Sars cov 2 2019 ncov spike protein

SARS-CoV-2 (2019-nCoV) spike protein was purchased from Sino Biological. Coenzyme Q10 (CoQ10), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), ADP, thrombin from human plasma, and N-acetylcysteine (NAC) were obtained from sigma-Aldrich (St. Louis, MO, USA). Collagen was obtained from Chrono-Log Corp. (Havertown, PA, USA). FITC mouse anti-human CD62P and FITC Mouse Anti-Human PAC-1 were purchased from BD bioscience (San Jose, CA, USA). Alexa Fluor^TM 488-conjugated fibrinogen was purchased from Thermo Fisher Scientific (Waltham, MA, USA). TMRM assay kit (mitochondrial membrane potential) was purchased from Abcam (Cambridge, UK). Prostaglandin E1 (PGE1) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). MDA, SOD, and T-AOC assay kits were purchased from Nanjing Jiancheng (Nanjing, China) and the 8-iso-PGF-2α assay kit was obtained from Andy gene (Beijing, China).

A single intravenous injection dose of CA521 was conducted in rhesus monkeys (N = 3, 2 females and 1 male, age 3–5 years, bodyweight 3.00–3.90 kg) and C57BL/6 mice (N = 4, 4 males, age 5–7 weeks, bodyweight 21–23 g) at 50 and 10 mg/kg. PK samples were collected at predose and 5 min, 30 min, 1 h, 3 h, 6 h, 24 h, 48 h, 72 h, 120 h, 168 h, 216 h, 264 h, 336 h, 504 h, 840 h, 1008 h postdose from money, and at predose and 3 min, 15 min, 30 min, 1 h, 2 h, 6 h, 24 h, 72 h,120 h,168 h, 240 h, 336 h, 504 h, 672 h, 840, 1008 h postdose. ELISA was used to determine the concentration of CA521^FALA in serum. In this method, SARS-CoV-2(2019-nCoV) spike protein (Sino Biological, 40592-V08H) was used as the capture reagent, and goat anti-human IgG, monkey ads-HRP was detecting agent. The quantification range of the calibration curve was 78.1–10,000 ng/mL for monkeys. The quantification range of the calibration curve was 39.06–5000 ng/mL for mice. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

A single intravenous injection dose of candidate antibodies (CA521^FALA, CA530^FALA, and CA304^FALA) was conducted in C57BL/6 mice (N = 12, 12 males, 4 males/group, age 5–7 weeks, bodyweight 21–23 g) at 10 mg/kg. Blood samples were collected at predose and 3 min, 15 min, 30 min, 1 h, 2 h, 6 h, 24 h, 72 h, 120 h, 168 h, 240 h, 336 h, 504 h, 672 h, 840 h, and 1008 h postdose from the mice via orbit vein bleeding. After placed at room temperature for 30 min, the blood samples were centrifuged at 13,500 rpm for 10 min at 4 °C, and supernatants were analyzed by ELISA. High binding ELISA plates were coated with 1 μg/mL SARS-CoV-2(2019-nCoV) spike protein (Sino Biological, 40592-V08H) at 4 °C overnight, and then blocked with 1% bovine serum albumin (BSA) in PBS at 37 °C for 1 h, followed by washing five times with PBST. Serially diluted antibodies (for standard curve) and 4 fold or 20 fold dilutions of serum samples were added to the plates and incubated at 37 °C for 1 h, followed by washing five times with PBST. Goat Anti-Human IgG-HRP (Southern Biotech, 2049-05) was used as the secondary antibody. The concentration of antibodies in serum was calculated according to the standard curve. The main PK kinetic parameters were calculated using Phoenix WinNonlin.