To Prepared Antigen And Polyclonal Antibody Of Abrin Toxin And Two Immunological Methods Establishmen

Abrin, which is similar to ricin, is one kind of plant toxin. It exhibits strong cytotoxic effect, and wouLd be used as bioterrorism agent which existed potential threat to public security. Therefore, it is important and necessary to establish and develop new method to detect abrin with high specificity and sensitivity.In this paper, the traditional method for extraction and purification of abrin was improved and optimized. New purification method was established. The abrin obtained using this new method was used as reference in relevant experiments. Two polyclonal antibodies from animal origin were prepared through expressing and recombinanting abrin A-chain protein in E. coli. The detection method based on colliodal gold immunochromatographic assay and double-antibody sandwich biotin-adivin ELISA assay was proved to be specific and sensitive in abrin detection.First of all, most contaminants in seeds were removed using affinity chromatography according to the specific binding capacity between abrin and galactose. Since toxic proteins always coexist with lectins in plant seeds, gel chromatography was carried out to remove lectins according to the molecuLar weight difference between abrin and lectin. The pure abrin obtained, which LD50 was 0.36 ug/kg, was used as reference.Then, abrin A-chain protein obtained through recombinant protein system in E. coli, further purified using metal chelating chromatography, was used to immunize white rabbit and Kunming mice. The rabbit serum polyclonal antibody and mice ascetic polyclonal antibody were collected and purified. Then the two polyclonal antibodies were used to recombinant abrin A-chainprotein. Combining the labeling technology, the colliodal gold immunochromatographic assay based on double-antibody sandwich and ELISA based on biotin-adivin were established in quantitive detection of abrin, respectively. The detection sensitivity of colloidal gold and ELISA couLd reach 600ng/mL and 0.6ng/mL, respectively. The linear range was 30ng/mL-600 ng/mL and 0.06ng/mL-600 ng/mL, respectively, while the recovery rate were 80%-110% and 73.17%-150%, respectively. Besides, both the variation coefficients were less than 15%.Finally, animal serum and several common foods were used to simuLate the environmental samples. Ricin and other common toxins evaluation showed that both methods possessed high specificity. The detection sensitivity of simuLated environment experiment was the same.