Preparation And Estblished Primary Immunoassay Of Monoclonal Antibodies Against Rhodamine B

Rhodamine B (RB) is a kind of triphylmethane dye with fresh peachblow color and produced synthetically. It was used as a food additive, but later it proved that Rhodamine B is potentially toxic, carcinogenic, and mutagenicity. The addition of RB in foodstuffs has been forbidden in China and European Union.The main methods for detection of RB are traditional physical and chemical instrument dectecting. They are spectrophotometry method, high performance liquid chromatographic method, ultra performance liquid chromatography method, liquid chromatograph mass spectrometer method, fluorescence probe method, Raman spectroscopy method and so on. Although those methods can satisfy the testing standards, they have disadvantages of need expensive instrument, specialized technical personnel, complicated sample preparation. Those disadvantages make the instrument dectecing method not suit to rapid detection of large quantities of samples. Immunological detection method has high selectivity and sensitivity, it is a simple, efficient, accurate, inexpensive detection method, and easy to carry. So it is suitable for rapid screening of mass samples.A derivative of Rhodamine B was synthesized following derivatization with N-hydroxysuccinimide anhydride (NHS) and conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) as immunogen or coating antigen, respectively. Using immunogen to immune BALB/c mices. Basing on the technology of preparation of monoclonal antibody and enzyme-linked immunosorbent assay, we establish three stable hybridoma cell lines that secrete monoclonal antibodies against RB. By using the monoclonal antibody, we established the indirect competitive ELISA method and colloidal gold immunochromatographic test strip for detecting illegal use of RB.1. Synthesis and identification of RB complete antigens.RB belongs to semi antigen, its molecular structure contains a carboxyl group by itself. Bovine serum albumin and ovalbumin were used as two protein carriers(BSA, OVA) respectively to couple with semiantigen RB by NHS method.The synthesized antigens were demonstrated to be successful by UV-scanning absorption and SDS-PAGE method. Finally, by immuning, fusion and ELISA method, the result show that the two kinds of antigen preparation was successful. The protein concentrations of RB-BSA and RB-OVA were 5.4mg/mL and 3.4mg/mL by BCA method.2. Preparation of monoclonal antibody against RB.Using RB-BSA as immunogen to immunized BALB/c mice, RB-OVA as coating antigen to coat ELISA plate. Seven days after fifth immunization, the titer of blood serum reached more than 1:16000. By using cell fusion technique, ELISA screening method and the method of limited dilution, three strains obtained sustainable secretion of the RB antibody hybridoma cell lines were obtained. They were named 2D1,4C3 and 7F11. Ascites of monoclonal antibodies were prepared by injecting cells of hybridomas into mice abdomen. The protein concentration of ascites were 20.6mg/mL,19.1mg/mL,22.4mg/mL, respectively. Titers of the monoclonal antibodies were 16000,64000,512000, respectively. Using HiTrap Protein G HP to purify 7F11 ascites. Identify the effect of purification by SDS-PAGE. The result show that complex proteins were significantly reduced. The cross experiment shows that the monoclonal antibody has 100% inhibition rate for RB, and it has no cross reaction with Rhodamine 6G, Rhodamine 123, Phenol red, Pararosaniline, Neutral red, Eosin Y, Orange Ⅱ, Methyl orange and gentian violet.3. The establishment of ELISA detection method.An indirect competitive ELISA method for detection of RB residues was established using purfied 7F11 monoclonal antibody. The best dilution concentration is 1:2000 and the best working concentration of purified 7F1I antibody is 1:64000. The standard curve of R to antibody-antigen reaction was prepared with the linear equation y=0.3056x-0.2357 (R2= 0.9903), the linear range was 6-5000ng/mL. IC50=254.5ng/mL, LOD under 6ng/mL, LOQ=12.5ng/mL.4. The addition and reclamation test of RB in the bacon.The addition and reclamation test of RB was carried on in the bacon. The sample was determined after pre-processing. The intervention disappeared when the extraction of liver and muscle sample was diluted 16 times. The standard curve equation was:y=0.3372x-0.3620 (R2=9913), the linear range was 12～5000 ng/mL IC50=359ng/mL, LOD under 12ng/mL, LOQ=23.4ng/mL. When adding 50,300,800ng/mL RB to the bacon, the recovery ratio was between 82% to 93%.5. Development of RB colloidal gold immunochromatographic strip.Three kinds of colloid gold with different diameter were prepared by trisodium cicitrate. According to the exosyndrome of color and UV-scanning curve, the colloid gold with 31nm in diameter was used to label antibody. After experiment, we confirm that the optimum pH for labeling was 8.1 and and the concentration of antibody was 5.2μg/mL. Using the most suitable pH and concentration of antibody to make immunogold. The immunogold was purified at high speed and low temperature. The best dilution choice of immunogold was 1:1.5, the optimum spraying quantity was 30μL/cm. The best spray volume of control line(Rabbit anti mouse IgG) and detection line(RB-OVA) of the cellulose nitrate film was 0.7μL/cm and 0.6 μL/cm. The sensitivity of strips was 400ng/mL.