2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts)

Costar half-area high binding assay plates (Corning #3690) were coated with purified protein at 100 ng/well in PBS overnight at 4 °C, and blocked with PBS buffer containing 3% milk powder (w/v) at 37 °C. For polyclonal phage ELISA, phages from each round of panning were incubated with immobilized antigen, and bound phages were detected with anti-M13-horseradish peroxidase (HRP) polyclonal antibody (Pharmacia). For the purified antibody binding assay, serially diluted antibody solutions were added and incubated for 1.5 h at 37 °C, and bound antibodies were detected with monoclonal anti-Flag-HRP antibody (Sigma-Aldrich). The enzyme activity was measured with the subsequent addition of substrate ABTS (Invitrogen) and signal reading was carried out at 405 nm using a Microplate Spectrophotometer (Biotek).

Serum samples were analyzed by ELISA for the specific detection of anti-rTSCRT and anti-TsCE IgG antibodies as described previously [18 (link)]. Biotin conjugated anti-hamster IgG (eBioscience, San Diego, CA), streptavidin-HRP and ABTS (Invitrogen) were used. All incubations were followed by three washings with PBS plus 0.05% Tween 20. Absorbance was measured at 405 nm. The cut-off value was defined as the mean of the optical density obtained with the serum from uninfected animals plus two standard deviations.

Oocytes extracts were incubated in a solution containing biotinylated synthetic peptide (Ser-Leu-Tyr-Ser-Ser-Ser-Pro.Gly-Gly-Ala-Tyr-Cys) peptide for 30 min at 30 °C (Cdc2 substrate; MESACUP Cdc2 kinase assay kit, MBL International Corp.). The mixture was then transferred to a microwell plate coated with anti-phospho-MV peptide monoclonal antibody (4A4). The retained biotinylated phospho-MV peptides were subsequently detected with streptavidin-HRP and ABTS (Invitrogen) measured in a plate reader at 405 nm (reference 490 nm).

Corning 3690 half-well 96-well plates were coated overnight at 4°C with 50 µl/well of 2 µg/ml eOD-GT8, eOD-GT8-KO, 426cTM₄ΔV1-3, 426cTM₄ΔV1-3-KO, 426c Env N276D trimer, 426c Env N276D-KO trimer or murine iv8 Fab in PBS. Plates were washed six times in PBS 0.05% Tween 20 (wash buffer). Plates were blocked with 100 µl/well PBS 5% milk (blocking buffer) for 2 h at room temperature (RT). Sera or monoclonal antibodies were prepared at 1:30 dilution or a concentration of 10 µg/ml in fresh blocking buffer, respectively, and further diluted in threefold or fivefold serial dilutions, respectively. A total volume of 50 µl was added to the plates and incubated for 2 h at RT. Binding was revealed by either anti-mouse IgG-HRP (Jackson ImmunoResearch) or anti-human IgG-HRP (Jackson ImmunoResearch) diluted 1:5,000 in wash buffer. Plates were incubated for 1 h at RT. Plates were washed six times in wash buffer. HRP activity was determined using ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate solution (Life Technologies), adding 50 µl/well. Plates were read at 405 nm on a FLUOstar Omega microplate reader (BMG Labtech). Data were analyzed with Microsoft Excel and GraphPad Prism 6.0.

96-well plates (Maxisorp, Nunc) were coated with purified merozoites at 5 × 10⁶ merozoites per well or recombinant merozoite surface antigens at 1–2 μg/ml^{24 (link)}. Briefly, plates were blocked with 1% casein, then incubated with serum samples (1/100–1/500) for 1 h, then washed with PBS-Tween 0.01%, followed by incubation for 30 min with purified C1q (10 μg/ml, Millipore) or normal serum (NS), heat-inactivated serum (HIS) from malaria naive donors, or C1q-depleted serum (Millipore) at 10% final concentration as a source of complement. After washing plates, C1q fixation was detected by incubating with rabbit anti-C1q antibodies (in-house) and then with anti-rabbit-IgG-HRP (Millipore), incubated for 1 h each. ABTS (Life Technologies) was added, incubated for 30 mins and absorbance quantified using a plate reader. All steps were conducted at room temperature (~21 °C). For studies of membrane attack complex (MAC) formation, normal serum was used as a complement source, and (rabbit-) anti-C5b/C9 antibodies (Millipore), and anti-rabbit-IgG-HRP (Millipore) were used for detection. IgG subclass ELISAs were done as previously described^{29 (link),46 (link),54 (link)} using mouse anti-human IgG subclass monoclonal antibodies (Thermo Fischer Scientific) and anti-mouse-IgG HRP (Millipore) detection antibodies. (Detailed protocols are available from the authors on request).

Supernatants of the control and 4- and 24- hour infection with CDTA treated and Δalr1 C. albicans from above experiment were either stored at -20°C or tested for levels of cytokines TNFα, IL-6 and IL-10 using an enzyme linked immunosorbent assay (ELISA) kit according to manufacturer’s (ABTS, Peprotech, USA) instructions [40 (link)].

Serum samples collected from the participants were used for autoantibody (ACPA and Anti-CarP antibodies) measurements. Determination of anti-CCP2 antibodies was performed using enzyme-linked immunosorbent assays according to the manufacturer’s instructions (Euro-Diagnostica, Malmö, Sweden). The cutoff value for positivity was set at 25 arbitrary units/ml according to the suggestion of the manufacturer. Determination of anti-CarP was made as described before (43 (link)). The plates were incubated with carbamylated or uncarbamylated fetal calf serum (half plate each), washed with PBS/0.05% Tween (Sigma, USA), and subsequently blocked for 6 h at 4°C with 100 μl of PBS/1% BSA (Sigma, USA). After washing, the wells were incubated with 50 μl serum 1/50 diluted in PBS/1% BSA/0.05% Tween. Antibodies were detected using horseradish peroxidase (HRP)-conjugated rabbit anti-human Ig antibody (ABCAM, UK), and HRP-conjugated HRP enzyme activity was visualized using ABTS (PeproTech, UK). As a standard, serial dilutions of a pooled serum sample from a patient with RA were used.