Submitted to: Food and Agricultural Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 12, 2010
Publication Date: May 12, 2011
Citation: Shelver, W.L. 2011. Generation of antibody and development of an enzyme-linked immunosorbant assay for the feed additive roxarsone. Food and Agricultural Immunology. 22(2):171-184. Interpretive Summary: This paper describes the development of a new method for measuring roxarsone, a compound added to chicken feed to reduce an infection called coccidiosis, to improve the health of young chickens, or to promote growth. Unfortunately, roxarsone contains arsenic and toxicity can arise either from the roxarsone or conversion to inorganic arsenic, a known toxic material. Once Roxarsone is administered, it can be distributed throughout the chicken’s body including the parts utilized for food. To insure safe food, a method for monitoring roxarsone in chicken meat capable of the large number of samples was needed. The method developed in this project meets these requirements.
Technical Abstract: Roxarsone (3-nitro-4-hydroxy-phenyl arsonic acid) has been used in the poultry and swine industries as a feed additive to treat coccidiosis and other intestinal disorders as well as to improve feed efficiencies and weight gain. In animals, roxarsone is eliminated mostly as parent compound which may subsequently be converted to inorganic arsenic that can leach into ground water or be absorbed by plants causing environmental and food-safety concerns. The goal of this study was to develop an enzyme-linked immuno screening method that allows efficient monitoring of roxarsone in chicken muscle. 3-Amino-4-hydroxy-phenyl arsonic acid (AHPA) was conjugated with keyhole limpet hemocyanin in the presence of bis-sulfosuccinimidyl-suberate which served as an immunogen in rabbits. The rabbit sera was very specific towards roxarsone with minor cross-reactivity toward 2-aminophenyl arsonic acid (7.3%, n=3), arsanilic acid (5.3%, n=3), AHPA (4%, n = 3), and phenyl arsonic acid (3%, n=3). Neither solid-phase nor liquid-liquid extraction resulted in satisfactory recoveries of roxarsone from fortified chicken muscle when a phosphate buffered saline with 0.05% Tween 20 (PBST) calibration curve was used. Muscle extract dilutions up to 1:50 did not produce calibration curves that overlapped with PBST curves based on the OD450nm. The mean IC50s for PBST, muscle 1:10, and muscle 1:20 curves were 11.7 ± 1.50, 13.1 ± 2.53, and 12.0 ± 2.45 ng/mL (n=6). Using a standard curve prepared in chicken muscle extract and diluted with PBST at 1:20, muscle samples fortified with roxarsone at 2.5, 5, 10, and 25 ng/mL had recoveries of 117, 98, 80, and 65 % having coefficients of variation of 36, 9.2, 10.2, and 11.5 respectively (n = 6).