Preparation Of Olaquindox Monoclone Antibody And Establishment Of Time-Resolved Fluorescence Immunoassay

One hapten of olaquindox (OLA-HS) was synthesized and then covalently coupled with carrier protein BSA and OVA, repectively. The□ousse were immunized with artificial antigens, then mouse spleen lymphocytes were fused with myeloma cells by cross-tumour technique. The hybridoma cell2B3was selected and cloned and then the monoclonal antibodies were obtained. Finally the study carried out a time-resolved fluorescence immunoassay (TRFIA) using the lanthanides europium as a probe on this basis, and then compared and validated with the conventional enzyme-linked immunosorbent assay (ELISA) and traditional instrumental analysis method (HPLC), the results of this study were good. The main contents and results of this study were as follows:1. Synthesis and identification of the artificial antigen for Olaquindox The hapten of OLA (OLA-HS) was synthesized by succinic anhydride method, and identified through thin layer chromatography (TLC), micro melting point apparatus and mass spectrometry (MS). Two artificial complete antigens of OLA (OLA-HS-BSA, OLA-HS-OVA) were prepared by active ester method (AE), and examined through ultraviolet spectrophotometry (UV) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) respectively. The UV scanning spectrum showed that the maximum absorption peak of synthesized substances was shifted, and the SDS-PAGE electrophoretic bands showed that the migration rate of OLA-HS-BSA and OLA-HS-OVA was slower than its each carrier protein BSA and OVA, which demonstrating the coupling of a certain number of hapten with carrier protein, these all are good proofs about the successful synthesis of the artificial antigen. The serum titer and sensitivity immunized by OLA-HS-BSA was measured through indirect ELISA and indirect competitive ELISA respectively, the results show that the titer of pAb for1st mouse is the highest, its IC50was183.3ng·mL-1. The high-titer, sensitive anti-OLA pAb had been produced.2. Preparation of monoclonal antibodies and identification of its immunological characterizationA positive cell line2B3secreting OLA-mAb was successfully screened by hybridoma technique. Massive OLA-mAb were induced from in vivo method and the ascites was purified by bitterness-ammonium sulfate. The results showed that the protein concentration was3.65mg·mL-1and all the isotypes of the mAb were IgG1and the light chain were κ, the mAb of2B3showed good sensitivity with IC50of33.96ng·mL-1to OLA. The rate of cross-reaction of OLA-mAb with carbadox was3.6%, and there was no cross-reactivity to other structural analogues. OLA-mAb of high-titer, sensitivity and specificity had been generated.3. The foundation of time-resolved fluorescence immunoassay (TRFIA)The key of this study was to establish a kind of direct competition TRFIA using Eu3+as a probe. First we got Eu3+-OLA-mAb for coupling certain number of Eu3+to OLA-mAb through a metal chelator cyclization diethylenetriamine five acetic anhydride (cyDTPA). Then determined the concentration of TTA and TOPO were0.45mol·L-1and0.5mol·L-1respectively, and the final PH was3.0. Finally this paper carried out series of optimization experiments to determine the optimal reaction conditions of TRFIA. The results were as follows:The optimal concentration of coating antigen was1.0μg·mL-1, the optimal dilution of Eu3+-OLA-mAb was1000x; The optimum coating buffer was0.05mol·L-1PH9.6CBS, the best ionic strength of Eu3+-OLA-mAb dilutions was0.01mol·L-1and its optimum PH was7.0, and the preferred organic solvent was "5%methnol-PBS". Finally we got a sigmoid curve, the detection limit for OLA was0.83ng·mL-1and its IC50was12.64ng·mL-1. The recoveries from feedstuff, pond water, tap water and ultrapure water were all among74.40%～138.25%when2.5ng·mL-1～130ng·mL-1OLA were spiked. The coefficient variation was below15%. The analytical sensitivity of TRFIA was higher than HPLC, so it has a great application value.4. The establishment of indirect competitive ELISAThis experiment established an indirect competition ELISA method and optimized its reaction system. Finally we got a sigmoid curve, its IC10and IC50were1.03ng·mL-1and21.83ng·mL-1respectively. The recoveries from feedstuff, pond water, tap water and ultrapure water were all among77.00%～127.05%when2.5ng·mL-1-50ng·mL-1OLA were spiked. The coefficient variation was below11%.