Abts substrate

We performed indirect ELISA experiments by coating microplates with 100 µL of a 10 µg/mL solution of rHev b 8, rZea m 12, rFra e 2, rArt v 4, rAmb a 8, rPhl p 12, and rBet v 2 at 37 °C using PBS, pH 7.4, incubated at 4 °C overnight. Previously, we determined the optimum mAb 2D10 concentration for recognition of rHev b 8, to be 2 µg/mL. We used albumin (BSA), glutathione S-transferase from Taenia solium, and Hev b 7 (patatin from H. brasiliensis) as negative controls. Between steps, wells were washed with 200 µL of 0.05% Tween 20 in PBS. The microplate was blocked with 150 µL, 1% BSA in PBS for two hours at 37 °C. Antibodies 1B4 and 2D10 were incubated at 2 µg/mL, diluted in PBS, and incubated at 37 °C for 2 h. The plate was then washed, and the secondary antibody, a goat anti-mouse IgG (Pierce Thermo-Scientific, Rockford, IL, USA, 31430) HRP-labeled (Pierce Thermo-Scientific, 31430), was added per well and set for 30 min at 37 °C. ABTS substrate (Life Technologies, 00-2024) was added and incubated for 30 min. Plates were read at 405 nm using a Cytation 3 plate reader (BioTek Instruments Inc., Winooski, VT, USA), and the absorbance of the negative control wells was subtracted from each value. Each condition was assayed in triplicate. The reported absorbance values and the corresponding standard deviation were averaged from three independent experiments for each condition.

Clarified supernatants (15 µg protein/mL; 100 µL) were prepared in coating buffer (containing 15 mM NaCO₃ and 35 mM NaHCO₃, pH 7.4), loaded onto a Maxisorp plate (Nalge Nunc, Rochester, USA) and incubated overnight at 4 °C. The plate was then blocked for 2 h in 1% w/v skim milk and incubated with primary antibody (3 h, 23 °C). Primary antibodies included goat anti-SOD1 (Santa Cruz, USA, #sc8637; dilution 1:200 v/v), goat anti-SOD2 (Sigma, USA, #S2147; 1:200 v/v) and goat anti-GPX1 (R&D Systems, USA, #AF3798; 1:500 v/v). Wells were then incubated with secondary antibody (1:5000 v/v dilution) (anti-mouse IgG-HRP and anti-rabbit IgG-HRP; 1 h at 20 °C). Next, a solution of ABTS substrate (Life Technologies, Carlsbad, CA) was added to each well followed by (1% w/v SDS) and monitoring at 410 nm using the FLUOstar Omega reader with values normalized to total protein.

For tmTNF-α binding by cell-based ELISA, tmTNF-CHO cells were fixed with paraformaldehyde and blocked with casein. Samples were treated and incubated with anti-human IgG − HRP Ab (Sigma-Aldrich, USA), followed by treatment with the fluorogenic substrate (Thermo Fisher Scientific, USA). Fluorescence intensity was detected by a microplate reader. For C1q binding, the TNF-α-coated microplate was blocked with gelatin. Samples and human C1q were sequentially treated, followed by anti-C1q-HRP Ab (Bio-Rad, USA). ABTS substrate (Invitrogen, USA) was added, and absorbance was measured by a microplate reader at 415 nm.

The binding capacity of anti-TIM-3 IgGs was measured by ELISA. ELISA was performed as described. Briefly, TIM-3-Fc was diluted to a final concentration of 2 mg/mL with PBS and plated on Corning half-area 96-well plates (Sigma-Aldrich) (50 μL for each well) at 4 °C overnight. The coating solution was removed, and the remaining protein-binding sites were blocked with 3% (w/v) bovine serum albumin in PBS at 37 °C for 1 h. Gradient concentration of IgG was added in triplicate. After reacting at 37 °C for 1.5 h, the plate was washed seven times with PBS 0.05% Tween20. Fifty microliters of horseradish peroxidase (HRP)-conjugated goat antihuman IgG fab antibody (Invitrogen) was added to each well and incubated at room temperature for 45 min. The plate was then washed three times with PBS 0.05% Tween20. Finally, ABTS substrate (Invitrogen) was added to each well for colorimetric development for 15 min. The absorbance was read at 405 nm with a plate reader.
For polyclonal phage ELISA, 1 × 10¹² phages from each round of panning were added to culture plates, incubated at 37 °C for 1.5 h. After washing three times, HRP-conjugated anti-M13-polyclonal antibody was added, and the plates were incubated at 37 °C for 45 min. After three washes with PBST, ABTS was added for color development. The absorbance at 405 nm was determined with a plate reader.

Goat anti-mouse total IgG_Fc-HRP (Jackson ImmunoResearch), goat anti-mouse IgM-HRP, IgG₁-HRP, IgG_2a-HRP, IgG_2b-HRP, IgG_2c-HRP or IgG₃-HRP (Southern Biotechnology Associates) were used as secondary antibodies (dilutions 1:1000). Antibody was detected using ABTS substrate (Fisher Scientific, NH, USA).

Serum samples were plated at an initial dilution of 1:100 and diluted serially 1:4 on Immulon II plates coated with NP₄-BSA (Biosearch Technologies) or purified PR8 virus, respectively. The serum titer was defined as the reciprocal of the last dilution that gave an O.D.>3x background. To measure Ig secretion in vitro, supernatants were plated undiluted and then diluted serially 1:5 on Immulon II plates coated with anti-mouse Ig_H+L (Jackson Immunoresearch). Ig concentration was determined by comparison to a standard curve of IgM (BD Bioscience). Goat anti-mouse IgM-HRP, IgG-HRP (Southern Biotechnology), and donkey anti-mouse IgG_H+L-HRP (Total Ig, Jackson Immunoresearch) were used as secondary antibodies. Antibody was detected using ABTS substrate (Fisher).