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<p>Abstract</p> <p>Background</p> <p>Veterinary drugs such as clenbuterol (CL) and sulfamethazine (SM₂) are low molecular weight (<1000 Da) compounds, or haptens, that are difficult to develop immunoassays due to their low immunogenicity. In this study, we conjugated the drugs to ovalbumin to increase their immunogenicity for antiserum production in rabbits and developed a protein microarray immunoassay for detection of clenbuterol and sulfamethazine. The sensitivity of this approach was then compared to traditional ELISA technique.</p> <p>Results</p> <p>The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC₅₀. Our microarray assay showed the IC₅₀were 39.6 ng/ml for CL and 48.8 ng/ml for SM₂, while the traditional competitive indirect-ELISA (ci-ELISA) showed the IC₅₀were 190.7 ng/ml for CL and 156.7 ng/ml for SM₂. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g) than the ci-ELISA (0.1 ng/g) for detection of CL residues.</p> <p>Conclusions</p> <p>The protein microarrays showed 4.5 and 3.5 times lower IC₅₀than the ci-ELISA detection for CL and SM₂, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique.</p>