棉花大丽轮枝菌(Verticillium dahliae)血清学检测--抗原处理的比较

分别以棉花黄萎病菌V20的菌丝研磨物、菌丝蛋白、超氧化物歧化酶酶带Ⅰ、超氧化物歧化酶酶带Ⅱ为抗原免疫家兔,制备了抗血清As1、As2、As3、As4。琼脂双扩散法(ADD)和间接ELISA方法测定其特异性,发现这4种抗血清既可与同种的抗原发生阳性反应,也可与不同种的抗原发生交叉反应。用异种抗原黑白轮枝菌和尖孢镰刀菌吸附这4种抗血清,排除其非特异性成分后,抗血清专化性明显提高,消除了交叉反应。特异性测定表明:经吸附后的4种抗血清可以准确地将大丽轮枝菌鉴定到种的水平,但仍不能区分其生理型和致病类型。另外,当分别使用菌丝研磨物或菌丝蛋白为特异性测定抗原时,ADD检测的结果虽然一致,但前者阳性反应沉淀线粗糙,轮廓不清晰,后者的沉淀线较细,且清晰、明显。因此适宜选择菌丝蛋白为抗原进行抗血清的特异性测定试验。

Production and characterization of polyclonal antibodies prepared against different antigens of Verticillium dahliae from cotton

The antisera As1, As2, As3, As4 with different specificity prepared against different antigens, induding mycelium, protein, band I of SOD isozyme, and band II of SOD isozyme of V20, a nondefoliating strain of Verticillium dahliae, were prepared. The specificities of four different antisera were tested by agar dimension diffusion (ADD) and indirect enzyme-linked immunosorbent assay (ELISA), respectively. All antisera showed nonspecific reaction with homogeneous and heterogeneous antigens. Antibodies specific for V. dahliae were purified from polyclonal antibodies by mixing them with the antigen V. albo-atrum and Fusarium oxysporum. After purified, the nonspecific polyclonal antibodies reacted with V. dahliae but not with other fungi commonly found in cotton diseases, although the antisera still can not determinate physiological type and pathotype of V. dahliae. The results from ADD were the same with it from ELISA. However, as the results of ADD, to a certain degree, the specialty of the antisera prepared against antigens of the proteins of V. dahliae was improved more than the antisera prepared against mycelial preparations, which was shown by the different quality of deposit lines of ADD. Therefore, we chose protein of mycelial as the antigen to do the specificity test.