Studies Of Immunoassays For Detection Of Ten Typical Organic Pollutants In Food Samples

Chemical pollution in food is mainly caused by pollution sources in agricultural cultivation,environmental pollution and the abuse and illegal use of additives.Herein,we investigated the rapid immunoassays for ten kinds of typical organic pollutants in food stuff,namely fipronil（FPN）,bifenthrin（BF）,dimethachlon（DMT）,profenofos（PFF）,benzo[a]pyrene（B[a]P）,chlordecone（CLD）,endosulin（EndS）,hexachlorocyclohexane（HCH）,phenacetin（PNCT）and acetaminophen（PAP）,through preparing specific monoclonal antibody（mAb）.Firstly,according to the characteristics of the molecular structure of the ten typical organic pollutants,using the target molecules,their analogues,and their metabolites as reactant,two or more haptens for every target were obtained through chemical reactions were characterized by high performance liquid chromatography-mass spectrometry（HPLC-MS）and proton nuclear magnetic resonance spectroscopy（¹HNMR）.Then,complete antigens were obtained through coupling haptens with carrier protein,such as bovine serum albumin（BSA）,and ovalbumin（OVA）,by means of carbodiimide method and identified and characterized by UV-visible spectrophotometry.（1）Using the target molecule itself as reactant,the haptens of FPN,PNCT and PAP were designed and synthesized.Through hydrolysis of cyanogen group in FPN molecules,a hapten containing carboxyl groups was prepared.Through the substitution reaction of phenol hydroxyl group in PAP molecule and ethyl 4-bromobutyrate,in addition of the hydrolysis of the ester,a hapten with3C spacer arm and carboxyl group was prepared for PNCT and PAP.（2）Using the metabolites of target molecules,the haptens of B[a]P,PNCT and PAP were designed and synthesized.The metabolites of B[a]P,namely tetrahydro-B[a]P-7-ol and tetrahydro-B[a]P-7-on,reacted with succinic anhydride（SA）and oxime reaction with carboxymethoxylamine hemihydrochloride（CMO）,repectively.Two kinds of haptens with 3C spacer arms and carboxyl groups were prepared.（3）Using structural analogues of target molecules,the haptens of BF,DMT,PFF,CLD,EndS,HCH,PNCT,and PAP were designed and synthesized.Through insertion reaction in imidazole of FPN structural analogue,another hapten containing carboxyl groups was prepared.The structural analogues of BF,namely2-methyl-biphenyl 3-formic acid and 2-methyl-biphenyl 4-acetic acid,both contain carboxyl group,and the position of carboxyl group and the distance from benzene ring are different,which are used as two kinds of haptens of BF.As another structural analogue of BF,2-methyl-biphenyl-3-methanol reacted with SA and caronic anhydride,leading to two kinds of BF haptens,respectively.1-（3,5-dichlorophenyl）-1H-pyrrolidone-3-formic acid is similar to DMT in structure and contains carboxyl group using as a hapten for DMT.Another hapten ofDMTwaspreparedbytheadditionreactionof1-（3,5-dichlorophenyl）-1H-pyrrolidone-2,5-dione with ethyl mercaptopropionate and hydrolysis reaction of ester.PFF structure analogues with 5C space arm and carboxyl group were prepared by multistep organic synthesis reaction as a hapten of PFF,and another structure analogues 4-bromo-2-chlorophenol reacted with ethyl 4-bromobutyrate,in addition of the hydrolysis of the ester,leading to another hapten of PFF.A hapten for CLD,EndS and HCH with 5C spacer arms and containing carboxyl groups was obtained by hydrolyzing the structural analogue of CLD,namely Kelevan.And another hapten for CLD,EndS and HCH with 5C spacer arms and containing carboxyl groups was obtained by amidation reaction of the structural analogue,namely chloric acid.The molecule structure of 3-acetyl paracetamol is similar to PAP.Another hapten of PNCT and PAP with 3C spacer arm and carboxyl group is prepared by oxime reaction of 3-acetyl paracetamol with CMO.Secondly,the female BALB/c mice were immunized with prepared immunogens.Then,the monoclonal hybridoma cell lines secreting specific antibodies were screened.there are seven kinds of mAbs angainst the ten target compounds were prepared:anti-FPN mAb 1B6,anti-BF mAb 2D9,anti-DMT mAb 1F4,anti-PFF mAb 4C9,anti-B[a]P mAb 4E8,anti-CLD,EndS and HCH mAb 4A2,anti-PNCT and PAP mAb 2D6.The subtypes of all of the above mAbs were IgG2b,as well as anti-PFF mAb was IgG2a.The affinity constant Ka values of all of the above mAbs were among 5.3×10⁸～9.6×10⁹L mol^-1.Thirdly,the indirect competitive enzyme linked immunosorbent assays（icELISA）for the detection of the target compounds were established.IC₅₀ value for FPN was 0.5 ng mL^-1,CRs with fipronil desulfinyl,fipronil sulfone,and fipronil sulfide were 26.5%,49.3%,and 20.6%,respectively.The IC₅₀ value for BF was 58.7 ng mL^-1,the CRs with other PYRs were negligible.The IC₅₀ value for DMT was 2.8 ng mL^-1,the CRs with other analogues were negligible.The IC₅₀ value for PFF was 27.6 ng mL^-1,and the CRs with other organophosphorus pesticides were negligible.The IC₅₀ value for B[a]P was 0.8 ng mL^-1,the CRs with other structural analogues,such as tetrahydro-benzo[a]pyrene-7-ol（B[a]P-7-ol）,pyrene,1-pyrenebutyric acid（PBA）,chrysene,anthracene,benz[a]anthracene,and phenanthrene,were 201%,24.7%,390%,35%,1.6%,12%,and 1.5%,respectively,and the CRs with naphthalene was negligible.The IC₅₀ value for CLD was 1.1 ng mL^-1,and the CRs with other analogues,such as EndS and HCH,were 10.6%and 3.5%,respectively.IC₅₀ values for PNCT and PAP was 3.5 and 34.8 ng mL^-1,respectively.Finally,the sensitive and rapid gold nanoparticle-based immunochromatographic strips（GNP-ICS）for the detection of the target compounds in food stuff were established.The vLOD values of FPN in spiked cucumber and egg samples were 0.3 and 0.5 ng mL^-1,respectively;both of the cut-off values were 10 ng mL^-1.The cut-off values of fipronil desulfinyl,fipronil sulfone,fipronil sulfide in spiked cucumber and egg samples were 50,25and 50 ng mL^-1,respectively.The vLOD and cut-off values for BF in spiked cucumber and cabbage samples were 50 ng mL^-1and 1.0×10³ ng mL^-1,respectively.The vLOD and cut-off values for DMT in both of spiked tomato and lettuce samples were 5.0 and 40.0 ng mL^-1,respectively.The vLOD values of PFF in spiked apple,orange,and kiwi samples all were 1.0×10³ ng mL^-1.The vLOD and cut-off values for B[a]P in spiked teaseed oil sample were 10.0and 100 ng mL^-1,respectively.The vLOD values of CLD,EndS and HCH in luttuce samples by GNP-ICS were 1.0,2.5,and 10.0 ng mL^-1,respectively,and cut-off values were 10,100,and 250 ng mL^-1.The calculated IC₅₀ values for PNCT and PAP by GNP-ICS was 3.6 and46.2 ng mL^-1,respectively;the vLOD and cut-off values for PNCT in spiked beverage samples were 2.5 and 50.0 ng mL^-1,respectively;the vLOD and cut-off values for PAP in drug samples by GNP-ICS were 25.0 and 500 ng mL^-1.Gererally,based on mAbs against the ten organic pollutants,such as FPN,BF,DMT,PFF,B[a]P,CLD,EndS,HCH,PNCT and PAP,the icELISA were constructed with high sensitivity and specificity.At the same time,the GNP-ICS constructed based on the above mAbs also showed high sensitivity and specificity in the detection of the target,and could achieve rapid,high-throughput and on-site detection of real samples.