Hrp labeled goat anti rabbit ig

BsAbs were tested for simultaneous binding to both antigens in a bispecificity ELISA. MaxiSorp plates (Thermo Scientific, Nunc, 430341) were coated with 50 µL/well BSA-DNP (10 µg/mL, Biosearch Technologies, D-5050-10, conjugation ratio 13) in coating buffer (0.1 M Na₂CO₃/NaHCO₃ pH 9.6) for 1 hour at 37°C. After every incubation, plates were washed three times with PBS/0.05% Tween 20. Plates were blocked with 100 µL/well PBS/1% bovine serum albumin (BSA) for 1 hour at 37°C. After washing, plates were incubated with 50 µL/well (bs)Abs (concentrations are indicated in figures) diluted in PBS/0.05% Tween 20/1% BSA (PTB buffer) for 1 hour at 37°C. Next, 1 µg/mL FH (CompTech, A137) or C4BP (CompTech, A109) diluted in PTB buffer was added. FH was detected using goat anti-human FH (Quidel, A312) and HRP-labeled rabbit anti-goat Ig (Dako, P0449), and C4BP was detected using rabbit anti-human C4BP (generated in-house by group of Anna M. Blom, Malmö, Sweden) and HRP-labeled goat anti-rabbit Ig (Dako, P0448), all diluted in PTB buffer. Plates were developed by incubating with 50 µL/well ABTS (Merck, A1888-5G) containing 1:2000 diluted H₂O₂ (Merck, 1072090250), and read at 415 nm using a microplate reader (BIO-RAD iMark).

Antigen-binding and anti-LS (scaffold) antibodies produced after vaccination were tested in the sera collected at different time points as well as in pre-challenge nasal swabs using ELISA. Costar high-binding 96-well ELISA plates were coated overnight at 4°C with 1 µg/ml of either recombinant LS, MERS-CoV S1 or S2 proteins in PBS. The plates were washed with PBS and blocked for 1 hr using 1%BSA/0.5%Tween-20/PBS. Following blocking, diluted samples (1:100 or serially diluted) were added and further incubated for 1 hr. The plates were then washed and and probed with an HRP-labeled goat anti-rabbit Ig (1:2000, Dako) secondary antibody. TMB was used for signal development and the absorbance of each sample was measured at 450 nm (OD₄₅₀).