SARS-CoV-2 DNA Monoclonal Antibody Safety and Pharmacokinetics
Okay, here’s a breakdown of the methods described in the provided text, focusing on the tools and software used for data analysis, and a summary of the key experimental procedures.
1. Data Analysis Tools & Software
* Excel: Used for initial data export and institution.
* Prism v.10 (GraphPad Software): The primary software for data analysis and graphing. Specifically used for:
* Analyzing ELISA data (AUC over time calculation).
* Fitting neutralization curves (nonlinear regression wiht Hill slope <0).
* Calculating ID50 and IC50 values.
* BioTek Synergy 2 Plate Reader: Used for reading absorbance (ELISA) and luminescence (pseudovirus neutralization assay).
2.Key Experimental Procedures
* Detection of DMAbs (in vivo binding too SARS-CoV-2 RBD) - ELISA
* Coating: 96-well plates coated with anti-YTE mAb.
* Blocking: Nonfat dry milk + Tween 20 in PBS.
* Serum Incubation: Sera diluted and serially diluted, incubated on coated plates.
* RBD Detection: Biotinylated SARS-cov-2 RBD proteins (Wild-type, Delta, BA.2, BA.4/BA.5) used for detection.
* Detection Reagents: HRP-conjugated streptavidin, Ultra TMB substrate, quenched with sulfuric acid.
* Reading: Absorbance read at 450/570 nm.
* Analysis: AUC (Area Under the Curve) over time calculated using prism.
* Pseudovirus Neutralization assay
* Pseudovirus Production: HEK 293T cells transfected with Spike-expressing plasmids and a luciferase reporter plasmid.
* Target Cells: huCHOAce2 cells.
* Neutralization: Serum/DMAb samples incubated with pseudovirus before adding to target cells.
* Incubation: 72-hour incubation.
* Luminescence Measurement: britelite plus substrate used, luminescence read on a BioTek Synergy 2 plate reader.
* Analysis: Neutralization curves fitted using Prism (nonlinear regression with Hill slope <0). ID50 and IC50 values calculated.
* Purification of DMAbs
* Capture: Anti-YTE-coated Dynabeads used to capture DMAbs from serum.
* Elution: Pierce IgG Elution Buffer used to elute purified DMAbs.
* Concentration: Amicon 30 kDa MWCO filters used to concentrate samples (for some cohorts).
In essence, the study uses a combination of ELISA and pseudovirus neutralization assays to characterize the antibody response to SARS-CoV-2, with Prism being the central software for data analysis.