Development Of McAb For Vomitoxin And Establishment Of IcELISA Kit And GICA Test Strip In Feed

Deoxynivalenol?DON?,also known as vomitoxin,is a highly toxic secondary metabolite,mainly produced by Fusarium culmorum and Fusarium graminearum,belonging to the B-group of trichothecenes.DON mainly contaminates wheat,corn and its by-products,so it widely exists in food and animal feed in nature.DON readily acts as an animal antifeedant and shows immunotoxicity,organ toxicity,inhibition of protein synthesis,and teratogenicity.It is also closely related to immunosuppression,keshan disease,esophageal cancer and other diseases.In addition,DON has strong thermal stability,and its toxicity cannot be destroyed by general processing and cooking.Therefore,DON pollution not only poses a great threat to animal health,but also affects human health.Most countries in the world regulate mandatory limits for DON,and the international agency for research on cancer classifies DON as a class III carcinogen.At present,the main physicochemical methods for DON contamination detection in food and feed have high precision and sensitivity,but the cost is high,which are not suitable for the general feed industry.Enzyme-linked immunoassay?ELISA?,as a traditional method,does not require special equipment,which is suitable for the field and high-throughput screening,and its development and application have grown rapidly in recent years.Objective:In this study,we can realize the detection of DON with high efficiency and low cost,and explore the establishment of a sensitive,fast and convenient immunological detection method to avoid the tedious physical and chemical detection and the low sensitivity of other immune detection methods.Through the molecular design and modification of small molecule hapten of DON?the preparation and characterization of artificial immunogen,the preparation of anti-DON monoclonal antibody by cell fusion technology and the identification of immunological characteristics,the developement of DON ELISA kit and colloidal gold strip to realize the preliminary application of the product.Aim to develop an anti-DON monoclonal antibody as the core reagent based on the preparation of DON artificial antigen,and the purpose is to develop a self-assembled kit and colloidal gold strip for the detection of DON content in food and feed,and provide technical support for the detection of DON pollution residues in food and feed in China.Methods:?1?According to the particularity of DON molecular structure,four methods of artificial antigen coupling were designed,and the artificial antigen and detection antigen were prepared respectively.Through the determination of UV,IR,SDS-PAGE and animal immunity,the best synthetic method of artificial antigen was selected.?2?Using the CDI method with the best immune effect to immunize the mice,selected the mice with the highest titer,the best sensitivity and the strongest specificity,prepared the hybridoma cell line by cell fusion technology,screened the hybridoma cell line by the heterogeneity detection technology,prepared the monoclonal antibody by the method of inducing ascites in vivo,and identified its immunological characteristics.?3?Using the self-made anti-DON monoclonal antibody to develop indirect competitive ELISA kit?ic ELISA kit?and direct competitive ELISA kit?dc ELISA kit?,then debugged and assembled the kits respectively,and analyzed the performance of the kits.Finally,the quality and performance of the two kits were compared.?4?On the basis of the self-made high affinity DON mAb,we prepared colloidal gold by sodium citrate reduction method,Mey's series stability method was used to optimize pH value of gold labeled antibody and concentration of DON mAb.And DON colloidal gold strip was assembled and its properties were measured.?5?Using HPLC to verify the developed dcELISA kit and DON-strip,and compared with commercial kits and test strips respectively.Then the results were compared and analyzed with the correlation between dcELISA kit and HPLC.Results:?1?Among the four methods of artificial antigen synthesis,CDI method is the best by the determination of UV,IR,SDS-PAGE and animal immunity.The titer of DON pAb produced by animal immunity is 1:?6.4×10³?,IC₅₀0 value is 47.75 ng/mL,and it has strong specificity.?2?The results showed the mAb had a good antibody titer by the identification of the immunological characteristics of the mAb.The titers of the supernatant and ascites were 1:?1.28×10³?and 1:?3.2×10⁵?respectively,IC₅₀0 was 9.84 ng/mL,and its affinity constant?Ka?is1.51×10⁹ L/mol,and it has strong specificity.?3?By measuring the performances of the developed kits,the results showed that the sensitivity of icELISA kit and dcELISA kit was 1.147 ng/mL and0.62 ng/mL respectively,which was higher than the sensitivity of the commercial kit?3 ng/mL?.The average recovery rate of the icELISA kit was 76.3%¹13.2%and the RSD of 3.9%¹3.2%,while the average recovery rate of the dcELISA kit was 77.1%¹07.0%and the RSD of 4.2%¹1.9%.?4?The results of UV and transmission electron microscopy showed that the preparation of colloidal gold was successful,and the particle size was 25.0±1.0 nm.By Mey's series stability method to determine the optimal pH value of gold standard antibody was 8.5 and the optimal concentration of DON mAb was 15?g/mL.The sensitivity of the strip was 5 ng/mL,and the sensitivity is higher than the commercial strip?25 ng/mL?,and it has strong specificity.?5?By comparing dcELISA kit?DON-strip and HPLC,the results showed the sensitivity of the strip was5 ng/mL,and the sensitivity of HPLC was 20 ng/mL.By determining the real sample,and the results showed that the average value of HPLC was 560.4¹049.1 ng/g?RSD was 12.4⁴3.4%,the average value of kit was 580.5¹020.3 ng/g?RSD was 13⁴3.8%.The correlation between dcELISA kit and HPLC was evaluated by comparative analysis,the regression equation was y=0.9793x+6.82,R²=0.9848.Compared with commercial kits,the sensitivity of commercial A1DON kit and A2 DON kit were 5 ng/mL and 3 ng/mL respectively.Compared with commercial strips,the sensitivity of B1 DON-Strip and B2 DON-Strip were 40 ng/mL and 25 ng/mL respectively.Conclusions:?1?According to the molecular structure of DON,four methods of artificial immunogen were successfully prepared in this experiment.CDI method was the best synthetic method of artificial immunogen.?2?The highest titer mice immunized by CDI was used for cell fusion.Therefore,a more sensitive anti-DON mAb was developed,which has good titer,strong specificity and high affinity.?3?Using the self-made anti-DON mAb,icELISA kit and dcELISA kit were successfully developed.Through comparison,dcELISA kit was obviously superior to dcELISA kit,and both of the kits were accurate,reliable,convenient and high-throughput.?4?Colloidal gold was successfully prepared by sodium citrate reduction method,the pH value of gold labeled antibody and concentration of DON mAb were optimized by mey's series stabilization method.And the strip had high sensitivity,high specificity,good repeatability.?5?The detection methods of dcELISA kit and DON-strip were verified by HPLC.The sensitivity of dcELISA kit is higher than that of two kinds of commercial kits,and the sensitivity of DON-strip is higher than that of two kinds of commercial test strips,and the coincidence rate of DON-strip is100%.Therefore,the dcELISA kit and DON-strip developed in this experiment can meet the requirements of higher sensitivity for actual sample detection,and can be widely used.