Immunoassay Of Fifteen Chemical Contaminants In Food Samples

In recent years,food safety problems have occurred frequently,posing a significant safety risk to public health.The development of fast,portable and low-cost testing methods will provide effective tools for national regulatory authorities to screen food safety.Therefore,in this study,highly sensitive and high-affinity monoclonal antibodies were prepared against five biotoxins（deoxynivalenol,tenuazonic acid,alternariol,amanitins,ergometrine）and ten compounds that are susceptible to abuse in food samples（higenamine,chlorpheniramine,carazolol,carbamazepine,repaglinide,tolbutamide,simvastatin,bezafibrate,saccharin sodium,and disodium nucleotide）and corresponding rapid detection immunoassays were developed based on these antibodies.1.Artificial antigen preparation（1）Haptens design:The haptens of deoxynivalenol,tenuazonic acid,higenamine,simvastatin and chlorpheniramine were designed and synthesized from the original drug,the parent nucleus structure,through nucleophilic substitution or oxime reaction and the introduction of carboxylate active groups with linking arms;alternariol,amanitins,ergometrine,bezafibrate,sodium saccharin,and disodium nucleotide as haptens using prodrugs or analogues with carboxyl,amino or hydroxyl active groups on their surface;starting from intermediates of drug synthesis,the haptens of carazolol,carbamazepine and tolbutamide were designed and synthesized by esterification with ethyl2-amino-2-methylpropionate,methyl 4-aminobutyrate,and n-butyl isocyanate,followed by alkali hydrolysis,and their structures were characterized by NMR and LC-MS.（2）Preparation of artificial complete antigens:The carboxyl,amino and hydroxyl reactive groups of the above-mentioned haptens were coupled to the free amino groups of the carrier proteins keyhole limpet hemocyanin（KLH）and bovine serum albumin（BSA）by carbodiimide,glutaraldehyde,formaldehyde and sodium periodate methods,respectively,to prepare artificial antigens.The characterization of complete antigens can be done with the support of UV-Vis spectrophotometry and denaturing electrophoresis.The successful synthesis of complete antigens is demonstrated by the characteristic absorption peaks of small molecule hapten-protein complexes in the scanning spectrum or by the red or blue shift of spectral peaks relative to the carrier protein,or by lagging behind the carrier protein in the electrophoretic bands due to slow speed.2.Preparation of monoclonal antibodiesComplete antigen’s prepared by selecting suitable carrier proteins were immunized multiple times by BALB/c mice,and mice with high potency and high inhibition rate of the target drug were selected for cell fusion.Screening of hybridoma cells using substrate domestication and multiple limited dilution cloning to obtain hybridoma cell lines secreting high performance antibodies:3F5（deoxynivalenol）,1E2（tenuazonic acid）,2B9（alternariol）,3B9（alpha-amanitin）,2E2（ergometrine）,3C10（higenamine）,4C5（chlorpheniramine）,3B6（carazolol）,2D4（carbamazepine）,4G7（tolbutamide）,3F8（simvastatin）,2B10（repaglinide）,4C5（benzafibrate）,1B11（sodium saccharin）and 3C3（disodium nucleotide）,the half inhibition rate(IC₅₀)of the monoclonal antibody is 0.2～21.2 ng/mL and the antibody affinity constant K_aof the monoclonal antibody is 10⁸～10¹⁰L/mol,all of which were high affinity antibodies.Cross-reactivity experiments showed that antibody 3B9 recognized both alpha-and beta-amanitin with a CR of 50.3%;anti-AOH m Ab has a CR with 13.8%to alternariol methyl ether;anti-EGM m Ab has a CR with 41.3%and 17.8%to LSA and LSD;anti-CRZ m Ab has a CR with 9.8%to carvedilol;anti-CBZ m Ab has a CR with 27.2%to carbamazepine-10,11-epoxide.3.Development of rapid immunochromatographic assayA rapid immunochromatographic method for the detection of biotoxins in food samples was developed based on monoclonal antibodies against biotoxins.The calculated limits of detection（c LOD）for DON,TEA,AOH,α-AMA,β-AMA and EGM were 0.16,0.03,0.08,0.02,0.05 and 0.11μg/kg,respectively.The cut-off values for DON,TEA,AOH and EGM in wheat and maize ranged from 10 to 100μg/kg;the cut-off values forα-AMA andβ-AMA in mushrooms were 50 and 100μg/kg,respectively.The prepared m Abs against susceptible compounds were further used in the development of a rapid immunochromatographic assay for the detection of 10 susceptible compounds in food samples.For the detection of HIG,CPM,CRZ,CBZ,TOL,SVT,RLN and BZB in functional drinks,the v LOD of the test strips ranged from 0.1 to 25μg/kg and the cut-off values from 2.5 to 200μg/kg.For tablet-pressed confectionery,the v LOD is 0.5 to 50μg/kg and the cut-off values is 10 to 800μg/kg.For the detection of SCS and DSN in dried mangoes,the test strips v LOD were 50 and 250μg/kg and the cut-off values were 500 and 1000μg/kg,respectively.In addition,the developed multi-ICA method was applied to the simultaneous qualitative determination of HIG,CPM and RLN in functional drinks with v LOD values of:10,5 and 10 ng/mL,and cut-off values of:200,100 and 200 ng/mL,respectively.In the quantitative analysis,the LOD values of HIG,CPM and RLN were 2.67,0.65 and 1.31 ng/mL respectively,with linear ranges of 5.88～86.62ng/mL,1.53～28.99 ng/mL and 3.16～64.90 ng/mL respectively.The recoveries of the actual samples were in the range of 89.2%～105.8%and the CVs were in the range of 2.8%～8.2%,which can meet the requirements of the real sample.In summary,the immunochromatographic detection technique based on the developed monoclonal antibody can be successfully applied to the detection of chemical contaminants in food,which improves the ability to screen chemical contaminants in the field and provides strong technical support for the rapid detection of chemical contaminants in food in the future.