The Studies On The Detection Of 11 Typical Pesticides In Fruits And Vegetables By Immunoassay

The frequent occurrence of excessive pesticide residues in fruits and vegetables has seriously threatened food safety and health of residents.With the continuous expansion of the scope of food safety supervision,traditional instrumental analysis technology is difficult to meet the needs of high-throughput on-site detection.It is urgent to develop a rapid,efficient and inexpensive detection method to provide an effective tool for grassroots food safety monitoring.Therefore,11 pesticides（methomyl,propamocarb,phorate,diethofencarb,difenoconazole,prochloraz,oxadixyl,etoxazole,pyridaben,forchlorfenuron and pendimethalin）detected at high frequencies or exceeding the standard in fruits and vegetables were taken as the research objects to design and synthesize haptens,prepare monoclonal antibodies,develop colloidal gold immunochromatographic detection methods.And then they were applied to the on-site rapid detection of the above pesticide residues in celery,cucumber,citrus and kiwifruit.1.Hapten was synthesized by introducing a benzene ring and m Abs was preparedAccording to the structural characteristics of methymol,propamocarb and phorate,the haptens were synthesized by introducing rigid structure benzene rings as connecting arms,which effectively exposed the specific chemical structure of each target pesticide.On this basis,complete antigens were prepared by conjugation to bovine serum albumin（BSA）,and hybridoma cells（methomyl 1D10,propamocarb 3G9,phroate 4C12）were obtained by mice immunization,serum screening,cell fusion and hybridoma screening.Further the corresponding monoclonal antibodies were obtained,with half inhibition rate(IC₅₀)was 49.83,3.24,8.61 ng/m L,respectively.The isotype of antibodies against methymol,propamocarb and phorate was Ig G2b,Ig G2b,Ig G2a.And the antibodies affinity constant K_D were 2.76×10¹⁰,3.64×10¹⁰,5.43×10¹⁰ L/mol,respectively.The above antibodies had cross reaction with their structural analogues less than 1%,showing that they were highly specific.2.Haptens were synthesized from the intermediate and m Abs were preparedBy analysis of the synthetic routes of difenoconazole,prochloraz,oxadixyl and etoxazole,screening for key intermediates in their synthetic routes,a carbon chain with a length of 3～6carbon atoms was introduced as a spacer arm to fully expose antigenic determinant,and complete antigens were prepared by coupling the introduced carboxyl group with the carrier protein BSA.Then highly specific monoclonal antibodies were obtained on this basis as difenoconazole 2F3,prochloraz 2F2,oxadixyl 4B8,etoxazole 2C6.The IC₅₀ was 0.64,0.88,3.68,4.04 ng/m L,respectively.The isotype of antibodies against them was Ig G2b,Ig G2a,Ig G2a,Ig G2a,respectively.And the antibodies affinity constant K_D was 6.30×10¹⁰,4.90×10¹⁰,3.56×10¹¹,3.00×10¹⁰ L/mol,respectively,suggesting that they were high affinity antibodies.And the cross-reactivity results showed that the above antibodies were highly specific.3.Haptens were derived from the original drug and its analogues and m Abs were preparedAccording to the structural characteristics of pyridaben,forchlorfenuron,pendimethalin and diethofencarb,haptens were derived from the parent structure or analogues,preserving the integrity of the original structure to the greatest extent and introducing carboxyl groups as active groups.On this basis monoclonal antibodies were prepared:diethofencarb 2H7,pyridaben 3C2,forchlorfenuron 4H5 and pendimethalin 1E3.The IC₅₀ was 2.36,0.13,0.46 and 2.93 ng/m L,respectively.The isotype of antibodies against them was Ig G1、Ig G2b、Ig G1 and Ig G2a respectively.And the antibodies affinity constant K_D was 2.96×10¹⁰、1.40×10¹¹、3.83×10¹⁰、1.85×10¹⁰ L/mol,respectively,suggesting that they were high affinity antibodies.The cross-reactivity results showed that the above antibodies had cross reaction with structural analogues less than 1%,showing that they were highly specific.4.Colloidal gold immunoassay method was establishedThe developed monoclonal antibodies were used as the core raw material.Colloidal gold immunochromatographic strips were constructed by optimizing the working concentration of antibody and antigen and the composition of the sample diluent,and used for rapid detection of the above pesticide residues in celery,cucumber,citrus and kiwifruit.Vegetable samples were extracted with methanol/water（v/v=1/4）solution and diluted twice with sample dilution（0.01M PBS,p H 7.4,containing 1%ON-870）and detected,fruit samples were extracted with methanol/water（v/v=1/4）solution and adjusted p H to neutral and then diluted three times after detection.All the test results can be interpreted by naked eye within 10 minutes,and the results showed that the visual limit of detection（v LOD）of difenoconazole,prochloraz,phorate,pendimethalin,pyridaben and methomyl were below 100 ng/g in celery;propamocarb,difenoconazole,prochloraz,diethofencarb,pyridaben and oxadixyl were 25 ng/g in cucumber;difenoconazole,prochloraz,etoxazole,pyridaben and forchlorfenuron were 20 ng/g in citrus and kiwifruit.Moreover,five detection lines were set on the immunochromatographic strips to realize simultaneous detection of five fungicides prochloraz,diethofencarb,difenoconazole,propamocarb and oxadixyl in cucumber samples under optimization of the concentration of colloidal gold-labeled antibodies and coating antigens.The v LOD values were 2,20,5,20,and2 ng/g,respectively.Quantitative analysis was realized with the colloidal gold reader,and the limit of detection（LOD）of the above five pesticides were 0.24,2.56,0.52,1.31 and 0.36 ng/g,respectively,which were all lower than the national maximum residue limit.In summary,11 highly specific antibodies were developed in this paper,and immunochromatographic strips which can meet the requirements of 11 pesticides in fruits and vegetables were established,the detection results of which were completely consistent with the detection of Instrumental analysis method,and it was suitable for on-site rapid detection and meet the surveillance requirements of the national maximum residue limit.It provides a reliable and effective detection tool for grassroots high-throughput rapid detection in the field of food safety in China.