Research On The Immunoassay Technology For The Deltamethrin Residue

In order to meet the need from rapid detection of pesticide residues in agriculture products and explore the basic rules of small molecuclar immunoassay, Deltamethrin was selected as the object of study. The results are as follows.1. Four artificial antigens of Deltamethrin were synthesized and polyclonal antibody was prepared. Four haptens Hp-1,Hp-2,Hp-3 and Hp-4 were synthesized by different chemical pathes and their structures were identified. The results of 1H-NMR and IR indicated that the haptens were synthesized successfully. Hp-2 and Hp-3 both were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) by different methods.The UV scanning spectrums showed that artificial antigens were synthesized successfully and the coupling ratio between haptens and proteins were about 8:1.Artificial antigen Hp-2-BSA were used to immunize rabbit to prepare polyclonal antibodies for Deltamethrin, two kinds of antisera were obtained (Ab0601 and Ab0602).Their titers were 1.6×10~5 and 3.0×10~5 determined by indirect ELISA. Hp-3-BSA were used to immunize mouse to prepare polyclonal antibodies for Deltamethrin, three kinds of antisera were obtained (Ab0701,Ab0702 and Ab0703).Their titers were 1.0×10~4,1.2×10~4 and 2.0×10~4 determined by indirect ELISA.The results of indirenct competitive ELISA showed that Ab0601 and Ab0602 were specific to Deltamethrin.The cross-reactive between Ab0602 and 3-phenoxybenzoic acid was 19.5%, the cross- reactive between Ab0602 and other four Pyrethroids were all lower than 1%.2. Immnuoaffinity column of Deltamethrin was prepared and the IAC-HPLC test method for Deltamethrin was established. The using condition of the column was confirmed by HPLC. Procedures for column using were as follows: 5mL 0.01M PBS (pH7.4) were used for balancing the column. PBS contained 1% methanol were used as the sample buffer, then the column was washed by 2mL ultrapure water and eluted by 1mL methanol, 20mL0.01M PBS (pH7.4) were used for regenerating the column.The column capacity was≥8μg , the column could be used only once. The IAC-HPLC test method for Deltamethrin was established. The regression equation of standard curve was Y= 15.291X +13.563, R=0.999, the linear concentrations of detection is ranged from 0.1μg/mL to 10μg/mL, the minimum detectable concentration was 0.1μg/mL. Cabbage for the test of recovery, the mean recoveries were found in a range from 78% to 82.1%,RSD 5.0～7.25%.3. Polyclonal antibodies that specific for five kinds of Pyrethroids were prepared. Hapten Hp-5 was synthesized from 3-phenoxybenzoic acid, Hp-5 was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) as antigen and coating antigen. Hp-5-BSA were used to immunize rabbit to prepare polyclonal antibodies.The results of indirect competitive ELISA showed that the polyclonal antibodies were specific for five kinds of Pyrethroids.