Antigen concentration and serum and conjugate dilution - The optimal concentration of TES antigen, using standard positive and negative sera, revealed an optimum of 0.1 µg/ml (equivalent to 0.2 ng per dot). The best cut-off for the detection of specific anti-Toxocara antibodies in sera was correspondent to 1/160 dilution. However, minimum concentrations of TES 0.01µg/ml (equivalent to 0.02 ng/dot) can be used to detect anti-Toxocara IgG at 1:320 serum dilutions. At all antigen concentrations, pooled normal sera gave negative results and nonspecific reactions were not found in either antigen or serum blank round areas. 1:1000 dilution of conjugate were found to be optimal for most preparations because they provide a clear discrimination of positive and negative reactions.
Incubation time - The pooled positive and negative sera were incubated with antigen for a period of time varying from 30 to 60 min, at room temperature. Best results could be observed after 45 min. The optimum incubation time for the peroxidase conjugate was also 45 min.
TES antigen fixation to nitrocellulose paper - In order to determine the most appropriate type of antigen dilution solution, the antigen was diluted in PBS or in buffered sodium carbonate bicarbonate solution (0.02 M Na2CO3; 0.03 M NaHCO3, pH 9.6) with or without 2% ovoalbumin (OVA). Sodium bicarbonate buffer with 2% OVA was the better antigen dilution solution since the positive dot increases color intensity without affecting the negative dot color in the same strip.
Sensitivity and specificity of the dot-ELISA assay - All 30 serum samples from patients with toxocariasis reacted positively, giving sensitivity of 100%. The specificity was checked on 60 sera. From 20 sera of patients with different parasitic infections, one from a strongyloides case, one from fascioliasis case and one from teniasis case reacted positively, but not a single positive reaction was observed among the sera from 40 apparently normal individuals, giving a specificity of 95%. When sera from all patients with different parasitic infections were repeatedly tested, the same cross-reaction was obtained.
Comparison of dot-ELISA with standard ELISA - All 30 sera investigated to detect IgG antibodies anti-Toxocara gave positive results by dot-ELISA and by standard ELISA. However, even after absorption with A. suum extract, standard ELISA showed positive results in 6 serum samples from the control group (3 from patients with teniasis, 1 with strongyloidiasis, 1 with fascioliasis, and 1 with cysticercosis) while only 3 serum samples from the same control group (1 patient with strongyloidiasis, 1 with fascioliasis and 1 with teniasis) were positive by dot-ELISA (Table I). Likewise, our results showed a good correlation between the visual grading of the dot-ELISA and the absorbance of ELISA (Table II).