Research Of Immunoassay For Thiamethoxam And Dextran In Sugarcane

As the second-generation neonicotinoid insecticide,thiamethoxam,with the advantages of broad spectrum and high efficiency,has been widely used during sugarcane cultivation.It has attracted more and more attention because of its high toxicity to bees.Sugarcane would produce dextran when it is exposed to mechanical damage,frost,pests and diseases,or placed for a long time after harvest,which caused negative impact on the sugar industry.Therefore,the detection of thiamethoxam and dextran in sugarcane has become increasingly important.Immunoassay has the advantages of rapid,sensitive,economical and high-throughput,and has been widely used in the fields of agricultural products,food and environment.However,there are few reports on the immunoassay for thiamethoxam and dextran in sugarcane,expecially for the immunoassay for the simultaneous detection of the analytes.This study aimed at the necessity of detection of thiamethoxam and glucan in sugarcane and the lack of research for the detection technology,carried out the preparation of thiamethoxam and glucan monoclonal antibodies,and the establishment of single analyte and double analyte immunoassay method.Specific research is as follows:The immune antigen and coating antigen of thiamethoxam were prepared by active-ester method,the conjugation ratio was 20:1 and 10:1,respectively.Then BALB/c mice were immunized by immune antigen to produce anti-thiamethoxam antibody.Enzyme-linked immunosorbent assay(ELISA)was used to determine the titer and competitive effect of tail blood supernatant..The mice,which showed good titer and competitive effect were selected to do the cell fusion experiment.After the fusion of mouse spleen cells and myeloma cells,a hybridoma that can steadily secrete the anti-thiamethoxam monoclonal antibody was selected,named as 6C7D12,its subtype was IgG3.The conjugate of dextran and carrier protein was immunized to BALB/c mice.After cell fusion experiment,three hybridomas that can steadily secrete the anti-dextran monoclonal antibody was selected,which were two subtypes,IgG1(3C6F7,3E5D2)and IgG2a(4B9A5).The anti-thiamethoxam antibody 6C7D12 was used to develop the indirect competitive ELISA(ic-ELISA)for the detection of thiamethoxam.After screening the coating antigen,the heterologous antigen was used for the development of ic-ELISA with the highest sensitivity.The optimized working conditions were 0.14 mol/L Na+,pH 7.0 and 5%methanol content.Under optimal conditions,the half inhibitory concentration(IC50),limit of detection(LOD)and linear range of ic-ELISA for the dection of thiamethoxam was 0.36 ng/mL,0.02 ng/mL and 0.02-5.88 ng/mL,respectively.Compared with the previous reported ELISA,this ic-ELISA showed the best sensitivity.The ic-ELISA showed no significant cross-reactivity(CR)with the analogues of thiamethoxam,except for clothianidin with slight CR,which indicated the ic-ELISA had good specificity.Recoveries and relative standard deviations(RSDs)of the ic-ELISA for the sugarcane spiked with 100-1000 ng/g thiamethoxam were 77.3%-104.3%and 3.5%-14.8%respectively,which showed the ic-ELISA was accurate and reliable.The ic-ELISA for the detection of dextran was established using monoclonal antibody 3C6F7.The optimal conditions of this ic-ELISA were:1.25 μg/mL coating antigen for the overnight coating at 4℃ 2%bull serum albumin(BSA)for blocking;the dilution ratio was 8000 for enzyme-labeled secondary antibody;the competitive reaction time was 1 h at 37℃ the incubation time for substrate was 10 min.The IC50,LOD and linear range of the ic-ELISA were 120.1 ng/mL,7.1 ng/mL and 19.53-1250 ng/mL,respectively.The CRs with sucrose,glucose,starch and doxandoglucan were less than 0.1%.Recoveries and RSDs of th e ic-ELISA for the detection of dextran in sugarcane were 88.0%-103.5%and 7.3%-9.1%,respectively.The ic-ELISA has good repeatability and stability,which can be used to detect dextran in sugarcane.The conjugate of horse radish peroxidase(HRP)and monoclonal antibody 4B9A5 was prepared.The antibody combination for sandwich ELISA was selected,The result showed 4B9A5-HRP was used as detection antibody,and 3C6F7 was used as capture antibody.After optimization,2.5 g/mL 3C6F7 was used for overnight coating at 4℃ 2%BSA was used for blocking;the dilution ratio was 8000 for enzyme-labeled antibody;the competitive reaction time was 45 min at 37℃ the incubation time for substrate was 10 min.Under the optimal conditions,the 50%saturation of the signal(SC50)and LOD of the sandwich ELISA for dextran were 60.0 ng/mL and 5.7 ng/mL,respectively.The linear range was from 7.8 to 500 ng/mL.There was no CR with other sugars.Recoveries of the sandwich ELISA for dextran in spiked PBS sample was 97.8%-108.3%,and recoveries and RSDs of the sandwich ELISA for dextran in sugarcane was 89.9%-95.9%and 3.2%-9.4%,respectively.The specificity,accuracy and sensitivity of the sandwich ELISA can meet the requirements of the detection of dextran in sugarcane.Based on the magnetic separation strategy,a multi-color upconversion fluorescence immunoassay for simultaneous detection of thiamethoxam and dextran was established.The artificial antigen of thiamethoxam and dextran were conjugated with magnetic nanoparticles(MNPs)as the separation elements.The antibodies of thiamethoxam and dextran were coupled to NaYF4:Yb,Er UCNPs with emission wavelength of 544 nm and NaYF4:Yb,Tm UCNPs with emission wavelength of 477 nm to prepared the signal elements,respectively.Due to the interaction between antigens and antibodies,signal and isolation elements would form immune-complexes.In the presence of thiamethoxam and dextran,the analytes compete with the separation elements to bind the signaling elements,thereby dissociating the immune-complexes.Under the action of the external magnetic field,the isolated signal elements are reduced,resulting in the decrease of fluorescence intensity.The quantitative detection of thiamethoxam and dextran was realized according to the relationship between the change of fluorescence intensity and the concentration of analyte.By optimizing the dosage of nanomaterials,incubation time,ion strength,pH value and methanol content in the system,the standard curve of this method was established.The IC50 and linear range for the detection of thiamethoxam were 0.46 ng/mL and 0.06-3.53 ng/mL respectively,while 49.33 ng/mL and 4.39-544.43 ng/mL for dextran.The assay showed no CR with the analogues of thiamethoxam and dextran,except for clothianidin with 8.7%CR.The recoveries and RSDs of the assay for sugarcane spiked with 20,100 and 400 ng/g of thiamethoxam were 82.9%-93.3%and 3.1%-6.5%,respectively.The spiked concentration of dextran was 20,40 and 80 ng/mg,recoveries and relative standard deviations(RSDs)were 87.5%-97.2%and 2.1%-4.6%,respectively.Compared with the traditional ELISA,this assay showed wider detection range and simpler operation procedure,which can meet the requirements of simultaneous quantitative detection of thiamethoxam and dextran in sugarcane.