Development And Application Of Immunoassay For Toosendanin

Toosendanin (TSN) is a triterpeniod extracted from Melai toosendan Sieb. et Zucc. which was used as a digestive tract parasiticide and agricultural insecticide in ancient China. TSN possesses unique bioactivities and is of considerable value in clinical medicine and pest control, and has been shown to be a selective presynaptic blocking agent and is also effective in inhibiting botulism. TSN was found to induce differentiation and apoptosis in several cell lines and to suppress proliferation of various human cancer cells. It was also found to inhibit feeding and developing of insects and induce serious damage of epithelial cells in the midgut. Due to its strong oral toxicity and inhibitory effect on the pests feeding behavior and considerable safety to environment, TSN has been commonly used in China as a biorational insecticide.In order to illustrate the mechanism of action and monitor the safety of TSN, a sensitive and reliable analytical method is required for detecting the chemical in insects, foods and various environmental samples. In this paper we report the successful design of a suitable hapten for the production of monoclonal antibodies to detect TSN specifically and the development of a simple and sensitive enzyme-link immunosorbent assay (ELISA) for the quantification of TSN. Meanwhile, the binding of TSN in epithelial cells of the midgut of Mythimna separata Walker (Lepidoptera: Noctuidae) treated with TSN was studied using post-embedding immunogold labeling method.1. The hapten, 28-hemisuccinyl-TSN (TSN-S) was synthesized by using esterification and charactered by HRMS, IR, 1H-NMR and 13C-NMR. TSN-S was conjugated with carrier protein bovine serum albumin (BSA) and the egg albumin (OVA) using the mixed anhydride reaction and active ester protocol, respectively. The conjugates (TSN-S-BSA, TSN-S-OVA) were identified by the scanning of IR.2. Monoclonal antibody was produced by conventional methods, including immunization, fusion, selection, clone, etc. After intraperitoneal injection with TSN-S-BSA as the immunoantigen, the mice which showed stronger immuno-reponse were selected to take advanced operation. The splenocyte were fused with SP2/0 myeloma, followed by selecting the hybrid cells on the selective media HAT and HT. After cloning and screening of the positive clones by ELISA with TSN-S-OVA as coating antigen, two hybridoma cell lines EE7 and FB9 were selected for their capability of producting monoclonal antibodies specifically against TSN. The titer of anti-TSN antibodies in the two cell lines suspension were 1:160 and 1:80, respectively. Both antibodies belonged to IgG and their subclasses were IgG2b. Cell lines EE7 and FB9 were inoculated in mice abdomen. The titer of ascites were 4×10-6 and 8×10-6, with the protein content of 4.25 and 3.32 mg/mL.3. Working concentrations of two monoclonal antibodies/coating antigen/second antibody (HRP-goat anti-mouse IgG) were chosen by competitive assays. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for TSN was development. Under the optimization condition, the assay based EE7 showed an IC50 value of 1.806 ng /mL, with a detection limit of 0.064 ng/mL. The assay based FB9 showed an IC50 value of 3.803 ng /mL with a detection limit of 0.632 ng /mL. Both assays displayed no cross-reactivity with closely structurally related compounds.4. The application of IC-ELISA based EE7 for TSN in the determination of this analyte in various samples including botanical TSN extracts, formulation, water, soil, vegetables and fruits was studied. Melai toosendan Sieb. et Zucc. bark and fruits of were ertracted using ultrasonic method and detected with IC-ELISA. The ELISA veracity was found to vary from 95.5 to104.2%, and the results indicated that method precision was below 6.0% (RSD). TSN was spiked into different type of water and soil at 1 and 10ng/g and the recoveries were discovered to vary from 81.4% to 95.9%, with the RSD below 9.2%. TSN was also spiked into 3 different agricultural commodities: cabbage, apple and cucumber at 20 and 200 ng/g and recovered at 87.9%~98.7%, and the method precision was found to vary from 0.9 to 7.0% (RSD, n = 6) on different days.5. The ultrastructure of the midget of 6th stage larvae armyworm was observed by transmission electron microscopy (TEM). There were no obviously changes of mitochondria and rough endoplasmic reticulum of larvae treated with TSN, while the membrane of midget epithelial cells were disordered markedly with changes as follows. Microvillus of columnar cells swelled, vacuolated and disappeared, the myeline figures appeared, secretion accumulated in goblet cells, chromatin condensed in nucleous, the smooth endoplasmic reticulum dilated, and the damage increased as the treated time was prolonged.6. The ultrathin sections of midget from the larvae were prepared for the immunoelectron microscopy. This was followed by incubations with anti-TSN monoclonal antibody and colloidal gold-labeled goat-anti-mouse immunoglobulin, in orderly. The results showed that TSN could mainly bind on the microvillus, especially on microvillus of columnar cells. There were few TSN binding on smooth endoplasmic reticulum. The binding of TSN on microvillus increased with the treated time. It was suggested that there could be receptors in the membrane of midget epithelial cells.