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

用血卵涡鞭虫可溶性抗原免疫BALB/c小鼠,经常规融合、间接ELISA方法筛选,将所得阳性克隆再经3次亚克隆后,共获得3株针对血卵涡鞭虫的单克隆抗体(2B2、3G4、4G7),单克隆抗体亚类鉴定表明,三者为IgG类抗体。用筛选的杂交瘤细胞株制备小鼠腹水抗体,其细胞上清及腹水效价分别为5.12×10-4和8.00×10-4。进一步利用单克隆抗体建立间接荧光抗体方法对单抗特异性进行鉴定,阳性虫体被染上黄绿色荧光,而正常梭子蟹血淋巴则未被染色。用单克隆抗体和多克隆兔抗血清以羊抗鼠HRP-IgG为酶标抗体,建立了检测血卵涡鞭虫的双抗体夹心ELISA方法,该方法对血卵涡鞭虫阳性标本检测符合率为100%。结果表明,制备的单克隆抗体效价高、特异性好,可用于血卵涡鞭虫的早期临床诊断。

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.