| Methamidophos,an effective and broad-spectrum organophosphorus insecticide,was widely used to control pests on rice,cotton,vegetables and other crops.The use of methamidophos provides unquestionable benefits in increasing agricultural production. However,as a high-toxicity pesticide,which can be absorbed by plants,disfused with flowing streams,it has the drawback of pesticide residues which remain on fruits and vegetables,constituting a potential risk to consumers,causing serious circumstance problems to the countryside.Therefore,we should continuously inforce the residue detection of methamidophos,especially the research of fast immunoassays.In this work,the single chain variable fragment(scFv) antibody specifically against methamidophos was produced by positive clone 28D4 isolated from phage-display scFv library.The competitive immunoassay of methamidophos was developed and applied to methamidophos detection of the field samples.Thus this study introduced a new way to immunoassay of pesticides residue based on scFv.1.Optimization and Characterization of ScFv Antibody Expressed by positive Clone 28D4The positive clone 28D4 isolated from phage-display scFv library was selected for further optimization for high-level protein production.The optimum process parameters for the expression of scFv are(a) a temperature of 30℃,(b) an IPTG concentration of 0.25 mM,(c) an induction time of 20 h.The scFv produced with 28D4 under the optimal conditions was purified and the yield was 0.95 mg/L,which is higher than that from unoptimized conditions(0.75 mg/L).The affinity constant of the scFv antibody against antigen was confirmed to be(3.14±0.99)×10~7 L/mol.The recombinant scFv antibody was specific for methamidophos,which exhibited 3.0%cross-reactivity with acephate,whereas reactions with other structurally similar organophosphates were below 0.1%. 2.The Development of an ELISA for Quantitation of MethamidophosAn indirect competitive ELISA was developed for quantitation of methamidophos based on HM3-OVA and its scFv antibody.It was eventually fixed for the ELISA assay conditions:coating of HM3-OVA is at 0.5μg/ml,the antibody working concentration is 320 times,the block of skimmed milk powder is 3%in the reaction system PBST,the icon concentration is 0.05mol/L,and the optional pH is 6.5.An assay curve was formed by the linking rate logit(B/Bo )and X,the logarithm of the methamidophos concentration(μg/mL ) as the two influencing factors.The ELISA showed a linear detection range from 1 to 500μg/mL and its equation of these two factors is as follows:Y=-1.0116X +2.0421,the R~2 reaches 0.9669,and the sensitivity reaches 4.45μg/mL,the mean inter-group CV and ex-group CV reaches 2.1%and 7.0%respectively.The average recovery of methamidophos added to tap water,rice grain and Chinese cabbage was 108.7%,89.8%and 93.2%by the ELISA.High correlations were observed between the standard curve and the sample recovery curves.3.The Applieation of an ELISA for Quantitation of Methamidophos residues in field samples8 out of 13 field rice grain samples,which collected from Jiangsu province in 2006, were observed methamidophos residues varied from 0.17 to 0.83mg/kg,and the average is 0.41±0.23mg/kg,were detected by ELISA.Only 1 field Chinese cabbage sample,which consisted of 5.9%of 17 samples collected from markets and the field of east suburb of Nanjing in December 2006,with methamidophos residue 0.395±0.013mg/kg,was also detected by ELISA.Meanwhile,these samples were also determined by GC.Compared with these two groups of results,a linear correlation(r=0.9986,n=9) was observed,and the values of these positive samples analysed by ELISA is equal to 1.0443 times of those values by GC and subtracting 0.007.In the test of dissipation behavior of methamidophos residue,methamidophos residue degraded as a expondist model with a simulate equation Y=0.56462e-0.113T(r=-0.9931,degration constant(k) was 0.113,half life was 6.13d) in outdoor Chinese cabbage in winter. |
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