P nitrophenyl phosphate tablets

ELISA plates (Maxisorb; NUNC 44-2404-21; Denmark) were coated overnight with 200 ng per well of purified recombinant proteins (MSP2-FC27, MSP2-3D7 or AMA1) in PBS, blocked with PBS–5 % skimmed milk powder, and used for standard ELISA [33 (link)]. After washing 3x with 0.1 % Tween/PBS, plasma samples were added in different dilutions. After another washing step, rabbit anti-human IgG conjugated to alkaline phosphatase (Sigma, A9544) was applied as a detection antibody. Finally, p-nitrophenyl phosphate tablets (Sigma, N2765) was used as a substrate. Color development was measured as the OD at 405 nm. All ELISAs were done in triplicates.

Early osteogenic differentiation was assessed on days 10 and 14 by alkaline phosphatase activity and measured using p-nitrophenyl phosphate tablets (Sigma-Aldrich), according to manufacturer’s instructions. Mineral deposition was assessed on days 10, 14 and 21 as previously described (Macri-Pellizzeri et al., 2018 (link)), using 1% Alizarin Red solution and imaged using an Eclipse TS100 inverted microscope (Nikon). Alizarin Red signal was quantified using an Infinite 200 microplate reader at 405 nm.

ELISA plates (Sumitomo Bakelite, Tokyo, Japan) were coated overnight at 4 °C with 100 μL/well of a solution of 0.5 μg/ml IgG F(ab’)₂ in 0.1 M carbonate/bicarbonate buffer at pH 9.6. After washing with 10 mM Tris buffer containing 0.9% NaCl with 0.05% Tween-20 (TBST) at pH 7.4, serum samples diluted 1:200 with TBST were added to the plate (100 μL/well). These were incubated for 2 h at room temperature (RT). After washing, 100 μL/well of alkaline phosphatase (ALP)-conjugated anti-human IgG Fc (Sigma-Aldrich) diluted 1:10,000 with TBST, or ALP-conjugated anti-human IgA (Sigma-Aldrich) diluted 1:10,000 with TBST was added and incubated for 1 h at RT. After washing, the AHAs were visualized with 1 mg/mL of p-nitrophenyl phosphate tablets (Sigma-Aldrich) in diethanolamine buffer at pH 9.8 for 30 min or for 2 h for IgA AHA measurement. Absorbance was measured at 405 nm using a microplate reader.
Levels of IgG AHA were calculated by a calibration curve using pooled human IgG purified using 40% ammonium sulfate and DEAE sephadex (Pharmacia Fine Chemicals, Uppsala, Sweden). We arbitrarily defined 1 mg/mL of the pooled IgG as containing 800 arbitrary units (AU)/mL of IgG AHA against IgG F(ab’)_2pepsin. We also used this calibration curve for measuring IgG or IgA AHA to other IgG F(ab’)₂ fragments.