Obtention of fully embryonated Toxocara canis eggs - T. canis eggs were obtained by dissection of gravid adult female worms and left to embryonate for 30 days at 28°C in a mixture of 2% formalin and 1% sodium hypochlorite solution. Embryonated T. canis were washed repeatedly with sterile distilled water and decoated for 20 min in 5% sodium hypochlorite solution at 37°C (Espinoza et al. 2003). The egg suspension was then washed repeatedly in sterile distilled water until all traces of chlorine had been removed.
Toxocara canis excretory-secretory antigens (TES) - Fully embryonated T. canis eggs were hatched by mechanical treatment with sterile glass beads for 15 min and the larvae were collected in a Baermann apparatus (Lescano 1991). The TES antigen were produced according to De Savigny (1975) modified by Bowman et al. (1987). The supernatant from the Toxocara larvae cultures was removed each week, then dialyzed and concentrated using PEG 20,000 (mol. wt) (Sigma, US). The protein content of the TES antigen was estimated (Lowry et al. 1951) and finally kept in aliquots at 20°C until use.
Ascaris suum adult stage extract - Adult worms were collected from swine intestine at local abattoir and washed in distilled water. Adult extract was prepared as described (Camargo et al. 1992) and protein concentration was estimated (Lowry et al. 1951) and finally kept in aliquots at 20°C until use.
Absorption with A. suum extract - Each human serum to be assayed was absorbed (v/v) with soluble A. suum extract containing 52 µg/ml of antigen in order to remove nonspecific antibodies that might cross-react with TES antigen. After incubation at 37°C for 1 h dilutions of the absorbed sera were made and added to the nitrocellulose (NC) strips or microtitration wells.
Human sera - A total of 90 serum samples from 3 groups of persons were tested by standard ELISA and dot-ELISA. Group A included 30 sera from patients with clinical diagnosis of toxocariasis. Group B consisted of 20 sera from patients infected with helminths: Ascaris lumbricoides (n = 3), Fasciola hepatica (n = 3), Taenia sp. (n = 3), Strongyloides stercoralis (n = 3), Hymenolepis nana (n = 3), Trichuris trichiura (n = 3), and larval stage of T. solium (n = 2). All infections were confirmed parasitologically except patients with cysticercosis, which were diagnosed by computed tomography findings and serological data. Group C comprised 40 serum samples from apparently normal children without evidence of toxocariasis.
Positive and negative reference samples were used to standardize the dot-ELISA. A positive reference serum was prepared by pooling 10 sera from patients with clinical diagnosis of toxocariasis with high (1024) and intermediate (512 to 256) titers in the ELISA test. A negative reference serum consisted of a pool of sera from clinically healthy and parasitologically negative children with no evidence of toxocariasis.
ELISA procedure - Standard ELISA was performed as reported by De Savigny et al. (1979) and Espinoza et al. (2003) with slight modifications. Briefly, 96-well microtiter polystyrene plates (Immulon 2 HB, US) were sensitized with a TES antigen solution (100 µl/well) containing 12.5 µg of protein per ml in 0.05 M bicarbonate buffer, pH 9.6, and then maintained for 18 h at 4°C in a moist chamber. The microtiter plates were washed 3 times with 0.15 M phosphate-buffered saline-0.05% Tween 20 (PBS/T) and then tested with a 1:128-diluted human serum sample (100 µl/well) in PBS/T containing 5% nonfat skimmed milk powder for 60 min at 37°C. The plates were washed 3 times with PBS/T and then 100 µl of a 1:4000 dilution of anti-human IgG-peroxidase conjugate (Sigma, US) were added, to each well. After 60 min the plates were again rinsed and to each well was added 100 µl of substrate solution, consisting of o-phenylenediamine (Sigma) 0.04 and 0.03% hydrogen peroxide diluted in 0.05 M citrate-phosphate buffer, pH 5.0, for approximately 30 min, then stopped by the addition of 2 N sulphuric acid and the plates were read at 492 nm, using an automatic microplate reader (Multiskan plus Labsystem version 2.01). The cut-off point was set at the mean optical density (OD) of the negative reference serum, plus 3 standard deviation (SD). Serum samples with OD > 0.26 (cut-off) were considered as reactives.
Dot-ELISA procedure - >The optimal conditions for dot-ELISA were standardized according to the procedures described by Boctor et al. (1987) with some modifications. Strips of NC paper (Sigma), 1 cm wide and 6 cm long were placed on a microtiter 96-well plastic plate and after a slight pressure round areas were marked. Two microliters of several different TES antigen concentrations (0.01- 40 µg/ml) were dotted on separate round areas. After drying, the free bindings sites on the paper were blocked by 18 h incubation at 4°C in 0.01 M phosphate-buffered saline (PBS), pH 7.2 containing 5% defatted dry milk. Then, the strips were washed 3 times (5 min/wash) with PBS containing 0.05% Tween 20. Twelve microliters of diluted serum (ranging from 1:40 to 1:320) were tested to determine the optimal working dilution. After 45 min at room temperature, they were washed as described above and incubated with 1:1000 dilution of goat anti-human IgG peroxidase conjugate (Sigma) for 45 min at room temperature. The strips were washed and soaked in freshly prepared substrate solution, which consisted of 0.5 mg of 3,3'-diaminobenzidine (Sigma) per milliliter and hydrogen peroxide (0.01%) in 0.01 M citrate buffer, pH 5.0, for 5 min. The reaction was stopped by washing with water and left to dry. The development of brown dots on NC was considered evidence of positively. The intensity of the color was judged by the naked eye, and numbers were on an arbitrary scale of 0; +; ++; +++; or ++++ in reference to the negative control (0).
Statistical analysis - For the determination of the dot-ELISA and micro-ELISA sensitivity, specificity and positive and negative predictive values, 2 ´ 2 tables were carried out.