C1058

A total of 2386 FDA-approved drugs were obtained from a compound library (HY-L022; MCE) and a self-built compound library. U2OS cells were used for the in-cell ELISA due to their good adhesion ability. U2OS cells were seeded in 96-well plates. When they reached 80% to 90% confluence, the cells were treated with each drug at a concentration of 2 μM or 10 μM for 24 hours. Then, the cells were fixed in 4% paraformaldehyde for 10 minutes and washed with PBS-Tween 3 times. The cells were blocked and permeabilized using blocking buffer (5% BSA in 0.1% Triton X-100) for 30 minutes at room temperature (RT). The cells were incubated with primary antibodies (AMFR, 1:20,000, 16675-1-AP; Proteintech) diluted in 1% BSA and 0.1% Triton for 3 hours at RT. Thereafter, the cells were incubated with secondary antibody (HRP-conjugated goat anti-rabbit, 1:5000, W4011; Promega) for 1 hour at RT. After 3 additional washes, binding was visualized with 2-component chromogenic TMB (PR1210; Solarbio) for 10 minutes. The reaction was stopped with stop reagent (C1058; Solarbio), and the signal intensity was measured at 450 nm with a FLUO star microplate reader (BMG Labtech) within 15 minutes. Eight DMSO-treated wells were used as controls for each plate. For each compound, treatment was performed in duplicate.

Sera were prepared from blood samples collected once a week from immunized or unimmunized mice via the medial canthus. The binding antibodies against EV-A71-VP1 were detected as described previously [29 (link)]. Briefly, 96-well plates were coated with recombinant protein, and blocked with 1% BSA (w/v) (A8010, Solar Bio, Beijing, China). Diluted sera were added to the wells, and a goat anti-mouse IgG H&L (HRP) (ab6789, Abcam, United Kingdom) was used as the secondary antibody. TMB substrate solution (Tetramethylbenzidine Single-Component Substrate solution, SE1005, Korad Bio, Beijing, China) was used for detection, followed by the addition of stop solution (C1058, Solarbio, Beijing, China), after which the absorbance at 450 nm was recorded using a SpectraMax i3x microplate reader (Molecular Device, San Jose, USA). Neutralizing antibodies against EV-A71 virus were detected using a previously described neutralization assay [22 ]. Briefly, sera were diluted in a gradient, mixed with virions of EV-A71 Isehara strain in 96-well plates and incubated for 2 h. Then, a suspension of human rhabdomyosarcoma cells (RD) was added and cultured for 7 days. The neutralizing antibody titre was defined as the reciprocal of the maximum dilution that inhibited cytopathic changes by 50% [42 ]. The same virus strain was used in the neutralization test and the in vivo challenge experiments.

Serum-soluble RGMa (sRGMa) expression was detected by a commercial RGMa enzyme-linked immunosorbent assay (ELISA) kit (DY2459-05, R&D, USA). The protocols were carried out in accordance with the manufacturer’s instructions. Briefly, the process was as follows: the 96-well plates were coated with capture antibody in PBS overnight. The plates were blocked with reagent diluent concentrates for 1 h. Subsequently, the standard protein and diluent serum samples were added to each well and incubated for 2 h. The detection antibody was then added to the reagent diluent and incubated for another 2 h. Following that, streptavidin-HRP was added to the plate for 20 min while avoiding direct light. The plates were then washed 3–5 times with wash buffer concentrates after each solution was incubated. After the streptavidin-HRP was removed and thoroughly washed, the TMB substrate (BU04, NEOBISCOENCE, Shanghai) was added to the well and incubated for another 20 min while avoiding direct light. Finally, a stop solution (C1058, Solarbio, Beijing) was used to stop action. All operations were performed at room temperature. The multimode Plate Reader (Perkin Elmer, 139959, Singapore) detected absorbance at 450 nm and calibrated at 540 nm.

The SARS-CoV-2 S trimer, RBD, S2 and nucleocapsid protein (N) (Acro Biosystems SPN-C52H9, SPD-C52H3, S2N-C52H5, and NUN-C5227, respectively) were coated on 96-well plates at 0.5 μg/ml in PBS overnight at 4 °C. After the plates were blocked with buffer (1× PBS with 3% BSA (Solarbio, A8020) and 0.05% Tween-20 (Sigma, P9416)) for 1 h at 37 °C, plasma samples were added and incubated for 1 h at 37 °C. The plasma samples were assayed at a 1:100 starting dilution and 7 additional threefold serial dilutions in blocking buffer (1× PBS with 1% BSA (Solarbio, A8020) and 0.05% Tween-20). The plates were washed 5 times with washing buffer and then incubated with anti-human IgG (Abcam, ab97225) or IgM (Abcam, ab97205) secondary antibody conjugated to horseradish peroxidase (HRP) in blocking buffer at a 1:50,000 dilution (and IgG) or 1:20,000 dilution (IgM) for 0.5 h at 37 °C. After the plates were washed 5 times, the HRP substrate TMB (Solarbio, PR1200) was added for 10 min, followed by the addition of 50 μl of 1 M H₂SO₄ (Solarbio, C1058) to stop the reaction. The absorbance was measured at 450 nm with an ELISA microplate reader (TECAN Infinite 200 PRO).

Human ACE2 (Sino Biological Inc, #10108-H05H) was coated in plates at 2 μg/mL in PBS at 4 °C overnight. After regular washing and blocking, 100 μl 0.5 μg/mL His-tagged WT RBD (Sino Biological Inc, #40592-V08B), 0.04 μg/mL Delta RBD (Sino Biological Inc, #40592-V08H90) or 0.01 μg/mL Omicron BA.1.1 RBD (Sino Biological Inc, #40592-V08H121) was added to each well. In addition, 100 μl diluted mAbs (12 μg/mL, 4 μg/mL, 1.333 μg/mL, 0.444 μg/mL, 0.148 μg/mL, 0.0494 μg/mL, 0.0165 μg/mL) were added and mixed. After a 1 h incubation at 25 °C, the plates were washed, and 100 μL of 0.1 μg/mL anti-His-HRP (Sino Biological Inc., #A5327) was added to each well and incubated for 1 h at 25 °C. After regular washing, 100 µL of TMB (Solarbio, #PR1200) was added and incubated for 15 min. The absorbance at 450 nm was measured immediately after stop solution (Solarbio, #C1058) was added.

To detect antibody titration in mouse sera, hybridoma supernatant and the medium of 293T cells were transfected with antibody expression vectors. Ninety-six-well binding plates (442402; Thermo Fisher Scientific, Waltham, MA, USA) were incubated with 100 μL of 1 μg/mL spike protein (40589-V08B1; sinobiological, Beijing, China) overnight at 4 °C. The following day, the plates were blocked with 100 μL of blocking buffer (2% BSA, 36101ES60; Yeasen, Shanghai, China, DPBS, 21600-044; Gibco, Carlsbad, CA, USA) for 1 h at room temperature after being washed with 200 μL PBST (PBS, 0.05% Tween-20). Serially diluted immunized serum samples or cell media were then incubated in the plates for 2 h at room temperature followed by incubation with a 1:5000 HRP-conjugated goat anti-mouse IgG(H+L) (SA00001-1; Proteintech, Wuhan, China) for 1 h. For detection, 50 μL 3,3′,5,5′-tetramethylbenzidine (TMB, 00-4201-56; Invitrogen, Waltham, MA, USA) was added to each well and incubated for 5–10 min. The colorimetric reaction was terminated using a stop buffer (C1058; SolarBio, Beijing, China) which was followed by OD450 detection using a microplate reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland).

Each well of a 96-well ELISA microplate (2506, Corning, Kennebunk, ME, USA) was coated with 100 μl (the concentration of antigen has been marked in the figure legend) of the corresponding antigen in PBS overnight at 4°C. After being washed three times with PBST (PBS, containing 0.05% Tween-20), the plate was blocked with 250 μl of blocking solution [3% bovine serum albumin (BSA) in PBST] at 37°C for 1 h. After the plate was washed three times with PBST, 100 μl of diluted antiserum or antibody in dilution buffer (3% BSA in PBST) was added into each well, followed by incubation at 37°C for 1 h. The plate was washed three times with PBST and then incubated with HRP-labeled anti-rabbit IgG antibody (7074s, CST, Danvers, MA, USA) or anti-mouse IgG antibody (7076s, CST, Danvers, MA, USA) diluted 1:3,000 with PBST at 37°C for 1 h. The plate was washed three times with PBST to remove unbound secondary antibodies. Finally, 100 μl of peroxide substrate solution (PR1200, Solarbio, Beijing, China) was added to each well, followed by incubation at room temperature for 10–20 min. Color development was ended by adding 100 μl of 2.5 M sulfuric acid (C1058, Solarbio, Beijing, China) per well, and absorbance was recorded at 450 nm with a microplate reader (Epoch, Bio-Tech, Minneapolis, MN, USA).