ELISA
The native excretory-secretory (E/S) antigen from the second-stage larvae of Toxocara canis was used to perform a manual enzyme-linked immunosorbent assay (ELISA) previously prepared (Zibaei et al. 2016). A brief of the ELISA method was as follows: 5 μg/ml of antigen in carbonate bicarbonate buffer (pH 9.6) was coated in 96 wells of microplate and incubated at 4°C. The next day, the microplates were automatically washed by ELISA washer with washing buffer (containing 0.05% Tween 20 in PBS). 5% skimmed milk in PBS was added to each well and incubated for 2 hours at room temperature to eliminate non-specific surface attachment and improve assay sensitivity. After washing according to the method, 100 μl of diluted serums (1:100 in PBST solution) were poured into the wells. Next, after washing, 100 μl of secondary antibody anti-human IgG conjugated with horseradish peroxidase (1:4000 in PBST) was added to ELISA plate wells and then incubated in a dark place at 37 °C for one hour. The plate was then washed as before, and 100 μl of the substrate (containing 0.4 mg/ml OPD, 0.3% H2O2 in 0.1 M citrate buffer, pH=5.6) was added to it then; the plate was placed in a dark place for 15 to 20 min at room temperature to visualize the antigen/antibody reaction. Finally, the stopping solution (sulfuric acid) was added (100 μl/well) to stop the reaction, and then the OD values were measured at an absorbance of 490 nm by an ELISA plate reader (ELx800, BioTek, USA). Each test included positive and negative controls, and the cut-off point was determined using the following formula: the mean of the negative controls’ OD value plus two standard deviations. Also, all positive sera were double-checked for final confirmation by the described ELISA method.