水产学报2011，Vol.35Issue(5)：780-786,7.DOI:10.3724/SP.J.1231.2011.17216
血卵涡鞭虫单克隆抗体的制备与初步应用
Preparation and preliminary application of monoclonal antibodies against Hematodinium sp.in Portunus trituberculatus
摘要
Abstract
The Portunus trituberculatus from Zhoushan is seasonally infected by a parasitic dinoflagellate of the genus Hematodinium sp.Dinoflagellates were found in haemolymph and tissues of infected crabs where the parasite proliferates and causes mortalities.Monoclonal antibodies (McAbs)used to detect infection include a morphological index ( pleopod diagnosis ) and several immunoassays.The monoclonal antibodies against Hematodinium sp.were developed by fusing SP2/0 cells with splenocytes of 8-week-old female BALB/c mice immunized with soluble antigen of Hematodinium sp..Indirect Enzyme-Linked Immunosorbent Assay (ELISA)was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones.After three cycles of subcloning, three McAbs against Hematodinium sp.were selected and designated as 2B2,3G4 and 4G7 respectively.The three McAbs in culture liquid were proved to have high ELISA titers and inducing ascites by using it.The ELISA titer in culture liquid and ascites were 5.12 × 10 -4and 8.00 × 10 -4 in the indirect ELISA ,respectively.By using the immunoglobulin subtypes kit, three McAbs were identified to be IgG.Identification of the monoclonal antibody specificity via indirect fluorescence-antibody examination showed that Hematodinium sp.was specific labeled with bright yellow green under UV, while the normal haemocytes remained dark.Through monoclonal and polyclonal antibodies,double antibody-based sandwich enzyme-linked immunosorbent assay detecting Hematodinium sp.was built.The positive compatibility rate of the double sandwich ELISA was 100％.Results showed that the monoclonal antibody characterized by high valence and high specificities,can be used to diagnose Hematodinium sp.in P.trituberculatus at an early stage.关键词
三疣梭子蟹/血卵涡鞭虫/单克隆抗体/间接荧光抗体法/双抗体夹心ELISA法Key words
Portunus trituberculatus/ Hematodinium sp./ monoclonal antibody/ indirect immunofluorescent assay/ double sandwich ELISA分类
生物科学引用本文复制引用
查智辉,施慧,许文军,谢建军,孙忠..血卵涡鞭虫单克隆抗体的制备与初步应用[J].水产学报,2011,35(5):780-786,7.基金项目
浙江省自然科学基金项目(Y3080181,Y3080317,Y3090402) （Y3080181,Y3080317,Y3090402）
2009浙江省大学生科技创新项目(新苗孵化项目) （新苗孵化项目）
2010浙江省大学生科技创新项目(2010R411037) （2010R411037）
浙江省科技厅项目(2009C32062,2010F20006) （2009C32062,2010F20006）
