Application of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Determination of Ractopamine in Incurred Samples from Food Animals

Abstract

A monoclonal antibody-based ractopamine immunoassay has been applied to incurred samples from sheep and cattle. Results obtained by immunoassay were compared with those from high-performance liquid chromatography (HPLC). Three sets of sample extracts containing primarily unmetabolized ractopamine were analyzed. Correlation of HPLC with enzyme-linked immunosorbent assay (ELISA) for beef liver samples gave an r(2) = 0.98 despite rather low ractopamine concentrations (range 1.1-13.4 ng/mL, n = 6). Ractopamine concentrations in cow urine samples treated by solid phase extraction, to remove ractopamine metabolites, also showed a high correlation between the HPLC and the ELISA results (r(2) = 0.95, range 1.0-275 ng/mL, n = 61). In contrast, HPLC and ELISA analyses of ractopamine in sheep urine were not well-correlated (r(2) = 0.58, range 0.85-51 ng/mL, n = 34). When ractopamine conjugates in urine samples were hydrolyzed with hydrolytic enzymes, ELISA and HPLC methods were highly correlated [r(2) = 0.94 for sheep (range 123-10 554 ppb, n = 60) and an r(2) = 0.98 for cattle (range 14-8159 ppb, n = 62)]. Tissues contained only minute amounts of ractopamine, and after 7-day withdrawal periods, less than 1 ppb of free ractopamine was detected. Ractopamine was rapidly metabolized in both cattle and sheep. The difference in ractopamine concentration of urine samples before and after hydrolysis indicated that only 1-5% of ractopamine was excreted unmetabolized. Results from this study indicate that the monoclonal antibody-based ELISA could be useful for a sensitive, quantitative, or qualitative ractopamine screening assay.

Substitution of Antibody with Molecularly Imprinted Film in Enzyme-Linked Immunosorbent Assay for Determination of Trace Ractopamine in Urine and Pork Samples

“…The limit of detection, namely IC 15 , calculated as the concentration of standard solution causing 15% inhibition of color development, was 0.01 μg L −1 . Compared with the literature (IC 15 , 0.76 μg L −1 ; IC 50 , 5.1 μg L −1 ), the assay IC 15 for RAC was lower than that obtained using monoclonal antibody, though the IC 50 was three times higher (Shelver and Smith 2002). This might be attributed to the specific method for preparation of the imprinted film, which seemed to result in a higher number of binding cavities being situated at or close to the film surface and thus accessible for RAC.…”

“…(Wang et al 2006b;Shelver and Smith 2004;Zhang et al 2009;Shelver and Smith 2002;Haasnoot et al 1994;Wicker et al 1995;Shelver et al 2000) The traditional liquid chromatography (LC), gas chromatography (GC) method or coupled with mass spectrometry (MS) were common methods because they could offer low detection limit and good accuracy and reproducibility, but expensive instruments and skilled operators were usually needed. Immunoassays, especially enzyme-linked immunosorbent assay (ELISA), have become a practical method for analysis due to its high-throughput, user-friendliness, and field portability.…”

“…Immunoassays, especially enzyme-linked immunosorbent assay (ELISA), have become a practical method for analysis due to its high-throughput, user-friendliness, and field portability. These important characteristics made immunoassays an attractive tool for trace analyte testing by regulatory agencies, though there were still some disadvantages existed in this mature approach (Shelver and Smith 2002). The reagent stability, difficulties associated with antibody production, together with the need to use laboratory animals were often considered as problems (Urraca et al 2007).…”

Molecularly imprinted polymer for the determination of trace ractopamine in pork using SPE followed by HPLC with fluorescence detection

“…The mixture was shaken (180 times/min) for different times (5,15,30,60,90, and 120 min) at room temperature and then separated centrifugally (4000 rpm) for 5 min. The unbound RAC in the supernatant was measured by UV spectrometry.…”

“…At present, RAC is commonly detected by HPLC [7,8], GC-MS [9,10], LC-MS [5, 11 -13], CE [14], and ELISA [15,16]. In the sample preparation step, the most common techniques are liquid -liquid extraction and SPE [1,5,7].…”