| The imidacloprid artificial hapten which was synthesized by chemical modifying Imidacloprid and the imidacloprid antigen from the hapten coupling with carrier protein by EDC method. Their structures were identified by HPLC-MS, 1H NMR, 13C NMR, IR and UV; which were showed that Imidacloprid artificial hapten and its antigen were synthesized successfully, and the coupling ratio was 18.5 : 1 to the hapten with the carrier protein in the antigen.In order to learn the preliminary immunological characteristics of imidacloprid, the experimental mouse were immunized with imidacloprid antigen. The satisfactory the titer of the antiserum was showed, that was 1∶1.28×104 belongs to the No. 5 mice and 1∶6.4×103 belongs to the No. 2 mice. The optimal working concentrations of coating antigen and antibody were 2μg/mL, 1∶3200 (No.2 mice) and 1∶6400 (No.5 mice) respectively by phalanx method. The blocking ELISA was developed and the result showed that the IC50 was 9.0×10-5 mg/mL to No.2 serum samples (from No.2 mice) and 1.0×10-4 mg/mL to No.5 serum samples (from No.5 mice). Furthermore, the cross reaction ratios which were No.2 serum sample against IMI and its structural analogous compounds (Thiacloprid, Acetamiprid, Nitenpyram, and Lothianidin) were 9.09%,1.00%,0.94% and 0.37% respectively.The higher detection sensitivity, specificity and affinity IMI-mAb was prepared with spleen cells from No.2 mice and commercial standard experimental myeloma cells by improved PEG-mediated cell fusion technique. In this method, the 51.6% success rate of cell fusion and 45.9% positive rate of hybridoma were detected by indirect ELISA and blocking ELISA in 14 d. And three ELISA-positive hybridomas which were 3C8 and 3E2 respectively were emerged in continuous three times limiting dilution method, and their characteristics are as follows:1. Higher detection titer (ascots titers of 3C8 and 3E2 were up to 1∶6.4×105 and 1∶3.2×105 respectively);2. Lower detection limited (IC20 of 3C8 and 3E2 against IMI were low to 7.48×10-6 mg/mL and 9.47×10-6 mg/mL respectively)3. Good detection sensitivity(IC50 of 3C8 and 3E2 against IMI were low to 4.92×10-5 mg/mL and 5.45×10-5 mg/mL respectively) ;4. Higher detection specificity (3C8 and 3E2 with IMI structural analogous compounds all have no cross reactivity, except Thiacloprid (the cross-reactive rate were all higher than 0.5%) ;5. Stronger affinity(association constant of 3C8 and 3E2 against IMI was 1.10×1012 L/moL and 2.36×1011 L/moL respectively).The IMI residue blocking ELISA which was built based on IMI-mAb by correlative experiment. The detection method has wide linear range(8.30×10-6 mg/mL5.32×10-4 mg/mL), the low limit of detection (8.00×10-6 mg/mL), the average recoveries of meeting the demand of pesticides residue determination(>75%) and high detection specific(except for thiacloprid).Comparing the properties of IMI residue blocking ELISA and GC-FID method, the result showed that the equivalent linear range, the limit of detection and sensitivity were exist in them. Furthermore, the basic demand of pesticides residue analysis couldn't meet, because of the relatively serious matrix interference and the lower recoveries.The IMI detection kit was assembled according to the built blocking ELISA method, and in 4℃during five months, the comparatively stable performance of the kit has been proved by correlative experiment. In addition, the kit operation specification was advanced.
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