Maxisorp microtiter plate wells

Wells of Maxisorp microtiter plate wells (Nunc) were coated for 16 h at 4 °C with 50 μL volumes of protein A-purified mAb PD7 at a concentration of 1 µg/mL in PBS. The wells were incubated with heat-treated protein solutions for 2 h, washed three times (5 min each) with PBST, and then incubated for 1 h with PD7-HRP conjugate diluted 1 in 1000 in PBST (equivalent to ~1 μg antibody protein/mL buffer). The wells were washed with PBST as described, given a final 5 min wash with PBS, and bound antibody visualised by incubating wells with tetramethyl benzidine (TMB) substrate solution for 30 min, after which reactions were stopped by the addition of 3 M H₂SO₄. Absorbance values were determined at 450 nm using a microplate reader (Tecan GENios, Tecan Austria GmbH). All incubation steps were performed at 23 °C in sealed plastic bags. The threshold for detection of the PD7 antigen in Afu-ELISA^® was determined from the means of controls (AMM or MEB only). These values were consistently in the range of 0.050–0.100. Consequently, absorbance values ≥ 0.100 were considered as positive for the detection of antigen. The limit of detection of the Afu-ELISA^® was determined from a calibration curve of known concentrations of heat-treated Asp f I diluted in PBST.

Nanobodies produced from PE or as rP from individual colonies were tested for binding to either SARS-CoV-2 S-2P or RBD protein. MaxiSorp microtiter plate wells (Nunc) were coated overnight at 4 °C with 100 ng/well of recombinant proteins (S-2P and yRBD) or irrelevant proteins as negative controls. After washing with PBST, wells were blocked with 10% skimmed milk powder in PBST and 100 μL of the PE or rP diluted ¼ was added to the wells. Nanobody-specific binding was detected by PEE as previously described [33 (link)]. In the case of rPE, HRP-labeled anti-M13 phage antibody diluted 1:3000 (GE Healthcare, Piscataway, NJ, USA) was used as a detection system. To determine specific binding, the OD₄₀₅ value of antigen-coated wells at least two times higher than the OD₄₀₅ value of the control wells, was considered positive. Plasmids from positive clones were transformed in DH5α cells and further characterized by a restriction reaction before sending samples for sequencing.