| Parathion-methyl(O,O-dimethyl-O-4-nitrophenyl phosphorothioate,PM),as a highly toxic organophosphate pesticide,has been widely used to control insect pests of agricultural products.The PM residues in food could enter into the human body through the food chain and make toxic effect to the nervous system,endocrine system,immune system and reproductive system of human.Therefor,the development of a rapid,simple and accurate method for PM residues analysis is necessary.Compared with the enzyme inhibition method and instrumental analysis method,enzyme-linked immunosorbent assay(ELISA)has the advantages of simple,cost-effective,specificity and high-throughput.In this study,a monoclonal antibody(MAb)and a single-chain variable fragment antibody(scFv)against PM were prepared and sensitive immunoassay methods for PM analysis were developed.The main results are as follows:(1)Firstly,BALB/C mice were immunized intraperitoneally with the PM immunogen,after cell fusion,hybridoma selection,monocloning,ascites fluid preparation and purification,a PM specific monoclonal antibody PM-4G6 was obtained.(2)The MAb PM-4G6 was labeled with horseradish peroxidase(HRP)by an improved sodium periodate method to obtain PM-4G6-HRP.An indirect competitive ELISA(IC-ELISA)based on PM-4G6 and a direct competitive ELISA(DC-ELISA)based on PM-4G6-HRP for PM were developed,respectively.Under the optimal ELISA conditions,the limit of detection(LOD,IC10)of the IC-ELISA was 0.6ng/m L,the IC50 was 6.6ng/m L and the linear range from 1.4ng/m L to 28.0ng/m L.The LOD of the DC-ELISA was 0.6ng/m L,the IC50 was 3.6ng/mL and the linear range from 1.2ng/m L to 12.4ng/m L.The cross-reactivity of the developed IC-ELISA and DC-ELISA for various organophosphorus pesticides was determined.Results indicated that the ELISAs showed specificity for PM with low cross-reactivity for fenitrothion,parathion,paraoxon-methyl and paraoxon.(3)The developed ELISAs were evaluated by the recovery study.For the IC-ELISA,the recoveries of PM from the spiked rice and wheat flour samples ranged from 85.2% to 128.9% with the CV(%)ranged from 2.6% to 7.1%.For the DC-ELISA,the recoveries of PM from the spiked samples ranged from 101.9% to 116.3% with the CV(%)ranged from 3.8% to 10.6%.The results demonstrated that the developed IC-ELISA and DC-ELISA were reliably for analysis of PM residues in cereal samples.(4)A phage-display scFv antibody library was prepared against a PM immunized BALB/C mouse.After panning and positive scFv antibodies selection,two scFv antibodies with different amino acid sequences were obtained(named PM-25 and PM-30).The gene of PM-30,which showed a higher sensitivity for PM,was inserted into pET-28a(+)vector and expressed in E.coli BL21(DE3).The optimal expression condition was inducing for 8h with 0.1mmol/L IPTG at 25℃.After expression,the scFv antibody was biotinylated with BirA.Then,the biotinylated sc Fv antibody was purified with yield of 59.2±3.7mg/L.(5)A sensitive IC-ELISA was developed based on the biotinylated PM-30 scFv antibody.The LOD of the IC-ELISA was 0.9ng/m L,the IC50 was 14.5ng/m L and the linear range from 5.9ng/m L to 191.4ng/m L.The cross-reactivity of the developed IC-ELISA for various organophosphorus pesticides was determined.Results indicated that the IC-ELISA showed high specificity for PM with low cross-reactivity for parathion,fenitrothion,paraoxon-methyl. |
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