Preparation,Characterization And Preliminary Application Of Monoclonal Antibodies Against Toxoplasma Gondii P30

Objective:The study was carried out to prepare monoclonal antibodies against P30 of Toxoplasma gondii and then to evaluate the monoclonal antibodies for the purpose of diagonosis of Toxoplasmosis at early stage.Methods:After induction of recombinant plasmid pET28b (+)-P30 into E. coli BL21 (DE3), P30 expressed was purified by Ni-NTA affinity chromatography and renatured by dialyzing method. Then the renatured P30 was quantified with BCA kit and were used to immunize BALB/c mice. According to the hybridoma technique of B lymphocytes, the immunized mouse spleen cells was isolated while attaining required serous antibody titer (1∶10 000 detected by ELISA) and fused with mouse MM SP2/0 cells by using polyethylene glycol (PEG), then hybridomas were cultured in HAT selection medium. During from the 10th to 20th postfusion days, ELISA method coating with recombinant P30 was applied to screen for positive clones. Sequentially the positive cell lines were propagated and tested repeatedly for assuring stability of positive clones. Hybridoma cell lines were established finally when subcloning wells were all positive. Using Pristane-primed BALB/c mice, production of the anti-P30 McAbs was made in vivo (ascites), which was induced by intraperitoneal injection of well-grown hybridomas and acquired from the 7th to 10th day. Following precipitaion with Ammonium Sulfate, the McAbs was purified by immunoaffinity chromatography. For McAb-isotyping, fast-strip method analysis were performed. SDS-PAGE was employed for analysis of ascites. Titers of McAbs from ascites and McAbs purified were measured by ELISA. The specificity of McAbs was analyzed by Western blotting. Karyotype analysis was carried out as usual and affinity constants were determined by means of ELISA. Antigen determinants were identificed by inhibitory ELISA. Eventually, CAg of T.gondii in mouse serum was detected by Double-McAb- Sandwich-ELISA, using 2H9 as coating antibody and 9D7 as labeling antibody. Results:1. The purity of the purificated and renatured recombinant P30 was above 90% with concentration of 512ng/mL.2. Two hybridomas (9D7, 2H9) secreting anti-P30 McAb continuously and steadily were obtained after repeatedly cloning and screening. The hybridomas grew well after long-term culturing and storage in liquid nitrogen.3. Ascites can be obtained from 90% mice, 6.5 ml each on average by in vivo inducing. After purified from ascites using affinity chromatography, the McAb concentration was 0.8～1.6 mg/mL.4. Fast-strip method analysis showed that McAb 9D7, 2H9 belongs to mouse IgG2a , IgG1 subclass, respectively, and for the light chains, both of them wereκ. The mean titers of 2H9,9D7 ascites were above 1∶10 000 and 1∶8 000 respectively, and the mean titers of McAbs were all above 1∶3 000 after purification.5. Identification of antigen determinants with inhibitory ELISA suggested that the two McAbs recognized different antigen epitopes of P30. And Karyotype analysis illustrated that the chromosome number of two anti-P30 hybridoma cell lines were all above 100. The result of Western blotting indicated that the McAbs can identify the P30 from lysed Toxoplasma gondii Tachyzoites or recombinant P30. The mean Affinity constants calculated for 2H9 and 9D7 were 7.64×10⁹ M^-1 and 6.13×10⁷ M^-1, respectively.6. Detected with Double-McAb-Sandwich-ELISA, the positive rates of CAg in mice infected with toxoplasma gondii were 20%, 50%, 60% for postinfection days 4, 5, 6 , respectively.Conclusion:1. The mouse anti-P30 McAbs were prepared successfully.2. The two McAbs had good performance of specificity and affinity.3. The McAbs may be utilited for the purpose of diagonosis of Toxoplasmosis at early stage.