Study On The Immunoassay Technology For The Fenvalerate Residue

In order to detect fenvalerate residues by immunoassay methods, two hybridoma cells were screened from the semi-solid HAT culture medium after immunization. With the further optimization of ELISA and spiked experiment, we established a rapid detection method for fenvalerate residues and it could be used for the analysis of fenvalerate contamination.1. This study firstly transformed -CN into -COOH in the middle of the molecular structure of fenvalerate, then synthesized a fenvalerate hapten with a connected arm of three carbon. Compared with the reported literature, this approach needed not protect the group (-NH2). By introducing the arms and active groups in the approximate middle of the molecular structure of fenvalerate, we intended to expose the acid party and the aromatic party to get the better immune effect.2. Conjugates were synthesized using the activated ester method. The UV spectrogram of the conjugate was found different from the hapten and carrier protein, showing that the artificial antigens were synthesized successfully. After immunization, the titers were 160000, 140000 and 110000, separately. The IC50 was 35.1μg/L for fenvalerate by competitive indirect ELISA, also showed less than 1% cross-reactivity with other pyrethroids.3. Two cloning hybridoma cells (5B10 and 4E3) were screened by cell fusion. The IC50 values for fenvalerate were 94.5μg/L and 19.2μg/L, respectively. The limit of detection (LOD) of MAb(4E3)was 1.02μg/L and the detection range was 3.81μg/L ~ 714.94μg/L, with less than 1% cross-reactivity. By determining the stability of the cells after cryopreservation, we found the survival rate of cells was about 40%.4. For immunological characterization of monoclonal antibodies, two MAbs were first determined with the following parameters: the concentration of OVA-hapten, the blocking buffer, the dilution of antibody, ionic strength, pH value and so on. The results were as follows (for MAb 4E3): the coating concentration of OVA-hapten (2μg/mL), 1%OVA blocking buffer, 1:32000 dilution for antibody, ionic strength 0.16M, pH 7.4, 5%methanol. After optimization, the IC50 was improved from 19.2μg/L to 17.4μg/L and the limit of detection was up to 1.02μg/L.5. After optimization, MAb (4E3) showed the best sensitivity to fenvalerate and little cross-reactivity with other pyrethroids. The IC50 values for fenvalerate was 17.4μg/L and the detection range was 3.81μg/L～714.94μg/L. The mean recovery rates for spike recovery experiments (rape, tea, green pepper, chinese cabbage) varied from 80% to 90%. Meanwhile, compared ELISA method with gas chromatography, it indicated that the ELISA method could be used for the analysis of fenvalerate contamination.