Hrp conjugated anti human igg

Cell lysates were prepared from the transfected cells with pWT, pBeta, and pGamma, and the cells transfected with the pVAX1 vector served as a negative control. The commercial S protein of wild-type SARS-CoV-2 (ACRO Biosystem, Beijing, China) served as a positive control. The primary antibody was an S-ECD/RBD monoclonal antibody (Bioworld, Nanjing, China), which is specifically recognized as the wild-type SARS-CoV-2 spike protein and has been shown to cross-react with the spike of other variants with a diverse binding affinity. The secondary antibody was HRP-conjugated anti-Human IgG (BD Biosciences, San Diego, CA, USA). ECL solution (BD Biosciences, San Diego, CA, USA) was used to visualize bands on the membrane.

SARS‐CoV‐2 S protein‐ and RBD‐specific IgG was determined by ELISA as described²⁴ with the following alterations. NUNC Maxisorp 96‐well plates (Thermofisher, Waltham, MA, USA) were coated with 2 µg/ml of S or RBD protein (Genscript, Leiden, Netherlands) at +4°C overnight. Plates were subsequently blocked (PBS, 2% BSA, 0.05% Tween 20) and incubated overnight with 1:50 diluted serum samples. Plates were washed and incubated for 1 hour with 1:1000 diluted HRP‐conjugated anti‐human IgG (BD, San Jose, CA, USA) and developed with ABTS (Sigma‐Aldrich, St. Louis, MO, USA). The optical density was measured at 405/492 nm with an Infinite F50 ELISA reader after 10 min (Tecan, Männedorf, Switzerland). Sera from historical controls were analyzed by ELISA and used to establish cut‐off levels.