Quantifying Chimpanzee Adenovirus Neutralizing Antibodies

Chimpanzee adenovirus ChAdOx1-specific neutralizing antibody titers were assessed using a secreted placental alkaline phosphatase (SEAP) quantitation assay as described^{6 (link)}. Briefly, GripTite MSR 293 cells (Invitrogen, catalog no. R795–07) were infected with the serial diluted serum in phenol red–free DMEM (Life Technologies, catalog no. 31053028) and the ChAdOx1-SEAP reporter virus in a 1:1 mixture for 1 hour before replacing with phenol red–free 10% FBS DMEM for 24 hours. For each sample, SEAP concentration was assessed in 50 μl aliquots of culture supernatant, with CPSD as an indicator substrate (Tropix Phospha-Light Chemiluminescent Assay Kit, Life Technologies, catalog no. T1017). Luminescence intensity was measured using a Varioskan Flash luminometer (Thermo Fisher Scientific). Serum dilution neutralization titers were measured by linear interpolation of adjacent values (to 50% inhibition) to determine the serum dilution required to reduce SEAP concentration by 50% compared to wells with virus alone.

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van Doremalen N., Lambe T., Spencer A., Belij-Rammerstorfer S., Purushotham J.N., Port J.R., Avanzato V.A., Bushmaker T., Flaxman A., Ulaszewska M., Feldmann F., Allen E.R., Sharpe H., Schulz J., Holbrook M., Okumura A., Meade-White K., Pérez-Pérez L., Edwards N.J., Wright D., Bissett C., Gilbride C., Williamson B.N., Rosenke R., Long D., Ishwarbhai A., Kailath R., Rose L., Morris S., Powers C., Lovaglio J., Hanley P.W., Scott D., Saturday G., de Wit E., Gilbert S.C, & Munster V.J. (2020). ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. Nature, 586(7830), 578-582.

Publication 2020

GripTite MSR 293 cells Tropix Phospha-Light Chemiluminescent Assay Kit Varioskan Flash luminometer

Adenovirus Assay Cells Chemiluminescent assay Chimpanzee Dilution Inhibition Light Luminescence Neutralizing antibody Placental alkaline phosphatase Placental alkaline phosphatase 1 Serum Virus

Corresponding Organization :

Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health, University of Oxford, Jenner Institute

Top 5 similar protocols

Variable analysis

independent variables

Serum dilution

dependent variables

Secreted placental alkaline phosphatase (SEAP) concentration

control variables

Cell line (GripTite MSR 293 cells)
Culture medium (phenol red-free DMEM)
Incubation time (1 hour)
Incubation time after replacing medium (24 hours)
Reagents (CPSD as indicator substrate)

controls

Positive control: ChAdOx1-SEAP reporter virus
Negative control: Cells with virus alone (no serum)

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