Determination Of Rhodamine B Residues By Immunoassay Methods In Food

Rhodamine B (RB) is a kind of triphylmethane dye with fresh peachblow color and produced synthetically. The addition of RB in foodstuffs has been forbidden in China and European Union. In this paper, the immunoassay for RB was established.According to the chemical structure of RB, we developed an immunogen RB-BSA (bovine serum albumin) and a coating antigen RB-OVA (ovalbumin) through a mixed anhydride method and carbodiimide method, respectively. The ultraviolet-visible spectrophotometer indicated that the conjugates had two characteristic absorption peaks of both hapten and protein, thus the coupling process was successful. The protein content of immunogen was detected by Coomassie blue staining, and the coupling ratio was 11.67. We prepared a polyclonal antibody to detect RB by immunizing New Zealand white rabbits. A sensitive and rapid indirect enzyme-linked immunoassay (ELISA) was established to detect the titre of antibody and inhibition rate, in order to screen a polyclonal antibody with high specificity. And the titre of antibody was up to 64000. ELISA was initially established, but effects of other factors on ELISA assay needed further optimization, including coating buffer, blocking solution, antibody dilution buffer, sample dilution buffer (pH, methanol, NaCl). The optimization condition was carbonate-bicarbonate buffer (CBS, pH 9.6) as coating buffer, 0.1% gelatin coating buffer as blocking solution, 0.1% gelatin and 0.05% Tween-20 phosphate-buffered saline (PBS) as antibody dilution buffer, 5% trehalose, 0.5% BSA and 0.05% Tween-20 PBS as second-antibody dilution buffer, pH 7.4 PBS (3.6% NaCl) as standard sample dilution buffer.The standard curve of RB was established under optimum experiment conditions. The limit of detection (LOD) of ELISA method was 0.1ng/mL, IC50 was 1ng/mL, and the linear range was 0.1～20ng/mL. The condensation of coating antigen and antibody was 2μg/mL and 3μg/mL, respectively. The antibody had high specificity, and the cross-reactivity rates with Rhodamine 6G, Crystal Violet, erythrosine and malachite green were all less than 0.1%. The recoveries of RB in spike chilli power were 67.87- 89.30% with coefficients of variation less than 15%, achieving the detection demand of RB residues.An immunoaffinity chromatographycolumn (IAC) purification method was developed based on RB antibody. Furthermore, purified samples were analysed by high performance liquid chromatography-mass spectrometry (HPLC-MS). The experiment was performed in the samples, and the recoveries were 65.4-95.3% which was corresponding to ELISA method. Above all, ELISA and IAC-LC/MS was an effective approach for the residues detection of RB in foodstuffs, providing a better technique for RB detection.