Alkaline phosphatase conjugated goat anti mouse igg

Antibodies to SARS-CoV-2 FL-S protein were determined by performing a standardised ELISA on serum collected 3-weeks after prime or prime-boost vaccination. MaxiSorp plates (Nunc) were coated with 100 ng/well FL-S protein overnight at 4 °C, prior to washing in PBS/Tween (0.05% v/v) and blocking with Blocker Casein in PBS (Thermo Fisher Scientific) for 1 h at room temperature (RT). Standard positive serum (pool of mouse serum with high endpoint titre against FL-S protein), individual mouse serum samples, negative and an internal control (diluted in casein) were incubated for 2 h at RT. Following washing, bound antibodies were detected by addition of alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma-Aldrich), diluted 1/5000 in casein, for 1 h at RT and detection of anti-mouse IgG by the addition of pNPP substrate (Sigma-Aldrich). An arbitrary number of ELISA units were assigned to the reference pool and OD values of each dilution were fitted to a 4-parameter logistic curve using SOFTmax PRO software. ELISA units were calculated for each sample using the OD values of the sample and the parameters of the standard curve.

Polyacrylamide gel electrophoresis was performed on pre-cast NuPAGE™ 4–12% Bis-Tris Midi Protein Gels (Invitrogen) polyacrylamide gels. Protein samples analysed under reducing conditions were incubated with 250 mM DTT before loading onto the gel. Gels were electrophoresed in NuPAGE™ MES SDS Running Buffer (Invitrogen). Total protein in a gel was visualised by Coomassie staining (Quick Coomassie, Generon). Proteins were blotted on nitrocellulose membranes using the BioRad TransBlot Turbo Transfer System (BioRad). Different mouse and rat antibodies at a concentration of 1 µg/ml were bound to the membrane using the iBind Flex western system (Invitrogen), alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, A8438, 1:1000) or alkaline phosphatase-conjugated goat anti-rat IgG (Sigma, A8438, 1:1000) were used for detection. Western blots were developed using SIGMAFAST™ BCIP®/NBT (Sigma).

The immunoreactivity of NHBA antigenic fragments against the 31E10/E7 mAb was tested using a previously described ELISA inhibition assay [26 (link)]. Briefly, the 31E10/E7 mAb (1 μg/ml) was preincubated with micromolar concentrations of competitors (represented by the indicated NHBA fragments or by the entire antigen fused to an histidine tail) prior to the addition to microtiter wells sensitized with the whole fusion antigen (NHBA-NUbp fused to an histidine tail or NHBA-NUbp-His, 5 μg/ml). Alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) was then added at a 1:5,000 dilution followed by p-nitrophenyl phosphate disodium salt (Sigma). Percent inhibition was calculated by comparing the absorbance value of wells with and without the inhibitors.

K_D values for complexes formed between mAb 12C1 and fHbp fragments at equilibrium in solution were calculated using an ELISA test, as described18 (link)22 (link). Briefly, the wells of microtiter plates were coated with 4 different concentrations (3.5, 7, 14 and 28 nM) of each antigen, which consisted of full-length fHbp or of the fragments listed in Table 1. In addition, 7 different concentrations (ranging from 7 to 480 nM) of the antigen were mixed with a fixed concentration of the 12C1 mAb (0.7 nM) and incubated overnight at 4 °C. On the following day, the plates were washed and 100 μl of the 8 antigen-antibody mixtures were dispensed into antigen-coated wells. After incubation at room temperature for 1 h, the wells were washed and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) was added, followed by p-nitrophenyl phosphate disodium salt, as described above. Under these conditions, absorbance is proportional to the concentration of free mAb 12C1 added to each well. The results for each antigen were analyzed using the plot described by Friguet et al. (ref. 18 (link); Supplementary Fig. 1), which provides a K_D value for the complex in solution. Results were expressed as means and standard deviations of the 4 K_D values determined for each of the 4 antigen concentrations used to coat the wells.