Hapten Designing Of Pesticide Parathion

The hapten designing and synthesis is the key step for development immunoassay of small molecule. The typical organophosphorus pesticide, parathion, was set as the object, and the structure activity relationship (SAR) of immunizing hapten was investigated to set up the immunizing hatpen designing model. Methods of molecular connectivity index (MCI) and molecular modeling were introduced into the immunizing hapten designing. The relationship between the heterologous degree of the immunizing hapten and parathion had been investigated according to Euclidean Distance. Then the different computed results were validated and compared with the conclusions which came from the dialysis and ELISA experiments. The results showed that the MCI was good at depicting the steric structure of the designed haptens, whereas the index was not good enough to depict the electronic structure. However, the electronic parameters (EP) which came from the molecular modeling could depict the electronic structure rather than the steric structure. So when the two methods were combined, the new method could represent the degree of similarity between the designed haptens and the analyte very well. Then the immunizing hapten designing model was set up successfully.Just the same as the immunizing hapten SAR investigation, MCI and EP were also applied to the competitive hapten SAR investigation. Then the triangular model was brought forward to compare the Euclidean Distance of the three compounds, which were the analyte, the immunizing hapten and the competitive hapten. The existed terms of the triangular model were as follows: CP>CI; Max(CP-IP),and CP>IP; Max(CI). Next, in combination with the heterologous ELISA experiment, the competitive hapten designing model was set up, and the critical term of the triangular model were also gained. That is, CI=2-4 and CP=1-4. Finally, hapten 0325 was chosen to validate the competitive hapten designing model and the critical term of the triangular model.Based on the immunizing hapten /competitive hapten designing model, the combination 0204/0322 was selected to delegate the optimal hapten. The linear equation of standard curve was as follows: y = 10.012Ln(x) + 32.768. The IC₅₀ and IC₁₀ was 5.60 ng/mL and 0.10 ng/mL, respectively. Then the influence of the optimal combination on the ELISA method were investigated and compared with the pioneer's results. The results showed the sensitivity (delegated by IC₁₀ value) of the heterologous ELISA was nearly 16-fold higher than that of the homologous ELISA. In addition, the sensitivity of the established heterologous ELISA was higher than that of all reported ELISAs, and it could be compared with that of the chromatography methods. There was little or no cross-reactivity to similar compounds tested except for the insecticide parathion-methyl, which showed a cross-reactivity of 7.8%. Moreover, the established immunoassay was applied for the residue determination of parathion-spiked samples, including water, soil, cucumber, rice and corn. The results showed that the immunoassay conformed to the demand of the guideline of the pesticide residue determination.At the last, to validate the immunizing hapten and competitive hapten molecular designing model, three pesticides were chosen. That is, triazophos, 2, 4, 5-trichlorophenol and fenthion. The experimental data were come from the published papers by other scholars. The validation results proved the molecular designing models were correct and could be applied for the pesticide hapten designing. The antibody with high affinity could be produced by the application of the immunizing hapten molecular designing model and scientific procedure. On the other hand, the optimal competitive hapten could be evaluated and designed by the application of the competitive hapten molecular designing model. Based on the two molecular designing models, the heterologous ELISA method could have high sensitivity, affinity and specificity.In the past hapten designing work, the adopted designing strategy were empirical and elective, and sometimes, the designing was only considered the difficulty in the hapten synthesis which resulted in there had not enough typical haptens, so that the common rules for rational hapten designing could not be found. The present investigation solved the problems. Applied the new procedure, the hapten could designed quantitatively and directionally. What the more importance is, the model of competitive hapten was built, which could be significant for improving the specificity of the immunoassay. Furthermore, the two models would be contributed to set up the basic theory of immunoassay for pesticide determination, which could have benefit to develop the immunoassay kit with decision-making power of intellectual property in our country.