摘要
采用A蛋白双抗夹心法,在包被抗血清前先用A蛋白包被微量测定板,利用A蛋白抗血清中免疫球蛋白的FC部位结合,以F(ab')2部位吸附抗原毒素,在被吸附的毒素上再加一层相同的抗血清和酶标记的A蛋白,以检测杂交竹梢枯病菌毒素蛋白。结果表明:A蛋白预包被能提高检测效价,降低本底值。抗原浓度对酶联检测有显著影响,纯毒素稀释倍数大于10-6,检测结果与对照接近,不适宜用于检测。OD值随抗原稀释倍数的增加下降,且下降幅度受测定抗血清浓度的影响;当检测液稀释1∶5(1∶50时,测定抗血清浓度在10-4(病株汁液)和10-6(含毒素发酵液)时可测出显著差别。抗血清浓度为10-4下,含毒素发酵液在标记A蛋白浓度为1∶10(1∶640范围内有较好测定结果,而病株汁液在标记A蛋白浓度为1∶10(1∶160才能测出。本研究首次将此技术用于真菌毒素检测中,该方法对杂交竹病株汁液中的致病毒素有高度的特异性和灵敏度,能早期诊断暗孢节菱孢菌引起的杂交竹梢枯病。
Protein A double sandwich method was used to detect the protein toxin of A.phaeospermum.Before the antiserum was coated,the microplate was coated with protein A;The antigenic toxin was adsorbed by F（ab＇）2 piece on use of linked FC piece of immunoglobulin in the antiserum of protein A,and the adsorbed toxin was coated again with the same antiserum and enzyme marked protein A.The result showed that the pre-coated protein A could improve the detected titer and reduce the background value.The concentration of antigen had a significant effect on ELISA,the result was similar to the level of control when the dilution multiple of toxin was more than 10-6,and it was not adapted for testing.OD value declined with the increase of the dilution multiple,and the rate of descent was affected by the concentration of antiserum.When the dilution multiple was 1∶5（1∶50,the significant difference was detected in the concentration of antiserums of 10-4（the juice of diseased plant） and 10-6（toxin containing fermentation liquid）.When the concentration of antiserum was under 10-4,it had a better result within the concentration of Protein A 1∶10（1∶640 in toxin containing fermentation liquid,and the juice of diseased plant was detected within the concentration 1∶10~1∶160.This method is firstly used in the fungus detection and it has high specificity and sensitivity to the pathogenic toxin of the juice of hybrid bamboo,so it can early diagnose hybrid bamboo blight caused by A.phaeospermum.
出处
《四川林业科技》
2012年第5期8-11,共4页
Journal of Sichuan Forestry Science and Technology
基金
国家自然科学基金资助项目(项目编号:31070578)
关键词
酶联免疫法
毒素
杂交竹
暗孢节菱孢菌
ELISA
toxin
Bambusa pervaiabilis × Dendrocalamopsis daii
Arthrinium phaeospermum
