| Food safety has been concerned world-wildly,especially in China,pesticide residue issue became a serious problem to human health,due to the intensive or improper using of pesticides in cropping system.Rice is the staple food crop in China,which require pesticides using to secure yield.For example pesticide Imidacloprid,a neonicotinoid pesticide,is used to control pests such as rice planthoppers,its residue in food could cause potential risk for humen health;another chemical that intensively used in rice is Triazophos,which is moderate toxic to mammals,and impact on central nervous function.triazophos application in vegetables has been forbidden by The Ministry of Agriculture of China.Therefore,it is very important to develop rapid detection method for on-site determination of Imidacloprid and Triazophos.Immunoassay have been wildly employed for the detection of various targets,for the specific binding interaction between antibody and antigen.Antibody based lateral flow assay(also known as colloidal gold immunochromatography)is one of rapid,easy operation kits,which has been used for various targets determination.In this study,two colloidal gold immunochromatographic test strips have been developed based on monoclonal antibody for the determination of imidacloprid and triazophos.For triazophos detection,a MRL based,multiple test lines strip has been developed,compared with traditional test strips,quantification have been much improved and directly linked to MRL based judgment.The main results are as followed.Carboxylated imidacloprid was synthesized and used as hapten,and coupled with carrier protein as immunogen.After BALC/c mouse immunization,the spleen cell was collected and fused with SP2/0 myeloma cell to generate hybridoma cell lines for Monoclonal antibody(Mab)production.Thereafter,lateral flow assay was developed for imidacloprid detection,meanwhile,a MRL based multiple test lines strip was established for Triazophos detection.(1)Hapten synthesis and conjugation.The-Cl group of imidacloprid C9H10CIN5O2 was replaced by ?-mercaptopropionic acid with chemical modification,obtained carboxylated imidacloprid using as hapten(QCP).Thereafter,the hapten was coupled with carrier proteins of BSA or OVA via active ester to obtain the immunization antigen(QCP-BSA)and the coated antigen(QCP-OVA).Artificial antigen were identified by UV spectrophotometry and matrix-assisted time-of-flight mass spectrometry.The results showed that the coupling ratios of the haptens and carrier proteins were 13:1 for QCP-BSA and 19:1 for QCP-OVA,respectively.(2)Monoclonal antibody development for Imidacloprid recognition.after five times immunization,The sera titer of BALA/c mice reached to 1.1×104,for mouse No.2B was 1:2×105.and the IC50 for imidacloprid was 80 ng/mL.Thereafter,mouse No.2B was sacrificed and spleen cell was collected and fused with SP2/0 myeloma cells to establish hybridoma cell lines.Limiting dilution was used to obtain subcell line clone.Hybridoma cell line No.6G11 with stable ability of antibody secretion was selected,and the the IC50 for imidacloprid inhibition reached to 10 ng/mL.(3)Mab based lateral flow assay for imidacloprid detection.Monoclonal antibody Labeled with colloidal gold for the test strips preparation.After optimization,the limit of quantification(LOQ)of lateral flow assay for imidacloprid detection reached to 0.05?g/mL.No cross reactivity was observed with thiamethoxam,thiacloprid and acetamiprid.Method validation was conducted on spiked rice.The results revealed that the recovery was 89-102%and relative standard deviation(RSD)of 6.4-13.4%,which consistent with the results of GC method.(4)A MRL based,multiple test lines lateral flow assay development.Two colloidal gold immunochromatographic strips with three test lines were developed for triazophos detection.Each test line present different MRLs of triazophos,and the limit of quantification of the test strip was 0.005 ?g/mL.(5)Method validation.Rice,cabbage,and apple were spiked with triazophos for the recovery test by using multiple test lines lateral flow assay.Different sample extraction procedures(dilution,solvent replacement)were employed for extraction.The results revealed that the recovery was 86.5-98.0%and relative standard deviation(RSD)of 1.7-3.2%,All results were consistent with the results of GC method.Several sample extraction or dilution treatments could effectively reduce matrix interference.The MRL based multiple lines test trips can precisely quantify and qualify whether triazophos was over MRL and its concentration range,lead to a direct judgment of food safety... |
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