Preparation And Preliminary Application Of A Broad-Specificity Monoclonal Antibody Against Ether Pyrethroid Pesticides

Pyrethroids are widely employed as one kind of important pesticide, but they are high toxic foraquatic organism, such as fish and shrimps. Besides, drug resistance has become an increasing concern.During these years, ether pyrethroid pesticides, as efficient and broad as ester pyrethroids, are the focusof researchers because of low toxicity and low resistance.Effective detective methods are necessary for the trade of products and the health of customers.Although they, for example GC-MS, are steady and accurate, instrumental analysis need complicatedpretreatment and cost a lot. Immunoassay has advantages because samples can be detected quickly andexactly with low cost, which is widely used in the detection of pesticide and veterinary drugs in a largenumber of samples. This paper conducted a study of enzyme-linked immunosorbent assay based onbroad-specificity monoclonal antibody for ether pyrethroid pesticides. The details and results are asfollows:1.2-（4-ethoxyphenyl）-2-methylpropanol reacted with sodium chloroacetate by nuclephilicsubstitution reaction. HaptenⅠwas identified by1H-NMR and13C-NMR, the product of the abovereaction, was conjugated to bovine serum albumin （BSA） and ovalbumin （OVA） by the active estermethod to get artificial immune antigen and coating antigen, respectively. The binding ratio of artificialantigens was detected by UV spectroscopy as14:1and35:1.2. Balb/c mice were immunized with immunogen HaptenⅠ-BSA to prepare antiserum whose titerreached2.56×10⁵. By using chessboard method the best concentration of antigen and antibody was1μg/mL and1.5×10⁴. Taking hapten as research object, the linear regression equation wasy=0.1705x+0.59826, R2=0.9922, IC50and IC₁₀ were265.3ng/mL and1.2ng/mL, respectively. Underthe action of PEG4000, the immuned mouse spleen cells were fused with SP2/0myeloma cells. Apositive hybridoma cell line named3H6was obtained by screening and limiting dilution.3. The mice ascites were purified by CA-AS method to obtain monoclonal antibodies with theconcentration of6.2mg/mL and the recovery is60.4%. The affinity constant of soluble antibody byBeatty method is1.6×10⁷L/mol. Meanwhile, the immunoglobulin was identified as IgG1, whose titerreached5.12×10⁵. The best concentration for antigen and antibody were1μg/mL and2×10⁴. Takinghapten as research object, the linear regression equation was y=0.16682x+0.60615, R2=0.9872, IC₅₀ andIC₁₀ were231.0ng/mL and0.9μg/mL, respectively. The antibody was able to cross-react toethofenprox, flufenprox, silafluofen, chlorfenprox, halfenprox, SSI-116, MTI-800, in the range between49.13%⁹2.02%. In addition, it hardly responded to the ester pyrethroids with the cross reactivity lessthan0.1%. The spiked samples were detected by ELISA based on the monoclonal antibody with therecovery of90.7%.This research provided the high quality monoclonal antibody and primary ELISA, which laidfoundation for development of commercial ELISA kits and immunosensors for ether pyrethroids.