Abstract: Objective To establish a double antibody sandwich ELISA method for detection of nucleoside triphosphate hydrolase-Ⅱ （NTPase-Ⅱ） protein of Toxoplasma gondii. Methods BALB/c mice were immunized with recombinant NTPase-Ⅱ （rTgNTPase-Ⅱ） protein of T. gondii. The hybridomas that secreted high titer of monoclonal antibodies （mAbs） with high specificity were screened and used to establish the double antibody sandwich ELISA for the detection of rTgNTPase-Ⅱ. In order to evaluate the sensitivity of the method, the concentration of whole-tachyzoite lysate and rTgNTPase-Ⅱ was detected, respectively. Serum samples from patients with malaria （7 cases）, schistosomiasis （12 cases）, paragonimiasis （14 cases） and cysticercosis （10 cases） were examined by the same method. Results Two hybridoma cell lines, MNT1 and MNT2, were developed for secreting mAbs against rTgNTPase-Ⅱ. Western blotting analysis showed that the two mAbs specifically recognized rTgNTPase-Ⅱ and whole-tachyzoite lysate. The MNT1 was used as coating antibody, and HRP-labeled MNT2 as secondary antibody. The double antibody sandwich ELISA detecting rTgNTPase-Ⅱ was developed with a minimum concentration of 6 μg/ml for whole-tachyzoite lysate and 1.5 μg/ml for rTgNTPase-Ⅱ. An overall specificity of 100% was determined. Conclusion The double antibody sandwich ELISA based on MNT1 as coating antibody and MNT2 as secondary antibody has a high specificity.

Key words: Toxoplasma gondii, Monoclonal antibody, Nucleoside triphosphate hydrolase-鄄Ⅱ protein, Double antibody sandwich ELISA