Study On Indirect Competitive Enzyme-linked Immunosorbent Assay(IC-ELISA) For Carabrone

Carabrone is a sesquiterpene lactone extracted from aerial portion of Carpesium abrotanoides, which showed high and relatively broad spectrum antifungal activities. On the research about the structure-activity relationship of carabrone and its derivatives, it was found that such compounds could be developed as potential fungicide. In order to illustrate the mode of action and monitor the safety of carabrone, an efficient, sensitive and reliable analytical method is required. In this paper, suitable haptens and antigens of carabrone used to immunize BALB/c mouse were designed and synthesized. A simple and sensitive enzyme-linked immunosorbent assay for the quantification of carabrone was also established. The main results are as follows:1. Haptens CG, CB and CD were prepared through the esterification of carabrone with Boc- glycine, Boc-β-alanine and N-Boc-γ- piperidic acid, respectively. And all of them were characterized by 1H NMR, 13 C NMR and MS. The haptens were conjugated to BSA and OVA by glutaraldehyde method to form artificial immunogens(CG-BSA, CB-BSA and CD-BSA) and coating antigens(CG-OVA, CB-OVA and CD-OVA).The conjugates were identified by IR, SDS-PAGE and MALDI-TOF/MS.2. Three types of anti-carabrone polyclonal antibodies were obtained after immunization of BALB/c mouse by antigen CG-BSA, CB-BSA and CD-BSA. The titers of antiserum were 128000, 34000 and 256000, respectively.3. The IC-ELISA for carabrone was established based on antibodies CG-Ab. The effects of ionic intension, organic solvent and pH value on the sensitivity of IC-ELISA were evaluated. The results of optimization tests showed that 10% methanol, 0.4 mol/L Na+ and pH of 7.5 were the optimum conditions. Under such conditions, the standard competitive curves of carabrone was obtained using(B/B0) % versus log concentration of carabrone. The linear equation was y=-27.076 x + 55.521 in the range of 0.05-50 μg/mL, the limit of detection(LOD) IC10 and IC50 for carabrone were 0.053 μg/mL and 1.60 μg/mL, respectively. The average recoveries of carabrone in tap water determined by IC-ELISA was 87.2%~102.8% in the spiked range of 0.5~10 μg/mL with CV of 1.3%~7.1%. And the recoveries of cabbage was 86.4%~107.1% in the spiked range of 3.0~30 μg/g with CV of 2.5%~11.3%.By the established IC-ELISA, the residue of carabrone could be monitored. The method was satisfied with the requirement of efficient and sensitive for the trace analysis of carabrone. Thus the present studies laid the foundation for the research of mode of action and the evaluation of environment toxicology of carabrone.