Alkaline phosphatase conjugated streptavidin

DEX was purchased from Sigma (St. Louis, MO). Recombinant human TNF, PTX3, mouse anti-human ERK1/2 mAb, affinity purified rabbit anti-phosphoERK1/2 (T202/Y 204) and ELISA kits for human PTX3 were purchased from R&D Systems (Minneapolis, MN). The p38 MAPK inhibitor, SB-203580[4-4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5- (4’-pyridyl)-1H-imidazole], the p42/ p44 ERK inhibitor, U-0126 [1, 4-diamino-2, 3-dicyano-1, 4-bis (2-aminophenyl-thio) butadiene], and the PI3K inhibitor, wortmannin, were purchased from Calbiochem (Mississauga, Ontario, Canada). All cell culture media (DMEM and F-12), antibiotics (penicillin, streptomycin), trypsin-EDTA, and cell culture reagents were obtained from Invitrogen Life Technologies, and FBS was from HyClone Laboratories (Logan, UT). The anti-human smooth muscle actin antibody (Ab) was obtained from DakoCytomation. Alkaline phosphatase-conjugated streptavidin was purchased from Jackson ImmunoResearch Laboratories. Unless stated otherwise, all other reagents were obtained from Sigma-Aldrich.

Ab titers for Cm-specific IgA, IgG1 and IgG2a were measured using an alkaline phosphatase-based ELISA as previously described [21 (link),29 (link)]. Briefly, microtiter plates were coated overnight with killed Cm elementary bodies (EBs). After blocking and washing, serially-diluted sera were incubated for 3 h at 37°C. Biotinylated goat anti-mouse Ab was added after washing. Following overnight incubation at 4°C, alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories; Bio/Can Scientific) was added for 45 min at 37°C. The enzyme substrate p-nitrophenyl phosphate (Sigma-Aldrich) (in 0.5 mM MgCl₂, 10% diethanolamine (pH 9.8)) was added and the reaction was allowed to proceed for 60 min. The plates were read by using an ELISA reader at 405 nm. Results were expressed as ELISA titers using the endpoint (cutoff at OD 0.5) of the titration curves.

384-well plates were pre-coated overnight at 4 °C with 12.4 μl/well, 2 μg/ml BG505 SOSIP 2JD6 nanoparticle produced in house^{34 (link)}. Plates were washed and blocked with 40 μl/well of PBS supplemented with 3% BSA at RT for 1 h and washed again. Mouse serum samples serially diluted (2×) with PBS-T and 1% BSA starting at 1:10 dilution were added (12.4 μl/well) and incubated at RT for 1 h. A P2A-LC tagged mouse VRC01 IgG monoclonal antibody standard starting at 1 μg/mL was also added (12.4 μl), serially diluted (2×), and incubated at RT for 1 h. Plates were washed and incubated with 12.4 μl/well biotin-labeled anti-2A peptide (3H4) mouse antibody (NovusBio, #NBP2-59627) at 1 μg/ml in PBS-T and 1% BSA. Plates were washed and the captured complex incubated with 12.4 μl/well alkaline phosphatase-conjugated Streptavidin (Jackson Immuno Research Labs, #016-050-084) at 1:3000 dilution in PBS-T and 1%BSA at RT for 1 h. Plates were washed and pNPP substrate was added as above. All plates developed for the same period of time before being read at 405 nm as above. P2A titers were reported as area under the curve for plots of Absorbance vs. log dilution factor for each sample. When maximum Abs 405 nm values were above 1.5, P2A-antibody quantities in serum samples were calculated by multiplying the sample EC50 with that of the mouse-VRC01 standard.

Individual wells of immunoassay plates were coated with influenza A H1N1 antigen (2 x 10⁵ FFU/well in 50-mM carbonate buffer, pH 9.6) and incubated overnight at 4°C. Wells then were incubated for 2 h with 1% BSA/PBS to block non-specific binding. Serum from experimental animals was diluted 1:20 (to detect IgG1 and IgG2a) or 1:10 (to detect IgA, IgE and IgM) and then further diluted in serial fashion in PBS/0.05% Tween-20/0.5% BSA. 10–20 μl of diluted serum in each well were mixed with 50 μl of biotinylated anti-mouse IgG1, IgG2a, IgA, IgE or IgM solution (BD Pharmingen) and incubated overnight, followed by addition of 50 μl of alkaline phosphatase-conjugated streptavidin (1:3000; Jackson ImmunoResearch, West Grove, PA, USA). Alkaline phosphatase activity remaining bound after washing was detected using phosphatase substrate (4-nitrophenyl phosphate; Sigma-Aldrich) with spectrophotometric monitoring of cleaved substrate at 405 nm.

Competition ELISA was performed as previously described (Sok et al., 2014 (link)). In brief, the antibody of interest was biotinylated following manufacture’s description (ThermoFisher). 96-well plates were coated with 6X His tag monoclonal antibody (Invitrogen) overnight. After washing, plates were blocked with PBS/3% BSA at RT for 1 h. BG505 SOSIP.664-His was added into wells at 1 μg/mL and incubated for 1 h. After washing, serially diluted competing antibodies in 1% BSA were added into wells and incubated for 30 min at RT. Biotinylated antibody was then added at the concentration of EC₇₀ and incubated at RT for 30-60 min. After washing, alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch) was added into wells at RT for 1 h. After final wash, phosphatase substrate (Sigma-Aldrich) was added into wells. Absorption was measured at 405 nm. Non-linear regression curves were analyzed using Prism 8 software to calculate EC₅₀ values.