Preparation Of The Single-chain Fusion Antibody And The Study On Immuno-Rapid Detection Assays For Sudan I And Tartrazine

SudanⅠwas used to the industrial dye. But now, it was used to be as one kind of food additive in some food industries. It was prohibited strictly to add to the food in China and EU, because it could produce the carcinogen by some corresponding enzyme in the body. Tartrazine was given approval in some food as one combination pigments. As an chemical group N=N compound ,it could be metabolized into an amines compound which was potential hazard to body. So it was severely restricted as an food pigment in our country. It was prohibited strictly to the flesh and fish ,and its dose was severely regulate in some applied varieties and range.As though the strict limit, SudanⅠand Tartrazine were used irrationality in some food industries widespread. Since 2005, several incidents had happened which the two kinds pigments were illegal added to the food, and which had made the people worried.The detection methods on SudanⅠand Tartrazine are relied on instruments. The HPLC and Lamina analysis\spectrophotometer are respective to detect SudanⅠand Tartrazine. There are some shortage on the detecting methods on instruments, such as wasting time, exertion, high cost, no ease to work et al. We study the new methods on detcting SudanⅠand Tartrazine by the immune-methods. The new immune-methods had few reports in our country and others. The new methods retrieve the shortage of the instrument, s methods. In this essay, we study the molecular constitution of SudanⅠand Tartrazine, synthesizing the analog of SudanⅠwhich coupled with carrier protein. We got the SudanⅠmonoclonal antibody(Sudan McAb) by hybridoma technique. According to the Tartrazine,s molecular constitution, we obtained the Tartrazine monoclonal antibody(Tar McAb) by hybridoma technique too. Two kinds of monoclonal antibody made it possible that the SudanⅠand Tartrazine were detected by immune- technique which we prepared the tacho-detecting kit and the tacho-detcting slip by gold colloid technique. It was possible that we got the single-chain antibody(ScAb) from two kinds of monoclonal antibody of SudanⅠand Tartrazine. The McAbs were linked by Linker in biotechnology to become the fusion gene. After expressing, we could detect the antibody which could identify the SudanⅠand Tartrazine. Then we can study the methods by which the SudanⅠand Tartrazine can be detected quickly.Synthesis of complete antigen of Sudan-Ⅰ. Based on the molecular structure of Sudan-Ⅰ, a analogy of Sudan-Ⅰwas synthesized . Compare to the molecular structure of Sudan-Ⅰ,a carboxyl group was induced to the counterpoint of chemical group-N=N in low temperature, then coupled with BSA through active ester method to be complete antigen. The completed antigen was dialyzed and identified by AGE,SDS-PAGE,UV-scanning. The coupling rate in average was 2-3. the blood serum of anti- Sudan-Ⅰwere obtained by immuniting the mice.Preparation of anti-SudanⅠmonoclonal antibody. Methods ASu, an analogue of SudanⅠstructure which was synthesized coupled with BSA,OVA through EDC to be complete antigen. After immunization of BALB/C mice with synthesized BSA-aSu,the simulated splenocytes were fused with SP2/O myeloma cells to produce hybridomas. The hybridom was identified by the routine methods. The hybridoma cell line of anti-sudanⅠwas obtained.The number of chromosome of the hybridoma cell line was from 86 to 90, the titer of ascites was 1.8×10~4. The affinity constant was 6.9×106L/mol. The anti-SudanⅠmonoclonal antibody was abtained successfully by synthesing the analogue ,and it can inhibit the sudan-Ⅰobviously.The establishment of sudanⅠcompetitive inhibition ELISA detection method. The competitive inhibition ELISA detection method was improved using sudanⅠmonoclonal antibody. The regression equations and correlation coefficients of the detection method is y=-38.541x+131.1,R2=0.9841; The lowest concentrations, which can effectively detect sudanⅠis 0.018μg/L. The simulate sample (add some sudanⅠ) was homogenated and dissolved by methyl cyaride. After methyl cyaride extraction, evaporation fixation, the indirect competitive ELISA were used to detect the sample. The recoveries are 84.72% . In addition, the sudanⅠcompetitive ELISA detection kit in plate were packed according to optimized condition of indirect competitive ELISA.Preparation of colloidal gold strip for rapid detection of sudanⅠ. A method for rapid detection of sudanⅠby colloidal gold strip was introduced. Four steps were involved in the process of the preparation ofthe strip, including production of colloidal gold, manufacture of immunogold, installation and analysis of the strip. With the strip, 50ng/L of sudanⅠwas detected in few minutes. Apart from very short time to get test results, the strip possessed benefits of user-friendly format and relatively inexpensive to make. So it is ideally suited for on site testing of sudanⅠ.The compounding of ScFv against sudanⅠ. The VH and VL genes of monoclonal antibody against sudanⅠwere amplified from the hybridoma cell line sudmab-1 by RT-PCR. They were linked using a flexible linker gene by SOE-PCR. The sudanⅠ-ScFv gene was cloned into PMD18-T vector and was sequenced. The ScFv gene consists of 665bp which encoded 212 amino acids including 15 linker amino acids. The VH and VL accord with the feature of the variable region gene of the mice antibody by blast analysis. They have the typical structure domain (IGV) of the immunoglobulin and have very high homology with many ScFvs on record by amino acid homology comparing analysis. Both VH and VL genes are confirmed as functionally rearranged mouse immunoglobulin variable region genes.The preparation and specialities study of Tartrazine monoclonal antibody . The spleen cells from the Balb/c mice hyperimmunized with a Tar-BSA conjugate were fused with SP2/0 myeloma cells. One hybridoma cell line was identified that secretes monoclonal antibody against Tar, designated 3D4 after subcloned for 3 cycles by indirect ELISA.The affinity constant was 4.8×106L/mol and the titer of the monoclonal antibody was 1.2×10~6 ,and it had no cross reacting with SudanⅠ-Ⅳ,sun set yellow. This proved that the Tar monoclonal antibody had upstanding specificity.The establishment of Tar indirect ELISA detection method. The indirect ELISA detection method was improved using Tar monoclonal antibody. The regression equations and correlation coefficients of the detection method is y= -40.134x+133.46 , R~2=0.9692; The lowest concentrations, which can effectively detect Tar is 0.36μg /L. According the kit instruction manual severity, we can detect the Tartrazine in food.The compounding of SCFv against Tar. The VH and VL genes of monoclonal antibody against Tar were amplified from the hybridoma cell line 3D4 by RT-PCR. They were linked using a flexible linker gene by SOE-PCR. The Tar-ScFv gene was cloned into PMD18-T vector and was sequenced. The ScFv gene consists of 747bp which encoded 248 amino acids including 15 linker amino acids and no terminator codon. The VH and VL accord with the feature of the variable region gene of the mice antibody by blast analysis. They have the typical structure domain (IGV) of the immunoglobulin and have very high homology with many ScFvs on record by amino acid homology comparing analysis. Both VH and VL genes are confirmed as functionally rearranged mouse immunoglobulin variable region genes.Clone and expression of the fusion ScFv against SudanⅠ-Tar. The SudanⅠ-scfv and Tar scfv were linked by double-enzyme cutting method to insert the express carrier of pET-22b and transformed into DE3. The molecular weight of the expressed fusion protein is about 50ku by IPTG induction. The expressed fusion protein has a favorable inhibitory with by indirect competitive ELISA. This will established the study foundation for using the fusion ScFv to construct the rapid detection which can screen two kinds of pigments simultaneously.In brief, the monoclonal antibodies against micromolecule SudanⅠ,Tartrazine were prepared in this experiment, and two sorts of competitive inhibition ELISA which were more rapid, sensitive and convenient were constructed. A new method for rapid detection of sudanⅠby colloidal gold strip was prepared which could detect sudanⅠin few minutes in the laboratory condition. At the same time, the VH and VL of the monoclonal antibody against SudanⅠ,Tartrazine were amplified and the SudanⅠ-ScFv,Tar -ScFv were packed by the SOE-PCR. We studied the SudanⅠ-ScFv which expressed in DE3.The expressing product could inhibit the SudanⅠby indirect competitive ELISA. SudanⅠ-ScFv,Tar -ScFv were linked by double-enzyme cutting method to insert the express carrier of pET-22b and transformed into DE3 and expressed by IPTG induction . The expression protein can inhibit the SudanⅠand Tartrazine with ELISA. The result will establish a study foundation for using the fusion ScFv to construct the rapid detection methods which can screen the two kinds of pigments simultaneously .