Standardised total anti-spike IgG ELISA65 (link) and anti-spike subclass and isotype ELISAs44 (link),66 (link) were performed. In brief, ELISA plates were coated with 2 µg/mL of full-length trimerised SARS-CoV-2 spike glycoprotein protein overnight at 4 °C and blocked with casein in PBS. Plasma samples were diluted in PBS and tested in triplicate. Goat anti-human IgG conjugated to alkaline phosphatase was added as the secondary antibody, and plates were developed using 4-nitrophenyl phosphate in diethanolamine substrate buffer. Plates were read at 405 nm, and standardised ELISA units (EU) were determined using a 4-parameter logistic model and various pre-determined control cut-offs (Gen5 v3.09, BioTek). Plate washing in-between each step was undertaken using 0.05% Tween-20 in PBS.Serology for IgG to SARS-CoV-2 nucleocapsid protein was performed using the Abbott Architect i2000 chemiluminescent microparticle immunoassay (Abbott, Maidenhead, UK) and carried out according to manufacturer’s instructions using serum. The manufacturer threshold for confirming detection of antibodies is ≥1.40 arbitrary units. Levels between 0.50-1.39 arbitrary units designate equivocal levels (Abbott Diagnostics Product Information Letter PI1060-2020). Values below 0.5 were set to half the LLOQ (i.e. 0.25).
Tomic A., Skelly D.T., Ogbe A., O’Connor D., Pace M., Adland E., Alexander F., Ali M., Allott K., Azim Ansari M., Belij-Rammerstorfer S., Bibi S., Blackwell L., Brown A., Brown H., Cavell B., Clutterbuck E.A., de Silva T., Eyre D., Lumley S., Flaxman A., Grist J., Hackstein C.P., Halkerston R., Harding A.C., Hill J., James T., Jay C., Johnson S.A., Kronsteiner B., Lie Y., Linder A., Longet S., Marinou S., Matthews P.C., Mellors J., Petropoulos C., Rongkard P., Sedik C., Silva-Reyes L., Smith H., Stockdale L., Taylor S., Thomas S., Tipoe T., Turtle L., Vieira V.A., Wrin T., Pollard A.J., Lambe T., Conlon C.P., Jeffery K., Travis S., Goulder P., Frater J., Mentzer A.J., Stafford L., Carroll M.W., James W.S., Klenerman P., Barnes E., Dold C, & Dunachie S.J. (2022). Divergent trajectories of antiviral memory after SARS-CoV-2 infection. Nature Communications, 13, 1251.
Other organizations :
Medawar Building for Pathogen Research, Oxford University Hospitals NHS Trust, Jenner Institute, University of Sheffield, Oxford BioMedica (United Kingdom), Health Protection Research Unit in Emerging and Zoonotic Infections at University of Liverpool, University of Liverpool, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle University, University of Birmingham, Sheffield Teaching Hospitals NHS Foundation Trust, Wellcome Centre for Human Genetics, Mahidol Oxford Tropical Medicine Research Unit, Mahidol University
Serology for IgG to SARS-CoV-2 nucleocapsid protein
control variables
Coating of ELISA plates with 2 µg/mL of full-length trimerised SARS-CoV-2 spike glycoprotein protein overnight at 4 °C
Blocking of ELISA plates with casein in PBS
Dilution of plasma samples in PBS
Addition of goat anti-human IgG conjugated to alkaline phosphatase as the secondary antibody
Development of plates using 4-nitrophenyl phosphate in diethanolamine substrate buffer
Plate reading at 405 nm
Standardisation of ELISA units using a 4-parameter logistic model and various pre-determined control cut-offs
Plate washing in-between each step using 0.05% Tween-20 in PBS
Use of the Abbott Architect i2000 chemiluminescent microparticle immunoassay for serology of IgG to SARS-CoV-2 nucleocapsid protein according to manufacturer's instructions
positive controls
Various pre-determined control cut-offs for standardised ELISA units
negative controls
None explicitly mentioned
Annotations
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