MAXISORP immunoplates (442404; NUNC) were coated with 0.125 μg of StrepMAB-Classic (2-1507-001;iba) at 4°C overnight and blocked with 2% skimmed milk in PBS (for plasma) or 2% BSA in PBS (for mAbs) for 1 h, plates were incubated with 50 μL of 10 μg/mL double strep-tag recombinant spike of SARS-CoV-2, SARS-CoV, MERS-CoV, OC43-CoV, HKU1-CoV, 229E-CoV and NL43-CoV. After one hour, 50 μL of serially diluted plasma or mAbs was added, followed by ALP-conjugated anti-human IgG (A9544; Sigma) at 1:10,000 dilution. The reaction was developed by the addition of PNPP substrate and stopped with NaOH. The absorbance was measured at 405nm. To determine the binding to SARS-CoV-2 RBD, SARS-CoV-2 NP, SARS-CoV-2 spike S1 (40591-V08H; Sino Biological Inc) and SARS-CoV-2 spike S2 (40590-V08B; Sino Biological Inc), immunoplates were coated with 0.125 μg of Tetra-His antibody (34670; QIAGEN) followed by 5 μg/mL of His-tag recombinant SARS-CoV-2 RBD, SARS-CoV-2 NP, SARS-CoV-2 spike S1 and SARS-CoV-2 spike S2. The plasma endpoint titers (EPTs) were defined as reciprocal plasma dilutions that corresponded to two times the average OD values obtained with mock. EC50 of mAbs were evaluated using non-linear regression (curve-fit), GraphPad Prism 8 software.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.