Application And Research Of Immunoassay To Determine Paraquat Residues In Foods

Paraquat (PQ) is a bisquaternary ammonium compound used as a herbicide word-widely. There is a potential for human exposure to PQ in general population through residues on food materials and water. However actual methods, such as HPLC, GC and UV, are time consuming and need expensive instruments. Thus, development of a rapid and convenient detection method for local PQ analysis is extremely desirable. In this paper, enzyme-linked immunosorbent assay (ELISA) and gold-immunochromatography assay (GICA) involving the use of colloidal gold-labeled polyclonal antibody (PcAb) specific to PQ as the marker were developed for the analysis of PQ in foods.First, N-(4-carboxybutyl)-N'-methylbipyridilium (PQ-h) was synthesized by using 4,4'-Bipyridine reacted with iodomethane by three steps. This experiment was conducted in dark under the protection of nitrogen and the products were identified through LC/MS, 1HNMR and IR, showing the purity and yield of the PQ-h were 96% and 69%, respectively. Then, immunogen (PQ-h-BSA) and coating antigen (PQ-h-OVA) were prepared according to the method of mixed anhydride. The ratio of PQ-hapten to BSA was 6:1.Next, the PcAb to PQ-h-BSA was produced by immunizing New Zealand rabbits, and the titre of the antiserum was 51200. Then, an indirect ELISA for the determination of PQ was developed and the optimal working concentrations of the coating antigen and antisera were determined to be 0.3μg/mL and 1:5000 respectively after several chess board tests. The IC50 and LOD of this method were 5.13 ng/mL and 0.005 ng/mL, respectively, and the cross reactivity between PQ and didquat was less than 0.1%. Besides, the recoveries of PQ in soybean were ranged from 70% to 80% and the relative standard deviation (RSD) was within 10%.Finally, colloidal gold was prepared through reducing HAuCl4·3H2O by sodium citrate and PcAb specific to PQ was conjugated to the gold nanoparticles under friendly and optimal condition as follows: optimum pH for conjugation was determined to be 8.5 and the least stable content of PcAb to colloidal gold was 8.1μg per milliliter gold solution. Then, a rapid, signal step and sensitive colloidal gold labeled immunochromatographic (GICA) test for detection of PQ in foods was developed and the optimum concentration of coating antigen and anti-rabbit IgG were 0.5 mg/ml and 0.11 mg/ml, respectively. This method is rapid, convenient and with high repeatability and stability, as the testing time is about 5 min, the lowest visual observation limit was 10 ng/ml and the shelf-life of immunostrips was at least five months at room temperature. Besides, the recoveries of PQ in soybean was good.In conclusion, the ELISA and GICA developed to determine PQ residues in foods have good practical value and provide an important experimental basis for the further study on ELISA testing kits and immunochromatography testing paper.