Infinite f50

According to the instruction (ACROBiosystems, Cat. No. EP111/EP-115, Beijing, China), various concentrations of OA were added to the wells in an ACE2 pre-coated plate followed by addition of HRP-SARS-CoV-2 Spike RBD. After incubation at 37°C for 1h, wells were washed and the substrate was then added. After incubation at 37°C for 20 min, the reaction was finally terminated by the addition of stop solution. The absorbance at 450 nm was measured immediately by the microtiter plate reader (Infinite F50, Thermo Fisher, China). The neutralizing antibody provided by ACROBiosystems was used as the positive control. The inhibition rate was calculated as the following formula: Inhibition rate = (1-OD Sample/ OD Negative Control) × 100%.

For cytotoxicity assay, ACE2^h cells were seeded into 96-well plates at a density of 1×10⁴ cells per well and then treated with different concentrations of OA (0.01, 0.1, 1, 10, 100 μM) for 24 h, then 10 μL of Cell Counting Kit (IV08-500, Invigentech, USA) solution was added to each well followed by 2 h of incubation. The relative cell viability was assessed by the detection of the absorbance at 450 nm using a microplate reader (Infinite F50, Thermo Fisher, China). The survival rate of ACE2^h cells was calculated as the following formula: Survival rate = [(OD _Treated –OD _Blank) / (OD _Control−OD _Blank)] × 100%.

Serum samples in NSG mice with Nalm-6 13-3B5* were collected at day 5 and day 15 after target cell injection in K₂EDTA tubes for MuSK antibody ELISA. To detect human anti-MuSK IgG4, the histidine-tagged recombinant extracellular domain of human MuSK (aa 24–495, R&D Systems, catalog no. 10189-MK) was coated on ELISA plates in PBS overnight at 4 °C at a concentration of 5 µg ml⁻¹. Plates were washed with washing buffer (Invitrogen, catalog no. 00-0400-59), and blocked with Pierce Protein-Free (PBS) Blocking Buffer (Thermo Scientific, catalog no. 37572). Mouse serum samples were evaluated at a dilution of 1:50 to 1:100 in comparison to a 13-3B5 purified recombinant human monoclonal IgG4 antibody as a reference standard for quantitation. Antihuman IgG (H+L) HRP (Bethyl, catalog no. A80-119P) was used to detect human antibodies. Plates were protected from the light and placed in the dark for 2 h. After washing plates three times, 100 μl of TMB (Thermo Scientific, 34028) was added for 30 min. Plate reading were conducted using ELISA reader (Tecan, Infinite F-50) within 15 min after adding stop solution (Invitrogen, SS04).