Development Of Specific Antibodies And Establishment Of IcELISA For Pirimiphos-methyl And Forchlorfenuron

Pesticides have been widely used in agricultural production to control insect,weed and regulate plant growth.However,due to excessive use,residues of pesticides have been greatly found in agricultural products and the environment,which has seriously caused the harm to the health of human and other animals.At present,the detection of pesticide residues mostly depends on instrumental methods,However,these methods need expensive instruments,and the sample pretreatment is tedious,in addition professional operators are needed,which are difficult to meet the needs of rapid detection on site.Immunoassay has the characteristics of simplicity,speediness and being suitable for rapid screening on site.It can be used as supplement of instrumental methods,and has become a research hotpot.In this study,the haptens and artificial antigens were designed and synthesized to preparation a polyclonal antibody against pirimiphos-methyl（Pr）,and an indirect competitive enzyme-linked immunosorbent assay（icELISA）for Pr was established.The haptens and artificial antigens of forchlorfenuron（CPPU）were synthesized and the monoclonal antibody was prepared,and an icELISA for CPPU was established.The specific research contents and results are as follows:（1）The pirimiphos-methyl artificial antigen were synthesized,and the specific antibody was prepared.According to the Pr molecular structure,different carbon chains and benzene ring structures were introduced into the phosphoryl chloride site respectively,and the structure was identified by MS.The artificial antigens（seven immunogens and three coating antigens）were obtained through active ester method,and identified by ultraviolet spectrophotometer.Female Balb/c mice were immunized with immunogens,according to the antisera screening,the titer of antiserum Pr1-THY was 1:16000,and the inhibition rate of 1μg/m L Pr was 76.63%.（2）An icELISA for the Pr was established,and the detection conditions were optimized.The optimal reaction conditions of icELISA were as follows:the coating antigen was diluted 4000 times（250 ng/m L）,the antibody dilution multiple is 16000,the reaction buffer is Tris-HCl,primary antibody reaction for 30 min,second antibody reaction for 30 min.The IC₅₀ was 65.53 ng/m L,the limit of detection was 0.89 ng/m L,and the linger range is between 16.05 ng/m L and 267 ng/m L.The cross reaction rates with six structural analogues were less than 10%.（3）The artificial antigens and monoclonal antibody against forchlorfenuron（CPPU）were prepared.The structure of CPPU was modified by Friedel-crafts reaction to get haptens,and identified by MS.Artificial antigens were synthesized through active ester method.Female Balb/c mice were immunized with immunogens.According to the antisera screening,the titer of antiserum CPPU-THY was 1:32000,and the inhibition rate of 1μg/m L CPPU was 83.30%.Through the cell fusion technology,the positive hybridoma cells were screened by microscopical cloning technique and limited dilution method,and a hybridoma cell line CPPU-a producing monoclonal antibody was screened.A large number of antibodies were prepared by mouse ascites.The subtype of the antibody is Ig G1,and the antibody was purified by affinity column chromatography.（4）An icELISA for CPPU was established,and the detection conditions were optimized.The optimal reaction conditions of icELISA were as follows:the coating antigen was diluted 4000 times（250 ng/m L）,the antibody was diluted 8000 times（1μg/m L）,the reaction buffer is PBS,primary antibody reaction for 30 min,second antibody diluted 5000 times.The IC₅₀ was 1.04 ng/m L,the limit of detection was 0.16ng/m L,and the linger range is between 0.31 ng/m L and 3.43 ng/m L.The cross reaction rates with six structural analogues were less than 10%.The recovery rates were from85.23%to 119.14%in cucumber and orange samples.The results of real samples showed high accordance with LC-MS.The results showed that the icELISA is reliable and can be used for detecting CPPU in real samples.