| Uniconazole and paclobutrazol are a class of regulators that inhibit plant growth,which are widely used in agricultural production.Researches show that long-term use and abuse of uniconazole and paclobutrazol will have an impact on human health.Therefore,the development of new technologies for the determination of uniconazole and paclobutrazol residues in food has an important practical significance for ensuring the quality and safety of food.In this paper,a monoclonal antibody against uniconazole was prepared,and on this basis,a new method for immunoassay of uniconazole in food was constructed.In the combination of the monoclonal antibody against paclobutrazole obtained in the laboratory in the early stage,the immune system of paclobutrazol in food was constructed.The established analytical method was used to detect uniconazole and paclobutrazol in food,and satisfactory results were obtained.The main research results are as follows:1.The uniconazole was modified by succinic anhydride method to obtain uniconazole hapten with a carboxyl.Then,the obtained uniconazole was conjugated with bovine serum albumin(BSA)and ovalbumin(OVA)by carbodiimide method to prepare a kind of new and effective immunogen and detection antigen,respectively.The structure of the uniconazole hapten was characterized by mass spectrometry,1H nuclear magnetic resonance spectroscopy and infrared spectroscopy.The structure of the artificial antigen was identified by ultraviolet spectrophotometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOFMS).Balb/c mouse were immunized by the prepared immunogen.The results suggested that artificial uniconazole antigen was successfully synthesized.The coupling ratio of the immunogen and the detection antigen was 21.8:1 and 6.0:1,respectively,and the immunized mice could produce corresponding responses.The serum titer and the IC50 were 80000 and 105.63 ng/mL,respectively,which greatly facilitated monoclonal antibody production and immunological activity research.2.Mice were immunized with immune antigens to obtain high titer mouse spleens.Spleen cells were fused with myeloma cells,and the positive screening was performed by indirect enzyme linked immunosorbent assay(ELISA)and indirect competition ELISA.After subcloning,18 cell lines that could secrete anti-uniconazole monoclonal antibodies were obtained.Considering the titer and specificity,the final selected cell line was 1B11-G11-D10-C11-A7.The cell line subtype is IgG2b and the light chain type is IgK.Intraperitoneal injection with Balb/c mice with the selected cell line was carried out to obtain the ascites.The antibody titer and IC50 were 128000 and 11.26 ng/mL,respectively.And the CR%with the paclobutrazol was 1.53%.3.On the basis of anti-uniconazole monoclonal antibodies obtained before,a monoclonal antibody based ELISA for the detection of uniconazole residues was established,through optimizing the reaction conditions,such as antigen coating solution,coating conditions,standard dilutions,competition mode,etc.The IC50 was 5.0 ng/mL,and the detection limit was IC20:1.47 ng/mL-IC80:97.25 ng/mL.The cross-reactions with uniconazole analogs were less than 2%,which suggested a high specificity.The spiked recoveries for soybean and navel orange were 86.0-114.7%and 86.6-100.5%,respectively.The results showed that the method established in our assay could be used for the rapid detection of uniconazole residue in food.4.The paclobutrazol detection antigen was prepared by the succinic anhydride method and the 4-(bromomethyl)-benzoic acid method based on the acquisition of monoclonal antibody against paclobutrazol.According to the performance analysis,it is found that the sensitivity of the ELISA system based on the 4-(bromomethyl)-benzoic acid synthesis antigen is 6 times higher than that of the ELISA system based on the succinic anhydride method,which provided a new method for improving the sensitivity of the paclobutrazol immunoassay system by modifying the coated antigen.On this basis,this study also established a rapid ELISA method for paclobutrazol analysis based on monoclonal antibody anti-paclobutrazol by optimizing the reaction conditions such as antigen coating solution,reaction time and competition mode.It has a high specificity to paclobutrazol with IC50 value of 54 ng/mL and detection limit of IC20:13.4 ng/mL-IC80:212.7 ng/mL.The cross-reactivity with paclobutrazol analogs were less than 2%.The spiked recoveries of apple and navel orange were 83.2-96.4%and 88.9-94.6%,respectively,which can meet the requirements of rapid analysis of paclobutrazol residues in food samples. |
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