双抗体夹心ELISA法检测海产蟹血卵涡鞭虫方法的初步建立

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。

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

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.