Preparation And Comparison Of High-specific And Broad-selective Monoclonal Antibodies Against Organophosphorous Pesticides

Antibody is the key component to develop immunoassay, which enables highly specific interactions with the antigen and/or the pesticide. The monoclonal antibodies (Mabs) screened by homologous indirect enzyme-linked immunosorbent assay (ELISA) is very suitable for application in homologous ELISA. But they have some disadvantages when used in heterologous ELISAs, such as most of the heterologous competitors, which were recognized by the Pab, could be not recognized by the Mab. The weak or no recognition to these competitors resulted in a limited application of the competitors, and Mab-based heterologous ELISA could be difficult to establish.In this study, a novel procedure for screening interesting Mabs by heterologous indirect ELISA was developed. Heterologous ELISAs were established in the process of screening Mabs against parathion. The results showed that these Mabs produced by heterologous screening method had broader-reactivity with twenty competitors than that of 5H7 (Mab produced by homologous screening method). That means it is easier to develop the heterologous immunoassays based on these new Mabs. In addition, compared with the previous ELISAs based on 5H7, higher sensitivity was obtained by these new Mabs. Furthermore, cross-reactivity results showed that two broad-selective Mabs (5F7 and 5D11) were produced during heterologous screening. Based on the Mab 5F7, a broad-selective heterologous ELISA was developed, and the IC50 values for parathion,methy parathion,fenitrothion,fenthion and phoxim were 7.06 ng/mL,32.34 ng/mL,164.84 ng/mL,500.94 ng/mL and 96.97 ng/mL, respectively. To validate the heterologous screening method for broad-selective Mab, a generic coating hapten (p-nitrobenzoic acid) was used to screen hybridomas and a broad-selective Mab 2H6 was selected. With the most suitable competitor, a sensitive and broad-selective ELISA was developed. The IC50 values were estimated to be 20.32 ng/mL for parathion,21.44 ng/mL for methyl-parathion,42.15 ng/mL for fenitrothion, and 58.85 ng/mL for isocarbophos. Spike recovery results indicated that the established ELISA could be applied to detection for the total quantity of an unknown mixture of related organophosphorus pesticides.On the basis of variable Mabs above, the amino acid sequences of the variable region for the Mab 5H7,5F7 and 5D11 were obtained. Then three-dimensional models for the three Mabs were constructed by using homology modeling method and further refined the coordinate by 1 ns molecular dynamics. In addition, several pesticides having similar chemical structures with parathion were docked to the constructed antibody structures. From these models, it appears that the bound pesticide may be "locked" in place by several intermolecular forces:van der waals force, hydrophobic interaction, hydrogen bond, andπinteractions. The specificity of the antibody mainly resulted from the key residues in CDR areas, especially in the HCDR3 area. Moreover, threonine (thr) in HCDR3 may be the most important residue for 5H7 having high-specificity to parathion, whereas the broad-selectivity of 5F7and 5D11 may be attribute to serine (ser) and tryptophan (trp) in HCDR3. The results from the binding mode analysis may be useful in rational hapten design, developing engineering antibody with high-specificity or broad-selectivity, and studying the theory of interaction between antibody and pesticide.