Quantifying SARS-CoV-2 Antibody Neutralization

The rACE2 displacement assay was performed as previously described (33 (link), 34 ), except assay plates were Maxisorp (ThermoFisher Scientific). Briefly, the 384-well plates were coated with RBD (supplied by NRC), blocked with BSA in PBS-T, incubated with 1:10 or 1:40 sera dilutions, then with biotinylated ACE2 (supplied by Jim Rini, University of Toronto), and treated with Streptavidin-Peroxidase Polymer Ultrasensitive (Sigma-Aldrich). Incubation with ELISA Pico Chemiluminescent Substrate and reading on the EnVision were performed as in direct detection. All values were normalized to sample-free blanks on the same plate. The resulting relative ratios (RR) were converted to IU/mL using the WHO International Standard 20/136 as a calibrant (33 (link)) and the following formula: log₂(IU/mL at sample dilution d)=RR/(−0.2308)+5.7898+log₂(d). Neutralization threshold was determined for the 1:10 dilution as 2×MAD (median absolute deviation) from the mean of blanks.

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Matveev V.A., Mihelic E.Z., Benko E., Budylowski P., Grocott S., Lee T., Korosec C.S., Colwill K., Stephenson H., Law R., Ward L.A., Sheikh-Mohamed S., Mailhot G., Delgado-Brand M., Pasculescu A., Wang J.H., Qi F., Tursun T., Kardava L., Chau S., Samaan P., Imran A., Copertino DC J.r., Chao G., Choi Y., Reinhard R.J., Kaul R., Heffernan J.M., Jones R.B., Chun T.W., Moir S., Singer J., Gommerman J., Gingras A.C., Kovacs C, & Ostrowski M. (2023). Immunogenicity of COVID-19 vaccines and their effect on the HIV reservoir in older people with HIV. bioRxiv.

Publication Preprint 2023

Maxisorp Streptavidin-Peroxidase Polymer Ultrasensitive

Ace2 Assay Dilution Elisa Peroxidase Polymer Sera Streptavidin

Corresponding Organization : University of Toronto

Other organizations : Maple Leaf Medical Clinic, McGill University, Centre for Health Evaluation and Outcome Sciences, HIV Legal Network, York University, Lunenfeld-Tanenbaum Research Institute, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Cornell University, University of British Columbia, Marymount University, St. Michael's Hospital

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Variable analysis

independent variables

Sera dilutions (1:10 or 1:40)

dependent variables

Relative ratios (RR) of ACE2 displacement
Neutralization threshold

control variables

Maxisorp (ThermoFisher Scientific) assay plates
RBD (supplied by NRC) coated on plates
BSA in PBS-T used for blocking
Biotinylated ACE2 (supplied by Jim Rini, University of Toronto)
Streptavidin-Peroxidase Polymer Ultrasensitive (Sigma-Aldrich)
ELISA Pico Chemiluminescent Substrate
EnVision reader
Sample-free blanks on the same plate for normalization
WHO International Standard 20/136 used as a calibrant

positive controls

Not explicitly mentioned

negative controls

Sample-free blanks on the same plate

Annotations

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