Development and application of polyclonal and monoclonal-antibody based enzyme-linked immunosorbent assays for the analysis of neonicotinoid insecticides imidacloprid and thiamethoxam
Development and application of polyclonal and monoclonal-antibody based enzyme-linked immunosorbent assays for the analysis of neonicotinoid insecticides imidacloprid and thiamethoxam
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2003-12
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Kim, Hee Joo
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Ling, Qing X
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Molecular Biosciences & Bioengineering
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University of Hawaii at Manoa
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Abstract
The sensitivity of previously developed enzyme-linked immunosorbent assay (ELISA) for imidacloprid was considerably improved with a purified polyclonal antibody and a direct competitive assay format (dcELISA). Under the optimized conditions, the half-maximal inhibition concentration (I50) and the limit of detection (LOD) were approximately 1 ug/L and 0.06 ug/L, respectively. Computational analysis suggests that the antibody specificity primarily relate to the dihedral angles, steric hindrance and electrostatic charges on the imidazolidinyl ring. The ELISA analysis of water samples fortified with imidacloprid showed a satisfactory correlation with the fortified levels. Two ELISAs were developed for thiamethoxam and imidacloprid with an antiserum and monoclonal antibody (MAb), respectively. Three antisera against thiamethoxam were raised from rabbits immunized with the hapten-KLH conjugate. Three imidacloprid specific MAbs were obtained from mice immunized with the hapten II-ovalbumin conjugate. Antisera and MAbs were characterized with indirect competitive ELISA (icELISA). Antiserum I with the lowest I50 value was selected for an ELISA for thiamethoxam and MAb E6F3 for imidacloprid. The antiserum I was specific for thiamethoxam and tolerant of up to 5% acetonitrile and 5% acetone. MAb E6F3 can tolerate up to 15% (v/v) acetone or 20% (v/v) methanol. Assay sensitivities of both ELISAs were not influenced at tested range of pHs and ionic strengths. Under the optimized conditions, Iso and limit of detection (LOD) were approximately 9.0 and 0.1 ug/L of thiamethoxam. Iso and the limit of detection were approximately 0.8 and 0.1 ug/L of imidacloprid in icELISA, and 0.3 and 0.03 ug/L in direct competition ELISA (dcELISA), respectively. Antiserum I and MAb E6F3 were specific to thiamethoxam and imidacloprid, respectively. The ELISA analysis of water and cucumber samples showed good correlation between the level determined and levels fortified. Affinity (1/Kd) of E6F3 determined with kinetic exclusion immunoassay (KinExA) for acetamiprid and clothianidin were similar, but 50-fold weaker than that of imidacloprid. MAb E6F3 had no measurable affinity for other neonicotinoids and analogs. Thiamethoxam specific MAbs were produced with the same hapten used for antisera for thiamethoxam. MAbs showed low Iso values and little cross reactivities to other neonicotinoids.
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xiii, 133 leaves
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering; no. 4396
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