Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases · 2010
[Application of double antibody sandwich ELISA for detection of nucleoside triphosphate hydrolase-II protein of Toxoplasma gondii].
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Hu Xin, Zhuge Qing-Yun, Li Ya-Fei, Pan Chang-Wang, Tan Feng
Аннотация
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, MNTI 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 microg/ml for whole-tachyzoite lysate and 1.5 microg/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.
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, MNTI 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 microg/ml for whole-tachyzoite lysate and 1.5 microg/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.