Sunrise absorbance plate reader

Intracellular GABA (GABA_i) and extracellular GABA (GABA_e), were measured as previously described [17] (link), [19] (link). Cultures were grown to mid-exponential phase (OD₆₀₀ = 0.35) or stationary phase (16 h) at 37°C with aeration in BHI medium. For exponential phase GABA measurements the cultures were reduced to pH 4.0, while for stationary phase GABA measurements, the pH of the cultures was lowered to 4.0 (EGDm) or 3.5 (10403S) with 3 M HCl. Different pH reductions for each strain were necessary to ensure optimal GABA production for each strain. Extractions were made after 1 h of acid treatment. Non HCl-treated cultures were used as negative controls. GABase from Pseudomonas fluorescens (Sigma Aldrich, Steinheim, Germany) was used in the enzymatic assay and increases in OD_{340 nm} were measured using a Tecan Sunrise absorbance plate reader.

For the analysis of the binding reactivity of anti-PfCyRPA mAbs with purified PfCyRPA, Nunc MaxiSorp^TM flat-bottomed 96-well ELISA plates (Thermo Fisher Scientific) were coated overnight at 4°C with 3 μg/mL of PfCyRPA protein produced either in HEK293 or High Five cells. The wells were then blocked with 5% (w/v) milk powder in phosphate-buffered saline (PBS) for 1 h at room temperature, followed by washing three times with PBS containing 0.05% (v/v) Tween-20. The plates were then incubated with serial dilutions of mAbs in PBS for 1 hat room temperature. After washing, the plates were incubated with goat anti-mouse (Sigma) conjugated to horseradish peroxidase (HRP) secondary antibody (Sigma) for 1 h at room temperature. Tetramethylbenzidine (TMB) was used as the substrate (KPL). The reaction was stopped after the appropriate time with 0.5 M H₂SO₄, and the absorbance was read at 450 nm with the Sunrise absorbance plate reader (Tecan). Data were processed and analyzed using GraphPad Prism 8. PfCyRPA concentration in the virosome lipid particles was determined by ELISA and native immunoblot. The final vaccine was prepared by dilution of the intermediate mixture to the required final antigen concentration for the animal study (2 g PfCyRPA per dose) and prepared in HN buffer (50 mM HEPES pH 7.4, 142 mM NaCl), followed by a final filtration step on a 0.22-µm PVDF syringe filter.