Prelimilary Research On The Development Of Enzyme-linked Immunosorbent Assay For Diosgenin

Diosgenin is extracted from the root and stem of dioscorea genus and it is a important precursor of steroid drugs. Diosgenin can be detected by means of colorimetry, TLC, HPLC, HPGC et al. Compared with these methods, enzyme-linked immunosorbent assay ( ELISA )is more sensitive and consume less samples. The disposal of samples is much easier. ELISA is especially applicable for large quantities of samples. In this paper, the development of ELISA for diosgenin was studied.Diosgenin, with low molecular weight, is not a antigen and cann't induce antibody in the body of animals. Then it need to be derivatized and conjugated to proteins to prepare the artificial antigen of diosgenin. In the presence of succinic anhydride and catalyst DMAP, diosgenin was derivatized into diosgenin hemisuccinate which had reactive carboxyl group. The termination of the reaction was measured by thin layer chromatogram(TLC). The optimal condition of the reaction was as follows: temperature 95℃, mole ratio of diosgenin and succinic anhydride 1 '. 4, mass ratio of diosgenin and catalyst DMAP 0.08 : 1. The product was characterized by means of MS, IR, ¹H-NMR and ¹³C-NMR spectra.Diosgenin hemisuccinate was attached to BSA by method of mixed anhydride and dicyclohexylcarbodiimide(DCC), to OVA by method of 1-Ethyl-3-(3-dimethyla-minopropyl) carbodiimide hydrochloride(EDC) and DCC. The conjugates were measured by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and the number of diosgenin residues per molecule of the conjugates DG-HS-BSA(mixed anhydride method), DG-HS-BSA(DCC), DG-HS-OVA(EDC) and DG-HS-OVA(DCC) was 28.2, 1.5, 7.1, 0.8, respectively. The results showed that mixed anhydride and EDC methods were much better than DCC method.DG-HS-BSA(mixed anhydride method) was injected in three male New Zealand rabbits and the immune serum was evaluated by an indirect Dot-ELISA. The results showed that antibody against diosgenin were induced in all the three rabbits. DG-HS-OVA(EDC) was used as coating antigen and a competitive inhibition enzymeimmunoassay technique was primarily developed for the detection of diosgenin. Results on different plated showed that the standard curve of diosgenin had a liner range from 25 ng/mL to 500 ng/mL.