Rapid and sensitive screening of sulfamethazine in porcine urine with an enzyme-linked immunosorbent assay and a field-portable immunofiltration assay.
An enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunofiltration assay (ELIFA) were developed for the screening of sulfamethazine (SMZ) in porcine urine. Incurred urine samples were measured by ELISA with a working range of 0 to 10 ng of SMZ per ml. The assay showed good accuracy and precision, with recoveries above 99.8% and intra- and interassay coefficients of variation (CVs) ranging from 2.6 to 5.6% and from 5.9 to 12.7%, respectively. Good agreement was observed when the results of the immunoassay were compared with those of liquid chromatography/tandem mass spectrometry analysis. For the ELIFA, a nylon membrane is placed on top of an absorbent material and covalently coated with rabbit anti-rat immunoglobulins. Free binding sites are blocked, and monoclonal anti-SMZ antibodies, SMZ standard or urine, and SMZ-horse radish peroxidase conjugate are subsequently dropped onto the membrane. During the assay, the reactants are drawn through the membrane because of its close contact with the absorbent pad. Finally, a substrate solution is added for blue color development. The blue spot produced can be visually evaluated or instrumentally measured (numeric deltaE*ab value), and the intensity of its color is inversely proportional to the analyte concentration. When a blue dot appears on the membrane, even if its color is less intense than that of the negative control, the sample is considered "negative," i.e., it is thought to contain a concentration of SMZ that is below the visual detection limit. If no color appears on the test membrane, the sample is considered "positive," i.e., it is thought to contain a concentration of SMZ that is equal to or above the visual detection limit. Validation of the assay showed good inter- and intra-assay precision (CV < 10%). Because samples can be analyzed after a simple dilution in <30 min with this assay format, it has strong potential for application in the field.
Crabbe
Patricia
P
Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Belgium. patricia.crabbe@rug.ac.be
Van Peteghem
Carlos
C
D000818:Animals; D000890:Anti-Infective Agents / Q000032:analysis / Q000652:urine*; D002853:Chromatography, Liquid / Q000662:veterinary; D004350:Drug Residues / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005374:Filtration; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013418:Sulfamethazine / Q000032:analysis / Q000652:urine*; D013552:Swine
D000890:Anti-Infective Agents; D013418:Sulfamethazine