Preparation Of Monoclonal Antibodies Against Acid Orange Ⅱ And Establishment Of An ELISA Method

Acid Orange Ⅱ is a chemical dye,mainly used as the coloring agent for textiles,leather products,plastics and wood products,in medicine it can also be used for tissue.Staining.unscrupulous traders often use it for food coloring because of its bright and easy to color.Studies have shown that acid orange Ⅱ has teratogenic effects on reproduction,and accompanied by moderate carcinogenicity,use it in food processing,may cause food poisoning,long-term eat these food and even cancer,it is forbidden to use it as a food additive.In 2011,the Ministry of Health clearly stipulated that acid orange Ⅱ and other synthetic pigments included in the non-food substances and easy to abuse the list of food additives,prohibited to add in the food.Mainly detection methods on the acid orange Ⅱ which has Reported in the literature,including the thin layer chromatography,high performance liquid chromatography,reverse high performance liquid chromatography,Ultra-high performance liquid chromatography,liquid chromatography tandem mass spectrometry,fluorescence spectroscopy,electrochemical analysis,etc.These methods require expensive equipment,more complex sample processing and related specialized technical personnel,it is not easy for the staff to quickly detect the acid orange Ⅱ in food.Immunological detection methods based on antigen-antibody binding specificity,with a high degree of selectivity and sensitivity,this detection method is simple,efficient and easy to carry,suitable for mass rapid screening of samples on-site.Currently,there are only a few polyclonal antibodies against acid orange Ⅱ,the specificity of its immunoassay method is low,much worse than the effect of a monoclonal antibody.In this study,using succinic anhydride method and halogenated carboxylic acid method synthesis of acid orange Ⅱ complete antigen,after dialysis and identification,obtain immunization and coating antigen.BALB/c mice were immunized in batches with synthetic immunogen,using monoclonal antibody technology,ELISA,preparation of mouse ascites and other technologies,a monoclonal antibody with strong specificity and high titer of acid orange Ⅱwas prepared,and on this basis establish ELISA detection method.1.Synthesis and Identification of AO II complete antigensThere are no perssad in the molecular structure of acid orange II that can be directly coupled to the protein,the phenolic hydroxyl groups were modified by two methods,sodium chloroacetate and the succinic anhydride methods,a spacer arm with a carboxyl group attached thereto.Then NHS method was used to connect the antigens and carrier protein,successfully prepared acidic orange Ⅱ’s immunogen and coating antigen.Two completely antigens were identified by UV spectroscopy and SDS-PAGE gel electrophoresis,preliminary determination coupled success.By animal immunization and ELISA determination,were detected to produce antibodies,indicating that the preparation of two antigens was successful.The protein concentrations of AO Ⅱ-BS A and AO Ⅱ-OVA were measured by BCA kit at 2.35mg/ml and 1.60mg/ml,the protein concentrations of AO Ⅱ-BSA(B)and AO Ⅱ-OVA(B)were 4.12 mg/ml and 3.11 mg/ml.2.Preparation of AO Ⅱ monoclonal antibodyUsing the Synthetic artificial antigen AO Ⅱ-BSA,AO Ⅱ-BSA(B)as the immunogen,immune BALB/c mice refer to the laboratory routine immunization program(two methods of synthesis,method A immunization 2,method B immunization 3).AO Ⅱ-OVA used as coating antigen,7 days after five immunizations serum titer of the situation 1:16000.By using cell fusion technology,ELISA screening method and limited dilution method for subcloning.And finally three hybridoma cell lines were successfully secreted against AO Ⅱ antibody,4D3、4F10 and 7E10.Preparation of mice induced using a monoclonal antibody ascites anti AO Ⅱ legal,4F10 titer is best up to 64000,protein concentration of 20.4mg/mL.Purified ascites4F10 by HiTrap Protein G HP purification column,ascites heteroaryl identified by SDS-PAGE purified protein significantly reduced.The results of the cross experiments showed that there was no cross reaction with Alkaline Yellow,Amaranth Red,Sudan Red I,Sub-Magenta,Rhodamine B,Carmine,Lemon Yellow,Sunset Yellow.The inhibition of acid orange Ⅱ 100%.3.Establishment of ELISA methodUsing the purified 4F10 monoclonal antibody to establish an indirect competitive ELISA method for the detection of AO Ⅱ residues.Optimal coating concentration of the antigen was diluted 1:4000,the optimal working concentration of antibody 1:24000,AO Ⅱ concentration of 20～2000ng/mL,the standard curve linear good,the linear equation was y=0.3363x-0.2732(R2=0.989),IC50 was 198.13 ng/mL,LOD was less than 16.69 ng/mL and LOQ was 52.22 ng/mL.4.The addition and reclaim test of AO Ⅱ in stewed meatThe addition and reclamation test of AO Ⅱ in stewed meat.The sample was determined after pre-processing.The results showe that the sample solution is diluted 16 times,the matrix interference disappeared.In the range of 20～1000ng/mL,the standard equation:y =0.3062x-0.2356,R2 = 990,IC50 was 251.55ng/mL,LOD less than 21.32 ng/mL and LOQ 67.28ng/mL.With 40.400 and 800ng/mL of AO Ⅱ standard solution to do the recovery experiment,the detection of rumble meat sample recovery rate of 84%to 92%.