrogen, Waltham, WA, USA) diluted 1:10,000 in conjugate buffer was applied for 1 h. Color was developed for 5 min using 3,3′,5,5′–Tetramethylbenzidine (TMB; T0440, Sigma-Aldrich, St. Louis, MO, USA) and OD was measured at 450 nm with a Hercuvan NS-100 microplate reader. Antisera from 8 fish per vaccine group were analyzed individually and the results were expressed as a mean OD ± standard error.

The binding ability of antibodies to WT SARS-CoV-2 S1 protein or mutated SARS-CoV-2 S1 protein was assessed by an ELISA with modifications [43 ]. Briefly, 50 ng of WT SARS-CoV-2 S1 protein or mutated SARS-CoV-2 S1 protein was coated in 100 μL per wells on ELISA plates (2592, Costar, USA) overnight at 4 °C followed by washing 3 times with phosphate-buffered saline with 0.05% Tween 20 (PBST). The ELISA plates were incubated for 1 h at 37 °C in 100 μL per well blocking buffer (PBST containing 5% FBS). Then, for 1 h at 37 °C, 100 μL of fivefold serially diluted parental mAbs or Bi-Nab or unrelated control anti-human PD-1 IgG in blocking buffer was added to each well. After 3 washes with PBST, the plate was incubated with HRP-conjugated anti-human IgG antibody for 1 h at 37 °C followed by washing with PBST. An addition of 50 μL of 3,30,5,50-tetramethyl-benzidine (TMB) (T0440, Sigma‒Aldrich, USA) buffer was added per well and reacted at 25 °C for 5 min and then stopped by 0.2 M H₂SO₄ stop buffer. The optical density (OD) value was measured at 450 nm using an iMark microplate absorbance reader (BIO-RAD, USA), and nonlinear regression was used to calculate the concentration for 50% of the maximal effect (EC₅₀).

RAW264.7 macrophage-like cells were treated with LPS and poly (1:C) HMW in the presence and absence of the necrotic cell lysates for 16–18 h. Supernatants were collected and centrifugated prior to storing at −20 °C for not more than three weeks. The concentration of IL6 in the supernatant was measured by immunoassay according to the manufacturer’s instruction (DY406, R&D Systems, Minneapolis, MN, USA). The immunoassay is a solid phase sandwich ELISA and the binding of the detection antibody linked to Streptavidin-HRP was identified by the colorimetric substrate tetramethylbenzidine (T0440, Sigma).