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.