Sera.  The sera from patients with acute Chagas’ disease, all from the states of Minas Gerais, Bahia, and Goiás, Brazil, were described in a previous study [14] except for serum samples collected during 1.9 month, 7.9 months and 15.15 years from an individual accidentally infected with T. cruzi, which were kindly made available to us for this study. In all patients,

T. cruzi was detected by microscopic examination of blood. The sera from chronic indeterminate disease and non-chagasic sera were also from previous studies [14]. Prior to use, the sera, stored in 50% glycerol at 4 °C, were centrifuged at 1,200 g for 10 min and diluted in appropriate buffers, as described later.

Ethical approval was obtained from the Human selleck compound Investigation Review Committee of Tufts Medical Center. ELISA assay.  Microtitre wells were coated overnight at 4 °C with recombinant extracellular domain (ECD) of human TrkA, TrkB and TrkC receptors fused to the Fc region of human IgG (400 ng/ml) (R&D Systems, Minneapolis, MN, USA) as described earlier [7], blocked with 5% goat serum (2 h, 37 °C), followed by chagasic sera diluted at 1:200 (unless otherwise indicated) in 5% bovine serum albumin/phosphate-buffered saline pH 7.2 containing 0.1% Tween-20, washed and developed with alkaline phosphatase Mitomycin C (AP)-labelled secondary relevant antibody. To determine the antibody titres against T. cruzi, trypomastigotes were obtained from cellular cultures, lysed by repeated cycles selleckchem of

freeze/thaw, cleared of debris by centrifugation (12,000 g, 10 min), layered on microtitre wells (500 ng/ml, 4 °C) and probed with chagasic sera, as described earlier. ATA isotyping was performed by ELISA with commercially available kits (Sigma-Aldrich, St Louis, MO, USA) based on mouse mAb to human IgG isotypes and goat antibodies specific to IgA and IgM. Antibody avidity.  This was determined as previously described [11, 15] except that we used ELISA instead of ligand blotting to obtain avidity measurements. In brief, microtitre wells were coated overnight with Fc chimera of Trk receptors-ECD (TrkA, TrkB, and TrkC) or control receptor (p75NTR), blocked with 5% goat serum (2 h, 37 °C), washed and incubated with sera (1:200, 2 h) without pre-incubation or after pre-incubation (4 °C, overnight) with various concentrations of soluble receptor-ECD and developed with AP-labelled secondary relevant antibody, as described earlier. Avidity to TrkA was also determined in an affinity-purified rabbit TrkA antibody (Abcam, Cambridge, MA, USA). Avidity measurements and plots were obtained with the prism 4.0 program (GraphPad Software, La Jolla, CA, USA).