Research And Application Of Detection Of Rhodamine B In Foods By Rapid Immunoassay

Rhodamine B(Rhodamine B, RB), commonly known as the pink flowers, is the pink and synthetic chemical dye. It has the impact of causing cancer and gene mutation. Due to the characteristics of the low price, the bright red and the strong stability, It was often used by the lawbreaker as the colouring agent of condiment, such as chilli powder, chilli oil and sausage,which had brought great harm of the people’s health and the social. So, it is very necessary to research the RB detection methods in food. For now, however, the literatures about the detection of RB in the food have not yet been reported. Although RB was listed as one of seventeen kinds of illegal food additive substance which announced as the first batch ones by the Ministry of Health, but there were still illegal application. So, it has great significance to research the RB rapid detection and identification methods. Due to the chemical structure of the RB which was not easy to compound of artificial antigen, this paper compounded of artificial antigen and prepared of the polyclonal antibody of RB by Rhodamine 123(Rhodamine 123, R123) which is the RB’s homologue. This paper preliminarily established the methods of indirect competitive enzyme-linked immunosorbent assay(ELISA) and colloidal gold immune chromatography analysis about RB. The two methods all had good specificity and sensitivity.This research respectively coupled R123 with bovine serum albumin(BSA) and ovalbumin(OVA) by glutaraldehyde method, which could obtain immunizing antigen R123-BSA and envelope antigen R123-OVA. After isopyknicly mixing immunizing antigen R123-BSA with freund’s adjuvant, this experiment was immune to 3 months’ big-eared white rabbit in New Zealand by the subcutaneous multipoint immune method to obtain polyclonal antiserum of RB. Through testing, the valence of antiserum was 1:4000, then, this experiment purified the rabbit’s antiserum by ammonium sulfate precipitation method to get the specific polyclonal antibody of RB. Based on the purified antibody, this research established the method of RB’s indirect competitive enzyme-linked immunosorbent assay. In the range from 0.001 ng/ml to 10000 ng/ml, the results showed that IC50 was 3.96 ng/ml, detection limitagainst RB was as low as 0.23 ng/ml, the cross reaction rate of Rhodamine 6G, Sudan red and Nacarat respectively was 55.93%, 8.82% and 0.51%, which indicated that this method had high specificity, moreover, the precision and sensitivity of the method were evaluated.For the actual samples tested, this research chose Chinese wolfberry, pepper, Chinese prickly ash to test, the recovery rate determination results were between 80.50% and 110.50% by eliminating the matrix effect. Moreover, ELISA was compared with the HPLC, and showed that the two methods were appearing a good linear relationship. The correlation coefficient R2 was 0.9695.The results showed that this ELISA kit can meet the needs of the actual testing, and it was suitable for immune rapid detection of RB in food.Based on the antibody labeled colloidal gold, this research established immune rapid detection technology of RB, with colloidal gold as tracer marker, with nitrocellulose membrane(NC membrane) as solid carrier which was separately coated with goat-rabbit Ig G(control line) and R123-OVA conjugate(test line). By optimizing the experimental conditions,this research developed the percolation type of colloidal gold immune rapid detection strip,and developed the standard color card for RB. The results showed that the detection limit against RB was as low as 0.5 ng/ml in the range from 0.01 ng/ml to 20 ng/ml. For the actual samples tested, this research chose Chinese wolfberry, pepper, Chinese prickly ash for the qualitative determination of RB, the matrix effect was eliminated by the method of diluting sample extract, moreover, the recovery rate determination results were good. This method was simple and the results were got through eyes, which could immediately realize the on-site rapid detection without special equipments. So, it is suitable for rapid and qualitative immune detection of RB in condiment and part of food.