ELISAs have been widely used for the determination of various contaminants such as toxins [14,15], drugs [16,17], pesticides [18] and illegal additives [11,12] in the biological, agriculture, and environmental fields. by high-performance liquid chromatography (HPLC) analyses. Keywords:chrysoidine, antibody, ELISA, soybean milk film == 1. Introduction == Chrysoidine (C.I. Basic Orange 2,Figure 1) is a type of industrial azoic dye [1]. Due to its good dyeing fastness, it is widely used for dyeing leather, paper, feather, grass, wood, bamboo,etc.[2]. Chrysoidine can cause acute and chronic toxicity to mammals when taken by oral Mouse monoclonal to EphB6 or skin route, or inhaled, and its median lethal concentration (LC50, 24 h) for fish was 0.5 mg/L [3]. Chrysoidine has also been recognized as a carcinogen [4] and its use in food has not been approved by any country. Unfortunately, it has been reported that soybean milk film, a popular soybean food consumed in China was adulterated with chrysoidine [5,6]. Moreover, chrysoidine has also been found in yellow-fin tuna and dried bean curd stick [2,7]. Therefore, control of this banned dye in food is very crucial and the development of a simple, economic, and rapid detection method is urgently needed. == Figure 1. == Thrombin Inhibitor 2 Structure of chrysoidine. The reported analyses of chrysoidine were mainly physio-chemical methods based on chromatography with various detectors [2,4,5,8,9]. The detection limit of chrysoidin in a HPLC-MS study was 0.25 ng/g [4], and 2.3 ng/g in a GC-MS method [8]. Chromatography methods can provide accurate and reliable results, but these methods are also expensive, laborious and time-consuming [10]. To date, immunoassay technologies, especially ELISA, are increasingly replacing traditional chemical analyses in screening of food contaminants and agrochemicals due to their sensitivity, time-efficiency and cost-effectiveness [11,12,13]. ELISAs have been widely used for the determination of various contaminants such as toxins [14,15], Thrombin Inhibitor 2 drugs [16,17], pesticides [18] and illegal additives [11,12] in the biological, agriculture, and environmental fields. However, to the best of our knowledge, there is no published literature on an immunoassay for the detection of chrysoidine. In this study, a highly sensitive and specific ELISA for the determination of chrysoidine was developed for the first time. Two chrysoidine haptens with different spacer arm lengths were synthesized and covalently coupled to different carrier proteins to produce both immunogens and coating antigens. The polyclonal antibody (pAb) to chrysoidine raised from immunized rabbits were characterized and used for a competitive ELISA. The developed ELISA was further employed to analyze spiked soybean milk film samples and validated by a HPLC method. == 2. Results and Discussion == == 2.1. Synthesis of Chrysoidine Haptens == For production of high quality antibodies and development of highly sensitive and specific immunoassays, it is important to design a proper hapten structure. It was proposed that both the conjugation position at the hapten molecule where the spacer is attached and the length of the spacer may play an important role for a successful antibody production [11,19], and that the molecular structure of the hapten should be left unchanged [20]. In this study two chrysoidine-derivatives with different spacer lengths were synthesized (Scheme 1). One derivative with one carbon-atom spacer length (Hapten 1) and the other with two-carbon-atom spacer length (Hapten 2) were modified at theparaposition of the azo bond. The structures of Hapten 1 and Hapten 2 were confirmed by thin layer chromatography (TLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) methods. == Scheme 1. == Synthesis of chrysoidine haptens. == 2.2. Synthesis of Immunogen and Coating Antigen == The chrysoidine derivative bearing a carboxylic acid group at the end of the spacer was Thrombin Inhibitor 2 activated by the active ester method and then covalently coupled with a carrier protein (BSA or OVA) [21]. The conjugates of hapten-BSA and hapten-OVA were used as immunogen and coating antigen, respectively.Figure 2shows the UV spectra of BSA, OVA, Hapten 1, Hapten 1-BSA and Hapten 1-OVA with absorption peaks of Hapten 1, BSA and OVA at 450, 280 and 280 nm, respectively. The absorption spectra of Hapten 1-BSA/OVA conjugates contain both absorption peaks of Hapten1 and BSA/OVA, but with somewhat red shift. The results indicated that the coupling of hapten to BSA and OVA was successful. Similar results were obtained with Hapten 2 conjugate (data not shown). == Figure 2. == UV spectra of BSA, OVA, Hapten 1, Hapten 1-BSA and Hapten 1-OVA. == 2.3. Optimization of icELISA Conditions == The prepared coating antigens were used for the.