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The interaction of FH or FH CCP fragments with IAV and pseudotyped lentivirus with was assessed by ELISA. Purified IAV (1 x 10⁴) or 10⁴ relative light units (RLU) of pseudotype lentivirus were adsorbed onto 96-well Maxisorp microtitre plates (Fisher Scientific) in carbonate-bicarbonate (CBC) buffer, pH 9.6 (Sigma) overnight at 4°C. Plates were then washed with PBST (PBS with 0.1% Tween-20). Wells were blocked for 1 hour at room temperature with 5% w/v BSA in PBS (blocking buffer). Purified FH or FH CCP fragments diluted in PBS were added at various known concentrations and incubated for 2 hours at room temperature. Wells were washed with PBST, and primary antibody, either the OX24 monoclonal anti-FH (purified from mouse hybridoma supernatant; MRC Immunochemistry Unit, Oxford), or a polyclonal goat anti-human FH (Complement Technology), diluted in blocking buffer, were added to the wells and incubated for 1 hour at room temperature. After washing in PBST, secondary antibody, rabbit anti-mouse-HRP (DAKO) or rabbit anti-goat (Abcam), diluted in blocking buffer, was applied for 1 hour at room temperature. Following incubation, wells were washed in PBST and the color was developed using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma). The reaction was stopped using 2 N H₂SO₄ (Sigma). The absorbance was read at 450 nm using microplate Reader ELX 808 (Bio-Tek).

After a checkerboard titration, 100 µl of each rEgAgB (1.5 µg/ml suspended in 100 mM carbonate-bicarbonate buffer, pH 9.6) was used to coat the wells of a flat-bottom 96-well microplate (Greiner Bio-One, Kremsmuenster, Austria) overnight at 4 °C. One hundred microliters of serum samples diluted 1:100 in phosphate buffered saline containing 0.05% Tween 20 (PBS/T) were incubated at 37 °C for 2 h, after which 100 µl of horseradish peroxidase-conjugated anti-human IgG antibody (1:4000 dilution in PBS/T; MP Biochemicals, Santa Ana, CA, USA) was further incubated at 37 °C for 2 h. Color reactions were developed with 100 µl of 1% o-phenylenediamine (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.03% H₂O₂ for 20 min in the dark. Reactions were halted by adding 50 µl of 2 N H₂SO₄ (Sigma-Aldrich). The absorbance was measured at 450 nm on an NEO microplate reader (Biotek, Winooski, VT, USA). Sera from healthy donors and PBS/T were used as negative and blank controls, respectively. All results were determined after correction with appropriate blank. Each sample was independently assayed in triplicate.

After a checkerboard titration, 100 μl of PwAWE and rPwYF (2.5 μg/ml each) suspended in 100 mM carbonate-bicarbonate buffer (pH 9.6) were coated to the wells of a flat-bottom 96-well microplate (Greiner Bio-One, Kremsmünster Austria) overnight at 4°C. Sera diluted to 1:200 in PBS containing 0.05% Tween 20 (PBS/T) or undiluted CSFs (100 μl each) were incubated for 2 h, subsequently with 1:2,000 diluted horseradish peroxidase (HRP)-conjugated anti-human IgG antibody (100 μl; heavy- and light-chain specific, MP Biochemicals, Santa Ana, CA, USA) for 2 h. The color reaction was developed with 1% o-phenylenediamine (100 μl; Sigma-Aldrich) supplemented with 0.03% H₂O₂ for 20 min in the dark. The reaction was stopped using 2 N H₂SO₄ (50 μl; Sigma-Aldrich). The absorbance (abs.) was measured at 450 nm on a NEO microplate reader (Biotek, Winooski, VT, USA). All results were determined after correction with PBS/T blank.

Levels of M. perstans-specific IgG4 and IgE were measured in individual serum samples using an extract of M. perstans worms following an established protocol for other filarial-specific Ig measurements [21 (link), 26 (link)]. In brief, 96-well polysorb plates (Thermo Fisher Scientific) were coated overnight at 4°C with 2.5μg/ml M. perstans extract in PBS (pH 9.6). After 3 washes with 0.05% Tween/PBS (pH 7.2) and once with PBS alone, plates were blocked with 200μl/well of 1% BSA/PBS for two hours at room temperature. Following further washing, 50μl/well of diluted sera was added (1:500 for IgG4 and 1:20 for IgE) and incubated overnight at 4°C. After washing, 50μl/well of biotinylated secondary antibodies (IgG4 (1:15000) or IgE (1:1000)) were added for two hours at room temperature and after another wash step streptavidin-peroxidase (50μl/well; 1:5000) was added for 45 minutes at room temperature (Roche Diagnostics, Mannheim, Germany). After the final wash, reactions were developed with TMB (tetramethylbenzidine, 50μl/well), (Sigma-Aldrich) and stopped with 25μl/well of 2N H₂SO₄ (Sigma-Aldrich). Following the manufacturer's instructions, total IgE and IgG4 levels were measured using specific kits purchased from eBioscience (Human IgE/IgG4 Ready-SET-Go). Absorbance levels and data analysis were performed as described above.