番茄黄化曲叶病毒双抗体夹心ELISA检测方法的初步建立

Establishment of Double Antibody Sandwich ELISA for Tomato Yellow Leaf Curl Virus

以纯化的番茄黄化曲叶病毒(TYLCV)外壳蛋白为抗原,免疫家兔制备并纯化出TYLCV的多克隆抗体IgG,以此抗体做包被抗体,并用碱性磷酸酶(AP)对其进行标记作为酶标抗体,从而建立了番茄黄化曲叶病毒的双抗体夹心ELISA(DAS-ELISA)检测方法。通过ELISA方阵试验确定该法的最佳工作浓度为酶标抗体(IgG-AP)作1∶400倍稀释,包被抗体浓度为6.25μg.mL-1;并且确定了抗原最低检出浓度为9.75ng.mL-1。采用该法对山东寿光的田间病样进行了定性和定量检测,结果表明建立的DAS-ELISA方法灵敏度高、特异性强,可用于番茄黄化曲叶病毒的常规检测。

The rabbit IgG of Tomato Yellow Leaf Curl Virus （TYLCV） antiserum was purified and conjugated with alkaline phosphatase （AP） by glutaraldehyde one-step method. The DAS - ELISA method for TYLCV detection was established. The optimal dilution of IgG-AP was 1 ： 400 and the optimal concentration of coated antibody was 6.25 μg· mL^（-1）, which was determined by phalanx titrimetry, at the same time the curve indicated that the lowest detection limit was 9.75ng· mL^（-1）. Field samples collected from Shouguang of Shandong Province were detected qualitatively and quantitatively by DAS - ELISA. All of these results showed that DAS -ELISA method was sensitive and specific, which could be easily applied in the field.