Study On Immunoassays To Detect Pirimiphos-methyl And Cadmium In Grains

Pesticide and heavy metals are two important pollutants that affect food security.Pirimiphos-methyl is commonly used in grain storage,which can influce the acetylcholinesterase activity and severely interfere the normal metabolism.Cadmium?Cd?is considered as an increasingly important environmental pollutant in foodstuff,especially in rice.It easily accumulates in human body by the food chain.Cd exposure is reported to decrease bone density and damage the kidneys and even the whole body.At present,the mainly analytical approaches applied to the Cd and pesticide determination in foodstuff are chromatography and chromatography tandem mass spectrometry,atomic absorption spectrum etc.Although instrumental methods are accurate and reliable,they are based on expensive instruments and professional operators,with the deficiencies of high maintenance cost,long time,operational complexity and low efficient in field testing.In contrast,immunoassay,based on antigen-antibody specific reaction,has the advantages of simple,rapid,specific,and low cost.In addition,it is a powerful tool for dealing with the on-site screening for a wide range of food commodities.In this study,several haptens and artificial antigens were synthesized against pirimiphos-methyl,and used to produce corresponding antibody.And based on specific polyclonal antibody,a rapid enzyme-linked immunosorbant assay?ELISA?was developed.In the meantime,an coating antigen was synthesized and a highly selective chemiluminescece enzyme immunoassay?CLEIA?was developed for the determination of Cd.The following are the main contents and results:?1?Preparation of haptens,artificial antigens and specific antibodies against pirimiphos-methylBased on the hydrolysate of pirimiphos-methyl [2-diethylamino-6-hydroxy-4-methylpyrimidine,Pr-OH],nine chemical compounds were selected or systhesized as haptens and conjugated to the proteins through active ester method,glutaraldehyde method,diazotization reaction and N,N-carbonyldiimidazole catalyst to obtain ten immunogens?Pr-1-BSA Pr-10-BSA and Pr-1-OVA Pr-10-OVA?.Balb/c mice were immunized with immunogens.After antisera screening,an antiserum produced by Pr-9-BSA exhibited a high affinity.The titer of the antiserum was 1:32000,and the inhibition rate to 1 ?g/m L of Pr-OH was 90%.A highly specific polyclonal antibody against pirimiphos-methyl was prepared successfully,which could be used for indirect detection.The effect of hapten structures against antibody properties was studied by molecular modeling.According to the results,it is estimated that the electronegativity of the pyrimidine ring and the conjugated patterns had intimate connection with antibody specificity.?2?Development of an indirect competitive ELISA for pirimiphos-methyl in cereal samples.An indirect competitive enzyme-linked immunosorbant assay?ic-ELISA?for the rapid determination of pirimiphos-methyl was developed,after the parameters that affecting the CLEIA such as coating antigen concentration,antibody concentration,reaction buffer system,reaction time,secondary antibody buffer,secondary antibody concentration and secondary antibody reaction time were optimized.The limit of detection(IC₁₀)was 3.2 ng/m L with a linear range(IC₂₀^IC₈₀)between 6.09 ng/m L and 90.07 ng/m L.The half maximal inhibitory concentration(IC₅₀)of the established method was 23.42 ng/m L.And the cross reaction rates of six analogues were lower than 0.1%.The coefficient of variation?CV?in inter-assay and intra-assay were less than 15%.The recovery rates of the spiked samples were between 71.8% and 105.7% after the pre-treament of hydrolysis reaction and elimination of the matrix interference.The results of real samples detecting showed high accordance with HPLC.This ic-ELISA provided a valid method of detecting pirimiphos-methyl in real samples.?3?Development of an indirect competitive CLEIA to determine cadmium in cereal samples.The artificial antigen?Cd-ITCBE-OVA?was prepared via the conection of Cd,1-?4-isothiocyanatobenzyl?ethylenediamine-N,N,N',N'-tetraacetic acid?ITCBE?and protein.Based on a monoclonal antibody that can recognize Cd-ethylenediaminetetraacetic acid?Cd-EDTA?,a chemiluminescent enzyme immunoassay?CLEIA?was developed for detecting Cd.The parameters that affecting the CLEIA such as coating antigen concentration,antibody concentration,reaction buffer system,reaction time,secondary antibody buffer,secondary antibody concentration and secondary antibody reaction time were optimized.The detection limit(IC₁₀)of the established Cd-CLEIA was 0.06 ng/m L,IC₅₀ was 0.50 ng/m L,and the linear range(IC₂₀^IC₈₀)was 0.16¹.56 ng/m L.The cross-reactivity with other 12 metals and EDTA was below 0.3%,and the CV of inter-assay and intra-assay were below 15%.The recovery rates ranged from 79.2% to 118.4% for five types of spiked samples,including rice,wheat,maize,glutinous rice and brown rice after elimination of the matrix interference.The CV was 6.0% 15.8%.This CLEIA provided valid method of Cd in real samples.