双抗体夹心ELISA法检测海产蟹血卵涡鞭虫方法的初步建立

Establishment of Double Antibody-Sandwich ELISA for Detection of Marine Crab Hematodinium sp.

以针对血卵涡鞭虫的特异性单克隆抗体为捕获抗体,纯化的抗血卵涡鞭虫的兔抗血清为检测抗体,初步建立了检测血卵涡鞭虫的双抗体夹心ELISA方法。方阵滴定确定最佳反应条件:包被的单克隆抗体浓度为10μg/mL,多抗兔血清工作浓度为14.3μg/mL,待检样品稀释度为1:800;HRP-羊抗鼠IgG按1∶20 000稀释。用建立的ELISA方法对86份已知背景样品进行检测,阳性检出为97.6%,血卵涡鞭虫抗原检测的灵敏度为100 pg/mL;与蟹血淋巴细胞、假丝酵母菌、溶藻弧菌、副溶血弧菌等样本均无交叉反应。结果表明,建立的双抗体夹心ELISA方法灵敏度高、特异性强,可用于诊断梭子蟹乳化病血卵涡鞭虫病的检测。

On the basis of monoclonal antibod against Hematodinium sp.to Portunus trituberculatus as capture antibody and the polyclonal antiserum as detector antibody,a double antibody-sandwich ELISA was developed to detect Hematodinium sp.The micrplates were coated with monoclonal antibody at concentration of 10 μg/ml and working concentration of the polyclonal antibody purified from polyclonal antiserum was 14.3 μg/mL,and the enzyme-conjugated antibody was 1：20 000 for this double antibody sandwich ELISA assay.By antibody-sandwich ELISA,the positive rates of the Hematodinium sp.was more 97.6,and the sensitivity of this method to detect the antigens of Hematodinium sp.was 100 pg/mL,no cross-reaction was found when pathogenic yeast（Candida olephila）,Vibrio alginolyticus,Vibrio parahaemolyticus,and the blood lymphocytes were tested by this technique.These data indicated that a sensitive,specific and reproducible sandwich ELISA for detection of Hematodinium sp.was developed.It provided an useful tool for diagnosis of Hematodinium sp.infection.