| Toxoplasma gondii is an obligate intracellular protozoan and has a worldwide distribution. Pigs play an important role in the process of the transmission of T. gondii.Immunochromatographic Assay, as a simple and convenient method of serological diagnosis, is not required to operate under the instrument and equipment, which is suitable for the preliminary screening of a large number of samples, especially suitable for livestock farms and field testing.GRA7 express constantly in host cells in both tachyzoite and bradyzoite. It can induce strong immune responses in the host with good immunogenicity and reactionogenicity. Therefore, GRA7 is a valuable antigen used for the diagnosis of T.gondii. In the present study, recombinant granule antigen protein GRA7 is used to establish Immunochromatographic Assay to lay a foundation for practical application.1. Development of the indirect ELISA using recombinant granule antigen protein GRA7 for detection of T.gondii specific IgG antibody in pig serum. Microplates were coated with GRA7 with a concentration of 5μg/mL in the condition of 4℃ overnight. Then blocked with nonfat milk at 37℃ for 1h. The best dilution of pig serums and IgG antibodies are 1:50 and 1:100 000 both for 90 min. TMB is added for 18 min and stopped by 2mol/L H2SO4. Receiver operating characteristic(ROC) analysis showed a relative sensitivity of 83.3% and specificity of 93.0% at the cut-off value of 0.16 for GRA7-ELISA. Area under the curve(AUC) showed GRA7-ELISA has a good stability. These results suggest that GRA7 is a potential serodiagnostic marker for T. gondii infection in pigs.4. Establishment of an Immunochromatographic Assay based on GRA7. The results showed that the best coating pH is 9.0 and the best coating protein concentration is 7.5μg/mL. Test line(T) was drawed by GRA7 at the concentration of 1mg/mL, and the control line(C) was also 1mg/mL. Pig serum samples were tested by GRA7-based colloidal gold strips, showing no significant difference with the reference ELISA. McNemar chi-square analytical method showed a Kappa value of Kappa=0.7570,(95%CI: 0.6480-0.8660). A substantial agreement of 89.2% showed a sensitivity of 83.0% and a specificity of 93.3%. The overall seropositive rate of the 176 pig was 27.8%. |
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