P nitrophenyl phosphate pnpp substrate

384-well ELISA plates (Corning, #3700) were initially coated with 12.4 μl/well streptavidin (Jackson Immuno Research Labs, #016-000-084) at 2  μg/ml diluted in PBS and incubated at 4 °C overnight. Plates were washed 3× with 100 μl/well PBS containing 0.05% Tween (PBS-T) and blocked with 40 μl/well PBS + 3% BSA at RT for 1 h before the addition of 12.4 μl/well biotin-labeled BG505 SOSIP (produced in house, Pugach et al., Journal of Virology, 2015) at 2 μg/ml diluted in PBS-T and 1% BSA. Mouse sera were serially diluted (3×) using PBS-T and 1% BSA starting at 1:10 and 12.4 μl/well incubated at room temperature (RT) for 1 h. After washing (as above), 12.4 μl/well alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) (Jackson Immuno Research Labs, #115-055-146) diluted 1:5000 in PBS-T, 1% BSA was added and incubated for 30 min at RT. Plates were washed and p-nitrophenyl phosphate (pNPP) substrate (Sigma Aldrich, #S0942) dissolved to 1 mg/mL in substrate buffer (10 mM MgCl₂ with 80 mM Na₂CO₃ and 15 mM NaN₃, pH 9.8), was added at 12.4 μl/well to visualize the binding of antigen-specific mouse IgG. Optical density (OD) at 405 nm was read on a SpectraMax Plus (Molecular Devices) plate reader, allowing the same amount of development time for each plate. EC₅₀ values were generated by fitting curves to plots of Absorbance values vs. the log of the serum dilution for each sample.

ALP activity was considered as a specific marker of osteoblast differentiation which was detected using ALP activity colorimetric assay kit (Sigma-Aldrich). In the beginning, mMSCs incubated with PMMA particles following transfection or treated with different concentration of RSV for 3 days were harvested and rinsed twice with PBS followed by protein extraction using lysis buffer. The remaining supernatant was used to measure total protein content using a BCA protein kit (Pierce Biotechnology, Rockford, IL, USA) in accordance with the manufacturer’s instruction after centrifugation, and the absorption at 405 nm in each well was determined using p-nitrophenyl phosphate (PNPP) substrate (Sigma-Aldrich). However, craniums collected from mouse calvarial model were decalcified using nitric acid-paraformaldehyde solution for 3 days and sectioned followed by dewaxing using gradient ethanol. After that, clear sections were washed twice with PBS and incubated with chromogenic substrates at 37 °C for 1 h. ALP-positive cells were counted under a light microscope.

ELISA was performed as previously described [36 (link)]. Briefly, plates were coated with 100 ng/mL of recombinant SARS-CoV-2 S glycoprotein (S2P, [37 (link)]) in carbonate bicarbonate at 4 °C overnight. Naive or vaccinated mouse sera were used at a dilution of 1:100 in TSTA. Anti-mouse IgG– or IgA–alkaline phosphatase (AP) conjugates were diluted to 1:1000 and used as secondary antibodies. P-nitrophenyl phosphate (pNPP) substrate (Sigma, St. Louis, MO, USA) was added after 3 washes, and the optical density was measured (OD of 405 nm). IgG or IgA values were determined by subtracting the value of blank wells plus 3XSTDEV from each sample.

The titration of specific antibodies against rPbHsp60 and rgp43 in the sera from patients with PCM was carried out with indirect ELISA. Sera from NC were used for control. Wells of flat-bottomed polystyrene plates (Corning, USA) were coated with 5 µg of rPbHsp60 or rgp43 diluted in 100 µL of 0.2 M carbonate buffer, pH 9.6, and incubated overnight at 4°C. Then, the wells were blocked with 3% gelatin in TBS-T for 1 h and incubated with 100 µL of serum diluted to 1:250 in a solution of 1% of gelatin in TBS-T, at 37°C. After 2 h, the plates were washed five times and the wells were incubated with 100 µL of alkaline phosphatase-conjugated goat anti-human immunoglobulin antibody (Merck Millipore) diluted to 1:2,000, at 37°C. After 1 hour, the wells were washed as described above and the antigen-antibody reaction was detected with a p-nitrophenyl phosphate (pNPP) substrate, according to the manufacturer's instructions (Sigma-Aldrich). The reactions were done in duplicates and the absorbance was determined at a wavelength of 405 nm. The cut-off limit was determined by receiver-operating characteristics (ROC) analysis and was defined as 0.68 absorbance at 405 nm.

96-well half-area plates were coated with 100 μg/mL heparin or MAD2-6 IgA, incubated at 37°C for 1 hour, and blocked with 1% BSA in PBS. Serially diluted P14-P19 PfCSP peptides and recombinant PfCSP (see table S2 for sequence information) were added to the plate and incubated for 1 hour at room temperature. Next, 1:500 goat anti-human IgG Fc-alkaline phosphatase (Southern Biotech 2048-04) was added to the plate for a 1 hour incubation, followed by p-nitrophenyl phosphate (p-NPP) substrate (Sigma). The plates were read with the EnSpire plate reader (Perkin Elmer) at 405 nm after 1 hour.
In competition experiments, plates were coated with 100 μg/mL MAD2-6 IgA or IgG overnight at 4°C and blocked with 1% BSA in PBS. A mixture of heparin (Fresenius Kabi 401586J) at a range of concentrations and 0.5 μg/mL of biotinylated P17 was added to the plates. Next, streptavidin-alkaline phosphatase (Southern Biotech 7100-04) was added to the plate, and the plates were developed and read after 30 minutes as described above.