Development Of An Enzyme-Linked Immuno-Sorbent Assay And A Colloidal Gold Immuno-Chromatography Assay To Detect Ractopamine In Procine Urine Samples

Ractopamine is a member ofβ-adrenergic agonists, which can enhance heart systole and dilate skeletal muscle blood vessel and bronchi smooth muscle. It is used to cure shock and bronchial spasm for animals and human. Ractopamine can decrease body fat and increase protein synthesis. However, when excessive ractopamine is accumulated in human bodies or animal tissues (including liver, kidney and lung) which contain high concentration of ractopamine are eaten by people, incidence of food poisoning will occur. RAC has been forbidden in animal feeding in Europe and in China, however illegal use of RAC has been reported constantly in China. The current detection methods of ractopamine are not rapid, cheap, stable, and accurate enough. In our study, two kinds of immunoassays had been developed for the rapid screening of ractopamine in porcine urine.Our work includes three parts as follows.1) Preparation of ractopamine conjugation antigensIn this study, after reacting with succinic anhydride in presence of pyridine for conjugating -COOH, RAC was conjugated with protein by the method of EDC-NHS. During conjugation, modified bovine serum albumin binding with more amino groups, was used to improve the rate of conjugation. The rate of ractopamine activation was 85%, and the parameters of ractopamine Immunogen conjugation were as follows: RAC hemisuccinate: EDC: NHS was 1:2:5; activation time was 15 min; the rate of ractopamine and modified bovine serum albumin was 20:1; conjugation time was 2 h; the final molecular rate of ractopamine and modified bovine serum albumin was 14.3; the rate of ractopamine Immunogen conjugation was 71.5%. Ovumalbumin was selected to prepare ractopamine coating antigen by the same parameters. The final molecular rate of ractopamine and ovumalbumin was 7.5, and the rate of the coating antigen was 37.5%.2) Development of ractopamine indirect competitive monoclonal antibody-based enzyme-linked immunosorbent assayractopamine (IC-ELISA) In our study, we reported the development of an indirect competitive ELISA for ractopamine. Mice immunized with ractopamine-modified bovine serum albumin were utilized for monoclonal antibody generation. The parameters were as follows: coating antigen was 0.6μg/mL; 1.18 mg/mL anti- ractopamine monoclonal antibody was diluted by 8000 times; 1 mg/mL goat anti-mouse IgG:HRP was diluted by 5000 times. The characteristics of the RAC indirect competitive ELISA were as follows: sensitivity was 0.1 ng/mL; IC₅₀ was 2 ng/mL; detection range was 0.1-1000 ng/mL; cross-reactivity to four other 6-adrenergic agonists was lower than 0.2‰. The recoveries obtained by spiking standard RAC into the RAC negative urine samples were from 70% to 106.2%.3) Development of ractopamine Colloidal Gold Immuno-ChromatographyAssay (GICA)Monoclonal antibody against ractopamine was used to bind with gold particles, and the prepared ractopamine-ovumalbumin was used as the coating antigen. The parameters of Colloidal Gold Immuno-Chromatography Assay for ractopamine were as follows: size of gold particles was 40 nm; monoclonal antibody against ractopamine labeling with gold particles was 10μg/mL; optical density value of colloidal gold was 5; coating antigen was 0.5 mg/mL. The GICA detection card was assembled by using these parameters. The characteristics of this GICA detection card were as follows: sensitivity was 5 ng/mL; detection range was 0-10000 ng/mL; cross-reactivity of the GICA detection card to four other 8-adrenergic agonists were lower than 5‰. One hundred porcine urine samples were detected. False positive and false negative rates were 0 and 3.2%, respectively. Matainence tests at 60℃in incubator indicated that the detection card was valid after being stored at room temperature over 12 months.In conclusion, two sensitive, specific and rapid immunoassays have been developed that can serve as the screening assay for ractopamine.