Establishment And Application Of Enzyme-Linked Immunosorbent Assays For Three Types Of Organophosphorus Pesticides

O-Methyl-O-(4-nirtopheyl)-N-(2-carboxyethyl)-phosphoramidothionte(H1),O-Methyl-O-(2-methyl-4-nirtopheyl)-N-(2-carboxyethyl)-phosphoramidothionte(H2) and O-Methyl-O-(3,5,6-trichloro-2-pyridinyl)-N-(2-carboxyethyl)-phosphoramidothionte(H3) three haptens for parathion-methyl,fenitrothion and chlorpyrifos-methyl were synthesized with 4-Amino-Butanoic acid and intermediates of three pesticides.O-Methyl-O-(4-nirtopheyl)-O-Butanoicacid(H4),O-Methyl-O-(2-methyl-4-nirtopheyl)-O-Butanoicacid(H5) and O-Methyl-O-(3,5,6-trichloro-2-pyridinyl)-O-(4-nirtopheyl)-O-Butanoicacid(H6) three haptens for parathion-methyl,fenitrothion and chlorpyrifos-methyl were synthesized with 4-Bromobutyric acid ethyl ester and intermediates of three pesticides.H1,H2 and H3 was conjugated to bovine serum album(BSA) with the active ester method to form immunogens(H1-BSA,H2-BSA and H3-BSA),respectively;H1, H2,H3,H4,H5 and H6 was respectively conjugated to ovalbum(OVA) with the active ester method to form coating antigens(H1-OVA,H2-OVA,H3-OVA,H4-OVA,H5-OVA and H6-OVA).BALB/c mice were immunized by intraperitoneal injection of multiple immunogen HA-BSA(mix equally with H1-BSA,H2-BSA and H3-BSA).Antiserum was determined by indirect ELISA,and the mouse which showed the highest titer was used for cell fusion to prepare monoclonal antibody.Spleen cells of immunized mouse were fused with SP2/0 murine myeloma cells by PEG.After several times of screening by ELISA and cloning by limited dilution,Four hybridoma cell lines which secreted monoclonal antibody(McAb) against the multiple immunogen were obtained.Among them,one hybridoma cell line against parathion-methyl(3H9/A3),two against fenitrothion(3H9/B3,6C8/H6) and one against chlorpyrifos-methyl(3G7/F5) was obtained,respectively.Four hybridoma cell lines were all found to secrete antibodies of IgG1,kappa type.The affinity constants of 3H9/A3, 6C8/H6 and 3G7/F5 were 1.23×10⁸L/mol,2.0×10⁷L/mol and3.09×10⁸L/mol respectively. The specificity test showed that the monoclonal antibody 3H9/A3 was highly specific to parathion-methyl;monoclonal antibody 6C8/H6 was highly specific to fenitrothion; monoclonal antibody 3G7/F5 was highly specific to chlorpyrifos-methyl.Specific monoclonal antibodies of three types of organophosphorous were obtained successfully, which laid the foundation for further study on screen the residues of organophosphorus pesticides.Further research was based on the monoclonal antibodies which were secreted by hybridoma cells 3H9/A3,6C8/H6 and 3G7/F5 specific to parathion-methyl,fenitrothion and chlorpyrifos-methyl respectively.ELISA assay conditions of parathion-methyl, fenitrothion and chlorpyrifos-methyl were eventually fixed on by study of several factors in assay buffer,including concentration of ionic,PH,organic solvent and so on.Standard curses were formed by Logit(B/B₀) and the logarithm of insecticides concentration as the two influencing factors.Parathion-methyl standard curve was linear between0.01μg/ml～10μg/ml,the equation was as follows:y=-1.02x-0.9894,the R² reaches0.9744,IC₅₀ was 0.107μg/ml.The recovery rates from soil,tap water and greengrocery were 84.3%,93.9%and 100.5%,respectively.Fenitrothion standard curve was linear between 0.001ng/ml～100ng/ml the equation was as follows:y=-0.4421x-0.5681,the R² reaches 0.9947,IC₅₀ is 0.052ng/ml.The recovery rates from soil,tap water and greengrocery were 84.5%,99.05%and 94.9%,respectively.Chlorpyrifos-methyl standard curve was linear between0.01μg/ml～100μg/ml,the equation was as follows:y=-0.9476x-1.0641,the R² reaches 0.9951,IC₅₀ is 0.075μg/ml.The recovery rates from tap water,soil and greengrocery were 85.1%,104.8%and 90.2%,respectively.In our work,three different monoclonal antibodies were obtained from one hydroma line by multiple immuogen,which provided a new way for obtaining several monoclonal antibodies of different pesticides in a short period.Meanwile,the monoclonal antibody-based rapid ELISA protocols were developed for detection of parathion-methyl, fenitrothion and chlorpyrifos-methyl,which paved the way for studying the pesticide residue kits and also provided a way for environment security determination.