| Dufulin is a new and efficient plant antiviral agent which was developed by Center for Research and Development of Fine Chemicals. Currently it was widely used in the tobacco mosaic virus(TMV), cucumber and tomato virus disease(CMV), southern rice black-streaked dwarf disease. It can be detect large quantities samples, and widely used in small molecule pesticide residues detection.Hapten of Dufulin(DHS and DHL) was synthesized successfully. The Dufulin were conjugated directly to carrier proteins(BSA/OVA) by ECD(Carbodiimide) method. Antibody were obtained by immunizing rabbits. Antibody was labeled by Horseradish Peroxidase(HRP) with sodium periodate method. The ELISA methods were established for detection of Dufulin after the reaction conditions were optimized, such as coating concentration and diluted multiple of antibody. Chiral recognition specificity of the antibody by cross reactivity between antibody and the corresponding isomer were studied. In order to reserch structure-activity relationship of antigen and antibody by cross-react that structural analogs of Dufulin with antibodies.(1) Design and synthesize hapten of Dufulin(DHS and DHL). Molecular structure of hapten were identified by nuclear magnetic resonance(1H-NMR, 13C-NMR), mass spectrometry(MS) and infrared spectroscopy(IR). Six kinds of immunogen X-DHS/DHL-BSA, S-(+)-DHS/DHL-BSA and R-(-)-DHS/DHL-BSA and coating antigen X-DHS/DHL-OVA, S-(+)-DHS/DHL-OVA and R-(-)-DHS/DHL-OVA were identified through full-wave UV spectroscopy. Six polyclonal antibodies(PcAb) against X-, S-, R-DHS/DHL were preparated. Serum were tested by indirect competitive ELISA method, the results showed that-high titer of antiserum is 1:80000 and minimum titer 1:10000. Antibody affinity constants was measured successfully. X-DHS/DHL. X-DHS/DHL Polyclonal antibody affinity constant(Ka) were 2.5×109 and 3.0×109 L/mol, respectively. S-(+)-DHS/DHL Polyclonal antibody affinity constant(Ka) were 2.2×109 and 1.8×109 L/mol, respectively. R-(-)-DHS/DHL Polyclonal antibody affinity constant Ka were 5.0×109 and 5.3×109 L/mol, respectively.(2) The limit of detection(IC20) and the 50% inhibition(IC50) for dufulin were 2.8 ng/mL and 37.5 ng/m L detected by the combination(DHS-PAb, DHS-OVA), and its linear equation was B/B0=-15.8lgC+68.4(R2=0.9986) in the linear range of 1-5000 ng/mL. Detection tomato, cucumber, rice and tobacco by ELISA. Intra-day and Inter-day fortified recoveries of Dufulin are 70-87.3% and 70.2-80.7%, respectively. Intra-day and Inter-day relative standard deviations(RSD) of dufulin are 7.1-11.2% and 8.4-12.1%, respectively. The results of the ELISA were verified by LC-MS/MS. LC-MS/MS method was established for the detection of Dufulin in tomato, cucumber, rice and tobacco. Intra-day and Inter-day fortified recoveries of Dufulin are 71.9-93.6% and 71.9-93.6%, respectively. Intra-day and Inter-day relative standard deviations(RSD) of dufulin are 2.9-8.5% and 6.9-15.2%, respectively.(3) Cross-reativety test of six kinds of polyclonal antibodies. X-DHS/DHLwith S-(+)-DHS/DHL and R-(-)-DHS/DHL of the cross-reactivity rate about 100%. Indicating that the antibodies of X-DHS/DHL can?t recognize the corresponding optical isomer. The polyclonal antibodies of S-(+)-DHS/DHL and R-(-)-DHS/DHL have corresponding chiral recognition site.Conclusion: Six kinds polyclonal antibodies were prepared successfully and studied differences between the antibody in chiral identification. Between antigen and antibodies cross-reaction of 8 kinds Dufulin analogs for studying mechanism of between antigen and antibody providing some support. Direct competitive ELISA method was established for the development of stable and commercialization of Dufulin kit laid foundation. |
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