Parasites and cells
Extracellular amastigotes (EA) from T. cruzi G (25), Y (26), CL (27), CL.B clone (28), MD (provided by Maria P. Deane from Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil), F (29), Sylvio X-10/4 clone (30) and Tulahuen (31) strains were isolated after differentiation of tissue culture-derived trypomastigotes in LIT medium as previously described (13) and intracellular (IA) parasites were purified from infected Vero cells after mechanical disruption and centrifugation through a 10/15% metrizamide gradient (32). We also examined, for comparison, amastigotes isolated from supernatants of infected Vero cells, which were designated tissue culture amastigotes (TCA). In several experiments we used a clone, designated D11, isolated in our laboratory from G strain by the method described by Lima and Villalta (33), and the clone CL.B. Some characteristics of the D11 clone resemble the parental strain. D11 causes subpatent parasitemias in outbred albino mice, and metacyclic trypomastigotes displaying the monoclonal antibody (mAb) 1G7-reactive antigen, gp90, are completely lysed by this mAb (10,34).
Vero cells (obtained from Instituto Adolfo Lutz, São Paulo, SP, Brasil) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Cultilab, Campinas, SP, Brasil), 10 µg/ml streptomycin, 100 U/ml penicillin, and 40 µg/ml gentamycin at 37oC in a 5% CO2 humid atmosphere. Cells were subcultured every 2/3 days and after infection they were kept in RPMI/2% FBS at 33oC to improve the yield of released trypomastigotes.
Monoclonal antibodies
mAbs were produced by immunizing BALB/c mice with amastigotes from clone D11 of the G strain. Their isolation and immunochemical characterization have been described elsewhere (35). Briefly, mAb 4B5 (IgG1) was obtained from mice immunized with psoralen/UV-inactivated (36) intracellular amastigotes, and mAbs 1D9 (IgG3) and 4B9 (IgG3) were isolated after immunization with mixtures of heat-inactivated intracellular and extracellular amastigotes of the same clone D11.
All antibodies were used as ascitic fluids previously titrated onto fixed parasites (see below) before routine use to determine the highest dilution that provided maximum absorbance at 492 nm (1:200 for anti-p30, 1D9, 4B5, and 4B9; 1:1000 for 2C2). mAb 2C2 (IgG2a), which is specific for the stage-specific 84-kDa glycoprotein of T. cruzi amastigotes (13), was kindly provided by Norma Andrews (Yale University School of Medicine, New Haven, CT, USA), and mAb anti-p30 (IgG2b), specific for a 30-kDa protein of L. (L.) amazonensis amastigotes (37), was kindly provided by Clara L. Barbiéri (Universidade Federal de São Paulo, São Paulo, SP, Brasil).
ELISA on glutaraldehyde-fixed amastigotes
Isolated parasites were washed 3 times in phosphate-buffered saline (PBS) and 50 µl containing 1 x 106 parasites was added to the wells of ELISA strip plates (Costar, medium or high binding capacity) and centrifuged at 800 g for 5 min. The supernatant was carefully removed by aspiration and the parasites were fixed with 0.25% (v/v) glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA) in PBS at room temperature for 20 min. After 4 washes in PBS to remove excess glutaraldehyde, the wells were blocked for at least 12 h at 4oC with 5% defatted powdered milk in PBS/0.05% sodium azide (PBS-M). At this stage, plates could be stored at -20oC for at least three months. Prior to use, the plates were warmed to room temperature for all subsequent steps: the PBS/milk was removed and the parasites were incubated with 50 µl of the antibodies diluted 1:50 in PBS-M for 1 h. After 3 washes with PBS, the bound Ig was developed by incubation with anti-mouse Ig coupled to horseradish peroxidase (Sigma Chemical Co., St. Louis, MO, USA) diluted 1:2000 (titrated dilution) in PBS-M. After 1 h the conjugate was washed 3 times with PBS and 50 µl of substrate solution (10 mg o-phenylenediamine (OPD) diluted in 25 ml 0.16 M citrate-phosphate buffer, pH 5.0, plus 10 µl of 30% H2O2) was added to each well as the substrate for the reaction. The enzymatic reaction was stopped after 4 min with 4 N H2SO4, and absorbance at 492 nm was determined with a Multiskan MS 352 reader (Labsystems, Helsinki, Finland). All readings were made in quintuplicate for each determination, in at least three independent experiments.
ELISA standardization
In order to assess the reliability, sensitivity and reproducibility of the ELISA in evaluating differences in the levels of expression of amastigote surface antigens, we performed several controls. First, we determined that a variation of up to 15% in the number of parasites/well did not significantly (less than 10% in absorbance values) influence the final values (Figure 1). This ensured that possible errors in parasite counting did not affect the final absorbance values. Second, we repeated the experiments with the different antibodies several times, using stored or freshly prepared fixed-amastigote plates, and always obtained consistent results where the relative values of absorbance of the different mAbs were the same in experiments carried out up to two months apart (data not shown). Third, the antibodies always retained their specificity and heterologous reagents always gave background values when applied to fixed amastigotes (data not shown). Fourth, using ELISA, we confirmed the results of Andrews et al. (20), who reported that the surface expression of Ssp-4 antigen on extracellular amastigotes is high after 24 h of differentiation and decreases if parasite culture in LIT medium proceeds for up to 72 h. However, the signal of an irrelevant mAb was not altered during this period (data not shown). The reduction in the expression of surface Ssp-4 is accounted for by phospholipase cleavage of the GPI anchor of the molecule, releasing soluble Ssp-4 into the culture medium (20).
Protein gel electrophoresis and immunoblotting
Sodium dodecyl sulfate electrophoresis (38) and immunoblotting (39) of protein amastigote extracts were performed on 10% resolving gels using a mini-PROTEANÒ II electrophoresis cell and a mini-trans-blot module (Bio-Rad Laboratories, Hercules, CA, USA), as previously described (10,35).
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