P nitrophenyl substrate

To evaluate the total mouse IgG concentration in the ascites fluids (AF) samples, ELISA plates (MAxisorp, Nunc) were coated with 120 ng (50 µl) goat anti-mouse IgG, F(ab′)₂ fragment specific in coating buffer (50 mM Na₂CO₃, pH 9.6) and incubated overnight at 4°C. Plates were then washed in PBST buffer and blocked for 1 hr at 37°C with 200 µl per well of 2% (W/V) BSA in Tris-NaCl pH 7.6 (TSTA). Following blocking and washing, plates were incubated with serial dilutions (50 µl, in duplicate) of the AF samples in TSTA (50 µl/well, in duplicate) for 1 hr at 37°C. After washing with PBST, plates were incubated with 30 ng (50 µl) alkaline phosphatase-conjugated donkey anti-mouse IgG diluted in TSTA for 1 hr at 37°C. Finally, plates were washed with PBST, and the color reaction was developed using p-Nitrophenyl substrate (Sigma). Absorbance was measured at 405 nm with Molecular Devices Spectramax M3 reader. The IgG concentration was determined by interpolation from a 1.56–100 ng/ml standard curve (fitted to four-parameter equation) of ChromPure mouse IgG whole molecule, by SoftMax Pro software. IgG concentration of all MAbs is presented in Table S1.

The activity of two glycolytic enzymes: α-glucosidase and β-galactosidase, using the method described by Dżugan et al. [42 ] was tested for honey samples. The reaction mixture contained 25 μL of 2 mM appropriate p-nitrophenyl substrate (Sigma Aldrich, USA) in 0.2 M citrate buffer (pH 5.5 for α-GLU and 4.0 for β-GAL) and 25 μL of honey aqueous solution (20% w/w) sample. In blank, 25 μL of suitable buffer was used instead of substrate solution. Samples were incubated at 37 °C for 90 min, and the reaction was then stopped by the addition of 250 μL of 0.5 M carbonate buffer at pH 10.5. Absorbance was measured on an ELISA reader (Clindiag Systems, Belgium) at a wavelength of 400 nm. Results were expressed as enzymatic units U/kg (μmol/min/kg) by multiplying the measured absorbance by a factor of 52.91.