Elisa imark microplate reader

The reactivity of serum samples towards peptide rMSP10 or PfMSP10 peptides was assessed by indirect ELISA. This assay was performed in Costar High Binding plates (Sigma Aldrich). Individual wells were coated with the recombinant protein or synthetic peptide, at 5 µg/mL, and blocked with phosphate buffered saline (PBS) containing 0.1% of bovine serum albumin (BSA). Serum samples were diluted 1:100 in 1.5% non-fat milk washing solution (0.15 M Na₂HPO₄, 0.15 M NaH₂PO₄, 0.44 M NaCl, 0.05% of Tween20, and 0.05% of BSA). IgG was detected using peroxidase-conjugated AffiniPure Goat anti-human-IgG antibody (Jackson Immuno Research, West Grove, PA, USA) diluted 1:2000. Later, the plates were washed and 100 μL/well of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BD OptEIA, San Jose, CA, USA) was added. The reaction was stopped using 50 μL/well of 0.25 M HCl, and the absorbance was read at 450 nm on an ELISA iMARK microplate-reader (BioRad). Positive controls included five samples from five P. falciparum-infected individuals and a ‘positive pool’ made of three samples. The positive pool control was used for the construction of a standard-curve (1:50, 1:100, 1:400, 1:1600, 1:3200, and 1:12,800) which was built for each experiment, yielding similar results and to allow us defined the correct serum dilution for the further assays (Additional file 1: Figure S3).

Total blood samples were collected into vacutainer tubes (cat. no. 366881, BD Biosciences, Plymouth, UK), allowed to clot for 1 h at 37 °C, and centrifuged for 15 min at 1700 rcf at 4 °C. Then, serum was aliquoted and stored at − 80 °C until use. Concentrations of human CD25/IL-2 Rα (cat. no. DY223, R&D Systems, Minneapolis, USA), CD27/TNFRSF7 (cat. no. DY382-05, R&D Systems, Minneapolis, USA), IL-17A (cat. no. DY317, R&D Systems, Minneapolis, USA), S100B (cat. no. DY1820-05, R&D Systems, Minneapolis, USA), CXCL9/MIG (cat. no. DY392, R&D Systems, Minneapolis, USA) and CXCL11/I-TAC (cat. no. DY672, R&D Systems, Minneapolis, USA) were measured using DuoSet ELISA kits (R&D Systems, Minneapolis, USA). CXCL10/IP-10 levels were evaluated with a BD Pharmingen kit (cat. no. 550926, San Diego, USA). The plates were analyzed in an ELISA iMark Microplate Reader (Bio-Rad, California, USA).