Antibody-Dependent Complement Deposition Assay

ADCD was conducted as previously described^{58 (link)}. Briefly, NVX-CoV2373 Spike protein was biotinylated using EDC (Thermo Fisher) and Sulfo-NHS (Thermo Fisher), and then coupled to red Neutravidin-conjugated microspheres (Thermo Fisher) or directly coupled to Carboxylate-Modified microspheres (Thermo Fisher). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum for 2 hours at 37°C, and then washed to remove unbound antibody. The immune complexes were then incubated with lyophilized guinea pig complement (Cedarlane) and diluted in gelatin veronal buffer with calcium and magnesium (Boston Bioproducts) for 30 minutes. C3 bound to immune complexes was detected by fluorescein-conjugated goat IgG fraction to guinea pig Complement Ce (MP Biomedicals). Flow cytometry was performed to identify the percentage of beads with bound C3. Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).

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Gorman M.J., Patel N., Guebre-Xabier M., Zhu A., Atyeo C., Pullen K.M., Loos C., Goez-Gazi Y., Carrion R J.r., Tian J.H., Yaun D., Bowman K., Zhou B., Maciejewski S., McGrath M.E., Logue J., Frieman M.B., Montefiori D., Mann C., Schendel S., Amanat F., Krammer F., Saphire E.O., Lauffenburger D., Greene A.M., Portnoff A.D., Massare M.J., Ellingsworth L., Glenn G., Smith G, & Alter G. (2021). Collaboration between the Fab and Fc contribute to maximal protection against SARS-CoV-2 in nonhuman primates following NVX-CoV2373 subunit vaccine with Matrix-M™ vaccination. bioRxiv.

Publication Preprint 2021

Sulfo-NHS red Neutravidin-conjugated microspheres Carboxylate-Modified microspheres lyophilized guinea pig complement calcium and magnesium guinea pig Complement Ce

Antibody Buffer Calcium Flow cytometry Fluorescein Gelatin Goat Guinea pig Immune complexes Magnesium Microspheres Neutravidin Nvx cov2373 Protein Serum Spike protein Sulfo nhs

Corresponding Organization : Novavax (United States)

Other organizations : Massachusetts Institute of Technology, Texas Biomedical Research Institute, University of Maryland, Baltimore, Duke University Hospital, Duke Medical Center, La Jolla Institute For Allergy & Immunology, Icahn School of Medicine at Mount Sinai

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Variable analysis

independent variables

Biotinylation of NVX-CoV2373 Spike protein using EDC and Sulfo-NHS
Coupling of biotinylated NVX-CoV2373 Spike protein to red Neutravidin-conjugated microspheres or Carboxylate-Modified microspheres

dependent variables

Percentage of beads with bound C3 detected by flow cytometry

control variables

Incubation of bead+protein conjugates with diluted serum for 2 hours at 37°C
Washing to remove unbound antibody
Incubation of immune complexes with lyophilized guinea pig complement and diluted in gelatin veronal buffer with calcium and magnesium for 30 minutes
Detection of C3 bound to immune complexes using fluorescein-conjugated goat IgG fraction to guinea pig Complement Ce
Flow cytometry performed using an IQue (Intellicyt) and analysis on IntelliCyt ForeCyt (v8.1)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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