A1888

Urinary properdin levels were assessed by a previously described sandwich enzyme-linked-immunosorbent assay (ELISA) (9 (link), 17 (link)), with a detection limit of 1.2 ng/mL, a plasma intra-variation of <17% and an inter-variation of <20%. In brief, 96-well ELISA plates (NUNC MaxiSorp^TM, Sigma-Aldrich, Saint Louis, MO, USA) were coated overnight at 4°C with monoclonal anti-human properdin (Hycult HM2282, Uden, the Netherlands). Urinary samples were diluted 5 times in DPBS with 0.1% Tween and bovine serum albumin (PTB) and incubated for 1 h at 37°C, followed by secondary antibody; polyclonal rabbit anti-human properdin-biotin (kindly provided by M. R. Daha, Leiden, The Netherlands) and detection with Streptavidin-HRP (Dako P0397, Glostrup, Denmark). Enzyme activity was detected using 2,2′azino-bis (3-ethylbenzo-thiazoline-6-sulphonic acid) (A1888, Sigma-Aldrich, Saint Louis, MO, USA). The optical density was measured at 415 nm using a microplate ELISA reader (Benchmark Plus, Bio-Rad, Veenendaal, The Netherlands). A standard curve was prepared using a serial dilution of zymosan activated serum in PTB with a known concentration of properdin. A reference sample, diluted in PTB with a known concentration of properdin was included as positive control. Potential background signal was assessed and corrected for, with PTB functioning as blank.

The antioxidant activity using the radical 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (A1888 Sigma-Aldrich®, Saint Louis, MO, USA) was measured by reacting 10 mL of a 7 mM ABTS solution with 10 mL of 2.45 mM (K₂S₂O₈) potassium persulfate (216224 Sigma-Aldrich®, Saint Louis, MO, USA). The mixture was agitated for 16 h in the dark. The cation radical was adjusted with 20% solution of ethanol (100983 Merck^®, Kenilworth, NJ, USA) to an absorbance of 0.7 ± 0.1 at a 734 nm wavelength; then 200 μL of the extract was added to 2 mL of adjusted ABTS. The reaction occurred for 6 min, after which the absorbance was read [25 (link)]. The results were expressed as mg of ascorbic acid equivalents (AAE/100 mL).

The 2,20-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (A1888 Sigma-Aldrich^®, Saint Louis, MO, USA) was used to measure antioxidant activity. A total of 10 mL of the 7 mM ABTS solution with 10 mL of 2.45 mM potassium persulfate (K₂S₂O₈) (Sigma-Aldrich^® 216224, Saint Louis, MO, USA) were reacted together. The mixture was stirred in the dark for 16 h. The ABTS radical was adjusted with a 20% ethanol solution (100983 Merck^®, Kenilworth, NJ, USA) to an absorbance of 0.7 ± 0.1 and a wavelength of 734 nm; subsequently, 200 µL of the extract was added to 2 mL of adjusted ABTS. The reaction occurred for 6 min [46 (link)]. A calibration curve was developed using ascorbic acid as a standard. The results were expressed in mg equivalents of ascorbic acid for 100 g dry forage (AAE/100 g DM).

This method was performed to analyze the potential of C. campinarensis EO to inhibit the 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Sigma-Aldrich; A1888, São Paulo, Brazil) radical, according to modification by Re et al. [55 (link)] of the experimental method proposed by Miller et al. [56 (link)]. Description of the method can be found in [54 (link)].

Antigen specific antibody (day 21 sera) was measured by ELISA as previously described4 (link)5 (link)6 (link). Briefly, 3 μg/ml of antigen (Intanza 2013) was diluted and used to coat the ELISA plates (Nunc Maxisorp, ThermoFisher) overnight at 4 °C. The plates were blocked with 0.4% BSA in PBS and used to determine the antigen specific antibody titres. Sera obtained were serially diluted and incubated for 2 hours in room temperature. Washing of plates were done 5 times using 0.02% PBST and secondary antibody, anti-mouse IgG HRP (G-21040, Invitrogen/ThermoFisher) at 1 μg/ml were added and incubated for 1.5 hours. Colour development was performed using ABTS (2,29-azino-bis3-ethylbenzthiazoline-6-sulfonic acid; A-1888, Sigma-Aldrich) as the substrate and measured at absorbance of 405 and 490 nm. Endpoint titres were calculated as described elsewhere27 (link). IgG1 and IgG2c isotypes responses were assessed using the same ELISA method (IgG1, ab97240; 1:4000, and IgG2c ab97255; 1:2000; Abcam).

Antioxidant activity was measured using the 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (A1888, Sigma-Aldrich, St. Louis, MO, USA) radical following [69 (link)] by reacting 10 mL of 7 mM ABTS solution with 10 mL of 2.45 mM (K₂S₂O₈) potassium persulfate (216224, Sigma-Aldrich, St. Louis, MO, USA). The mixture was stirred for 16 h in a container in complete darkness. The absorbance was adjusted with 20% ethanol to obtain a value of 0.7 ± 0.1. A total of 200 μL of sample was added to 2 mL of ABTS solution and allowed to react for 6 min; absorbance was measured at 734 nm in a spectrophotometer (JENWAY, Model 6705, Dunmow, UK).

The Trolox equivalent antioxidant capacity (TEAC) assay was used in this study with some modifications^{31 (link),36 (link)}. Briefly, lyophilized microalgal samples were extracted with 75% ethanol using a mini-beadbeater (BioSpec Products, USA) at the highest speed for 5 cycles, 20 s each, and cooled down on ice for 2 min between two cycles. Under dim light, ABTS (A-1888, Sigma-Aldrich, USA) and potassium persulfate (216,224, Sigma-Aldrich, USA) were dissolved in water to generate the blue-green ABTS^·+ (7 mM ABTS and 2.45 mM potassium persulfate). This mix was allowed to react in the dark at room temperature for 16 h and its absorbance at 734 nm (OD₇₃₄) was adjusted to 0.70 ± 0.05 before use. To build the standard curve of OD₇₃₄ against Trolox quantity, different amounts of Trolox (238813, Sigma-Aldrich, USA) were mixed with the freshly prepared ABTS^·+ solution to reduce the color, and the resulting OD₇₃₄ readings were recorded. The antioxidation capacity of the samples was expressed as equivalent amount of Trolox per mg dry biomass.