Title: Development of a roxarsone immunoassay for chicken muscle Author
Submitted to: Pacifichem Symposium
Publication Type: Abstract Only
Publication Acceptance Date: May 19, 2010
Publication Date: December 15, 2010
Citation: Shelver, W.L. 2010. Development of a roxarsone immunoassay for chicken muscle. [abstract] International Chemical Congress of Pacific Basin Societies (Pacifichem), December 15-20, 2010, Honolulu, HI. Presentation 265. Technical Abstract: Roxarsone (4-hydroxy-3-nitrophenylarsonic acid) has been used in the poultry and swine industries as a feed additive to improve feed efficiencies and weight gains, and to treat coccidiosis. In animals, roxarsone is eliminated mostly as parent compound, which may subsequently be converted into more toxic forms that can leach into ground water causing environmental concerns. Our goal was to develop an enzyme-linked immunoassay screening method that allows efficient monitoring of roxarsone in chicken muscle. 3-amino-4-hydroxy-phenylarsonic acid was conjugated with keyhole limpet hemocyanin in the presence of bis-sulfosuccinimidyl-suberate to serve as an immunogen for rabbits. The coating antigen was generated in a similar method except using bovine serum albumin as carrier protein. Optimization of the ELISA was performed by varying coating antigen concentration, varying primary and secondary antibodies dilution, and the use of different blocking protein solutions (bovine serum albumin, skim milk, and fish gelatin). Under optimized conditions, the sera was very specific towards roxarsone with minor cross-reaction toward arsanilic acid (3.2%, n=3) and 3-amino-4-hydroxy-phenylarsonic acid (4.2%, n = 3). Use of SPE or liquid-liquid extraction did not result in satisfactory recovery of spiked roxarsone from muscle when a PBST calibration curve was used. Muscle extract dilutions up to 1:50 did not produce calibration curves that overlapped with PBST. At muscle extract dilutions of 1:20, the IC50s were similar to the PBST curve, while at 1:10 dilutions the IC50s were slightly higher than those of PBST or 1:20 dilutions. The mean IC50s for PBST, muscle 1:10, and muscle 1:20 were 11.7, 13.1, and 12.0 ng/mL while the OD450nm were 1.61, 0.87, and 0.99 respectively (n = 6). A matrix matched standard curve with chicken muscle extract, diluted with PBST at 1:20, was spiked with roxarsone at 2.5, 5, and 10 ng/mL with spiked recoveries of 112, 97, and 78% with % CV at 40, 9.9, and 10.6 respectively (n = 5).