Phase 1: Antibody Preparation and Characterization -- Days 1-7
Prepare wild-type anti-SARS-CoV-2 monoclonal antibody and half-life-extended variant (YTE mutation: M252Y/S254T/T256E in Fc region). Characterize antibodies by analytical SEC-HPLC, SDS-PAGE, and mass spectrometry to confirm purity >95% and correct molecular weights. Perform binding kinetics analysis using SPR (Biacore 8K) with SARS-CoV-2 spike protein. Measure neutralization potency against Omicron BA.2 using pseudovirus neutralization assay on HEK293T-ACE2 cells. Prepare antibodies at 2 mg/mL in sterile PBS for injection.
...Phase 1: Antibody Preparation and Characterization -- Days 1-7
Prepare wild-type anti-SARS-CoV-2 monoclonal antibody and half-life-extended variant (YTE mutation: M252Y/S254T/T256E in Fc region). Characterize antibodies by analytical SEC-HPLC, SDS-PAGE, and mass spectrometry to confirm purity >95% and correct molecular weights. Perform binding kinetics analysis using SPR (Biacore 8K) with SARS-CoV-2 spike protein. Measure neutralization potency against Omicron BA.2 using pseudovirus neutralization assay on HEK293T-ACE2 cells. Prepare antibodies at 2 mg/mL in sterile PBS for injection.
Phase 2: Pharmacokinetic Study Design -- Days 8-10
Randomize 8-10 week old male and female TKI mice (n=8 per group per timepoint, total n=96) into treatment groups: vehicle control, wild-type antibody (10 mg/kg i.v.), and YTE-mutant antibody (10 mg/kg i.v.). Dose selection based on human equivalent dose calculations. Collect blood samples via submandibular bleeding at 1, 6, 24, 48, 72, 120, 168, 240, and 336 hours post-injection. Process samples immediately and store plasma at -80°C.
Phase 3: Antibody Concentration Analysis -- Days 11-21
Quantify circulating antibody concentrations using validated sandwich ELISA specific for human IgG. Coat plates with anti-human Fc antibody (Jackson ImmunoResearch) and detect with HRP-conjugated anti-human IgG. Use purified antibodies as standards (0.1-10 μg/mL range). Calculate pharmacokinetic parameters (Cmax, t1/2, AUC, clearance) using Phoenix WinNonlin non-compartmental analysis. Statistical analysis by two-way ANOVA with Sidak's multiple comparisons test.
Phase 4: Viral Challenge Preparation -- Days 22-24
Prepare SARS-CoV-2 Omicron BA.2 viral stock (target titer 10^6 PFU/mL) propagated in Vero-E6 cells. Verify titer by plaque assay and confirm variant identity by RT-PCR sequencing of spike gene. Prepare challenge dose of 10^4 PFU in 50 μL sterile PBS for intranasal administration. Test viral stock for mycoplasma contamination and endotoxin levels (<1 EU/mL).
Phase 5: Protection Efficacy Study -- Days 25-35
Administer antibodies (10 mg/kg i.v.) or vehicle control 48 hours before viral challenge. Use n=12 mice per group based on power analysis for detecting 1.5-log difference in viral loads (power=0.8, α=0.05). Challenge mice intranasally with SARS-CoV-2 Omicron BA.2 under isoflurane anesthesia. Monitor mice twice daily for clinical signs using standardized scoring system. Sacrifice mice at 3 days post-challenge for peak viral replication analysis.
Phase 6: Viral Load Quantification and Analysis -- Days 36-42
Harvest lungs aseptically and homogenize in 1 mL sterile PBS using BeadBug homogenizer. Clarify homogenates by centrifugation (10,000 x g, 10 min). Extract viral RNA using QIAamp Viral RNA Mini Kit. Perform RT-qPCR targeting SARS-CoV-2 N gene using CDC N1 primer/probe set. Quantify infectious virus by plaque assay on Vero-E6 cells with 1% agarose overlay. Calculate viral loads as log10 copies or PFU per gram lung tissue. Analyze by one-way ANOVA followed by Tukey's multiple comparisons test.