El800 microplate reader

The DPPH radical scavenging assay was carried out using an EL800 microplate reader (Bio-Tek Instruments, Winooski, VT, USA) [56 (link)]. Briefly, 180 μL of freshly prepared DPPH solution (0.32 mM in methanol) was mixed with 20 μL of sample (dissolved in methanol, extract 250 µg/mL; compounds 62.5–1000 µM) in a 96-well plate. The mixture was incubated for 20 min in the dark at 25 °C, and then, the absorbance was measured at 570 nm. Trolox was used as a positive control, which showed a linear radical inhibition curve within final concentrations of 1.56 µg/mL (6.25 µM) and 25 µg/mL (100 µM). Results are expressed in µg Trolox equivalents/µg extract or µM Trolox equivalents/µM compound. The DPPH radical scavenging activity (%) is calculated using Equation (1): $\%inhibition=\left(1-\frac{A_{sample}-A_{blank1}}{A_{control}-A_{blank2}}\right)\times 100\%,$
where A_sample is the absorbance of DPPH solution with a sample, A_blank1 is the absorbance of the test sample without DPPH, A_control is the absorbance of DPPH solution without a sample, A_blank2 is the absorbance of methanol, without DPPH or a sample.

Serum CatB-specific IgG1 and IgG2c levels were assessed by ELISA as previously described [12 (link)]. Briefly, Immulon 2HB flat-bottom 96-well plates (Thermo Fisher) were coated overnight at 4ºC with rCatB (0.5 μg/mL) in 100 mM bicarbonate/carbonate buffer at pH 9.6 (50 μL/well). The plates were washed three times with PBS-Tween 20 (PBS-T: 0.05%; Fisher Scientific) and were blocked as above for 90 min. Serial serum dilutions in duplicate were incubated in the plates for 2 hours. Control (blank) wells were loaded with PBS-T. After washing three times with PBS-T, goat anti-mouse IgG1-horseradish peroxidase (HRP) (Southern Biotechnologies Associates, Birmingham, AL) and goat anti-mouse IgG2c-HRP (Southern Biotechnologies Associates) were added to the plates and incubated for 1 hour at 37°C. After a final washing step, TMB substrate (50 μL/well; Millipore, Billerica, MA) was used for detection followed by 0.5 M H₂SO₄ after 15 min (25 μl/well; Fisher Scientific). Optical density (OD) was measured at 450 nm with an EL800 microplate reader (BioTek Instruments Inc.). The results are expressed as the mean IgG1/IgG2c ratio of the endpoint titers ± standard error of the mean. Endpoint titers refer to the reciprocal of the highest dilution that gives a reading above the cut-off calculated as previously described [31 (link)].