Establishment And Preliminary Application Of A Detective Method Of ELISA For Sudan Red

Sudan Dyes and Para Red are two kinds of dyes which are usually used in industrial products. Both of them belong to lipotropy small-molecular compounds with an azo group. Sudan Dyes have four types, includeⅠ,Ⅱ,ⅢandⅣ. Sudan I is also called Oil Yellow. Its chemical formula is 1-phenylazo-2-naphthalenol, and its molecular fomula is C16H12N2O. Para Red is also called Para-position Anilline Red. Its chemical formula is 1-(4-nitro-phenylazo)-2-naphthalenol, and its molecular fomula is C16H11N3O3. The molecular structure of Para Red is simillar to that of Sudan I.Since there are azo groups in the structure of Sudan I and Para Red, the human bodies will incline to develop cancers after people eat the foods which contain these two kinds of dyes, especially liver cancers. And these two dyes are not in the list of the legal food additives according to the'Hygienic Standards for Uses of Food Additives in P.R.C.'(GB 2760-1996), which were promulgated in China in 1996. Because of their strong ability of dyeing to remain the foods in fresh and bright-coulored for a long time, they are used as food additives in many kinds of foods illeagally. Therefore, It is essential and urgent to find a quick and effective method to detect whether there is Sudan I or Para Red in the foods.The purpose of this thesis is to establish a competitive ELISA method that can detect Sudan I and Para Red synchronously in foods of water-solubility type. And the method is on the base of many other techniques, such as the preparation method of complete antibodies, the preparation method of antiserum of animals, the preparation method of monoclonal antibody and the preparation method of ascites.This paper can be devided into three parts:1. Preparation of the immunogen and coating antigenThe structure, which is similar between Sudan I and Para Red, was coupled with two carrier proteins, BSA and OVA, respectively. The immunogen (SudanⅠ-BSA) and the coating antigen(SudanⅠ-OVA) were obtained through the'Carbodiimide Method'. The productions had been identified by observation, ultraviolet detection and raman detection.2. Preparation of the monoclonal antibodyAfter the BALB/c mice were immunized, the splenocytes of the mouse whose antiserum had the best quality were taken out and fused with Myeloma cells SP2/0. The cells were then cultivated in the HAT selective culture medium and the survival cells were just the fusion cells. Through the filtration by the' Limiting Dilution Assay', hybridoma cells which could secret the most monoclonal antibodies, specifically against SudanⅠwere acquired. The hybridoma cells were then injected into other normal BALB/c mice abdominal cavity to prepare the ascites. The ascites was purified by the'Caprylic Ammonium Sulfate Precipitation'3. Establishment of detection methodA competitive ELISA for SudanⅠand Para Red was established after optimizing the conditions of the experiments. SudanⅠor Para Red was distilled from tomato sauses by the'Methyl-Cyanide Extraction Method'. Compared with the standard curve, the recovery rate could be figured out.There are four important conclusions in this paper:1. The coupling experiment was successful. It was confirmed to obtain the immunogen(SudanⅠ-BSA) and the coating antigen(SudanⅠ-OVA);2. The hybridoma cells secreting monoclonal antibodies specifically against Sudan I were obtained successfully through several filtrations of fusion cells. We also acquired the ascites, which was purified by caprylic ammonium sulfate precipitation and the antibody titer was 1:128000;3. A competitive ELISA for detecting SudanⅠand Para Red was established successfully. The IC50 for SudanⅠand Para Red were 0.8dg/L and 4μg/L, respectively. The cross-reactivities rate of SudanⅡ,Ⅲ,Ⅳwas 1.24%-2.12%, and the rates of other four favorite dyes were lower than 0.2%;4. The recovery rate of the detection of SudanⅠor Para Red distilled from tomato sauce by the methyl cyanide was 69.8%-118%. Within the error permission range, this competitive ELISA can be used as a detective method. This sensitive and specific method can provide more guidances both in theories and practices for establishing a quick and primary immunoassay device to detect Sudan I or Para red in the foods of water-solubility type.