For ELISA experiments with NTD-targeted mAbs, 384-well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 2 μg/mL of S glycoprotein in 20 mM HEPES pH 8 and 150 mM NaCl. For ELISA experiments with RBD-targeted mAbs, 384-well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 4 µg/mL of hACE2-His in 20 mM Sodium Phosphate pH 8 and 100 mM NaCl. The antibodies that were used were purified previously13 (link),36 (link). Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher Scientific 37532) for one hour at 37 °C. Plates were slapped dry and mAbs were serially diluted in TBST with an initial concentration of 50 µg/ml. Plates were left for one hour at 37 °C and washed 4X with TBST, then 1:5000 Goat anti-Human (Thermo Fisher Scientific A18817) was added. Plates were left for 1 h at 37 °C and washed 4x with TBST, and then TMB Microwell Peroxidase (Seracare 5120-0083) was added. The reaction was quenched after 4 min with 1 N HCl and the A450 of each well was read using a BioTek plate reader.
Magaret C.A., Li L., deCamp A.C., Rolland M., Juraska M., Williamson B.D., Ludwig J., Molitor C., Benkeser D., Luedtke A., Simpkins B., Heng F., Sun Y., Carpp L.N., Bai H., Dearlove B.L., Giorgi E.E., Jongeneelen M., Brandenburg B., McCallum M., Bowen J.E., Veesler D., Sadoff J., Gray G.E., Roels S., Vandebosch A., Stieh D.J., Le Gars M., Vingerhoets J., Grinsztejn B., Goepfert P.A., de Sousa L.P., Silva M.S., Casapia M., Losso M.H., Little S.J., Gaur A., Bekker L.G., Garrett N., Truyers C., Van Dromme I., Swann E., Marovich M.A., Follmann D., Neuzil K.M., Corey L., Greninger A.L., Roychoudhury P., Hyrien O, & Gilbert P.B. (2024). Quantifying how single dose Ad26.COV2.S vaccine efficacy depends on Spike sequence features. Nature Communications, 15, 2175.
Other organizations :
Fred Hutch Cancer Center, Henry M. Jackson Foundation, Walter Reed Army Institute of Research, Kaiser Permanente Washington Health Research Institute, Emory University, University of Washington, Pitzer College, University of North Florida, University of North Carolina at Charlotte, Johnson & Johnson (Netherlands), Janssen (Netherlands), Howard Hughes Medical Institute, South African Medical Research Council, Perinatal HIV Research Unit, University of the Witwatersrand, Janssen (Belgium), Fundação Oswaldo Cruz, University of Alabama at Birmingham, Universidad Nacional de la Amazonía Peruana, Hospital Ramos Mejía, University of California, San Diego, St. Jude Children's Research Hospital, University of Cape Town, Desmond Tutu HIV Foundation, University of KwaZulu-Natal, Centre for the AIDS Programme of Research in South Africa, National Institute of Allergy and Infectious Diseases, National Institutes of Health, University of Maryland, Baltimore
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