[Objective]Fasciola gigantica excretory and secretory antigen monoclonal antibodies were prepared for further study on diagnosis and control of Fasciola gigantica diseases.[Method]The monoclonal antibodies of Fasciola gigantica excretory and secretory antigen were made by hybridoma technology and its biological characteristics were identified by ELISA,thiocyanate elution.[Result]After indirect ELISA selection and three times of subcloning,five hybridoma cell lines(6D3,6B4,7D2,7D1,and 7D4) of monoclonal antibodies against excretory-secretory antigen of Fasciola gigantic were obtained.The numbers of chromosome of hybridoma cell lines,ranging from 90 to 110,were larger than that of the parent cells.The prepared five McAbs were found to be the isotopes of Ig G2b,Ig M,Ig M,Ig G1 and Ig M,respectively,with κ-light chain.The supernate antibody titers of 6D3,6B4,7D2,7D1,and 7D4 were 1:400,1:3200,1:25600,1:6400,and 1:6400,respectively;the ascites titers of five hybridoma cell lines were 1:104,1:104,1:105,1:107,and 1:106,respectively.Epitope mapping indicated that 6D3 and 7D1,6D3 and 7D2,7D1 and 7D2,7D4 and 7D1,and 7D4 and 7D2 acted on different epitopes;6D3 and 6B4 probably acted on the same epitopes,or had steric hindrance on the same epitope;7D4 and 6D3,6B4 and 7D4,6B4 and 7D1,and 6B4 and 7D2 might have steric hindrance.Relative affinity sequence of the five lines was 6B47D47D16D37D2.All of the five cell lines could excrete monoclonal antibodies steadily.[Conclusion]It was concluded that the produced monoclonal antibodies against excretory-secretory antigen could be applied in the follow-up research of Fasciola gigantica diagnosis and immune mechanism.

To investigate immunomodulatory effect of Asparagus cochinchinensis polysaccharide(ACP),the murine splenic lymphocytes cells were incubated in RPMI-1640 tissue culture medium supplemented with ConA(final concentration 5 mg/L).ACP was added at concentrations of 0,50,100,200 or 400 mg /L on 24,48,72 h,respectively.The cell proliferation were observed.The results showed that ACP was able to stimulate proliferation of splenic lymphocytes within 50 to 400 mg/L.

The BALB/c mice were immunized with variant surface glucoprotein of Trypanosoma evansi.The spleen lymph cells were fused with mouse SP2/0 myeloma cells,two hybridoma cell lines 3D7 and 5B9 secreting monoclonal antibodies against variant surface glucoprotein were determined by indirect ELISA.Their titers in cell culture supernatant were 1∶6400 and 1∶12800,and titers of ascites were 1∶105 and 1∶106,both 3D7 and 5B9 belongs to IgG1 with κ chain.

A dominant VAT(variant antigen type) of Trypanosoma evansi was usued as solube antigen to establish an indirect ELISA for Trypanosoma evansi in water buffaloes. The results indicated that the established method had very high sensitivity,specitivity,and good effects were obtained by using this method to detect experimentally infected samples. This research set up a base for further establishing an indirect ELISA for Trypanosoma evansi in clinical use.

Healthy tilapia Oreochromis niloticus averaging(100±10)g in body weight were vaccinated by killed Streptococcus iniae cells as immunogen via oral administration,immersion,and intraperitoneal injection.The white blood cell count(WBC),differential leucocyte count(DLC),phagocytes activities,bacteriolytic activities,lysozyme activities,superoxide dismutase(SOD) activities and antibody activity(OD450 nm) were determined in the blood collected from the caudal veins of three fish in each group randomly sampled 0,3,7,10,14,and 21 days after immunization.The test fish were challenged by intraperitioneal injection(IP)(1.0×107 colony-forming units(cfu)/fish)of S.iniae 22 days after immunization.The tilapia immunized with vaccine showed significantly higher WBC,and the amount of lymphocyte,neutrophilic granulocyte and monocyte than those in the control group(P<0.05).There were significantly higher serum bacteriolytic activities,lysozyme activities and antibody activity(OD450 nm) in the vaccinated fish than those in the fish in the control group(P<0.05) while the immunized fish showed no significant differences in the superoxide dismutase(SOD) activities compared with the control group(P>0.05).The higher relative percent survival(RPS) was found in the fish immunized by IP and immersion,and the highest immune ability was observed in the tilapia treated by IP.

通过饱和硫酸铵盐析结合 Sephadex G-200柱层析的方法,纯化制备了健康非免疫状态下奥尼罗非鱼的免疫球蛋白,进行变性还原条件下的聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹试验对血清的免疫球蛋白进行初步分析,发现SDS-PAGE电泳条件下血清重链分子量为85 kD,轻链分子量为30 kD.如果罗非鱼血清免疫球蛋白在自然状态与其他硬骨鱼类一样也为四聚体,那么其总分子量的理论值应为920 kD.