Immunochemical determination of oxytetracycline in fish: comparison between enzymatic and time-resolved fluorometric assays.
An indirect competitive enzyme-linked immunosorbent assay (ELISA) with photometric detection of horseradish peroxidase (HRP) activity, was developed in plate to detect oxytetracycline (OTC) in Gilthead sea bream (Sparus aurata) samples. The results were compared to those obtained by time-resolved fluoroimmunoassay (TR-FIA) using a secondary antibody with coproporphyrin of platinum (II) (PtCP) as marker. The limits of detection obtained in fish extract were 16 and 0.08 microg kg(-1) for photometric and fluorometric detections, respectively; therefore, they were suitable for fish quality control according to the maximum residue level established by the European Union. An extraction procedure using methanol:water 70:30 (v/v)+1 mL EDTA 0.1 M, and different clean-up procedures based on solid-phase extraction (C(18), polymeric reversed phase, SCX, Si) was assayed. The matrix effects were overcome by means of an average tetracycline-free fish extract calibration curve used for quantification. The OTC optimized ELISA can also be applied to determine tetracycline and chlortetracycline residues with good results. Thus, the developed immunoassay could be considered as a generic assay for the most used tetracyclines in aquaculture antibiotic treatments. In order to confirm the utility of the developed immunoassay as a semi-quantitative methodology, fish samples obtained from different supermarkets were analyzed. Results correlate well with those obtained with a reference HPLC method.
Cháfer-Pericás
Consuelo
C
Instituto de Reconocimiento Molecular y Desarrollo Tecnológico, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
Maquieira
Angel
A
Puchades
Rosa
R
Miralles
Javier
J
Moreno
Amelia
A
Pastor-Navarro
Nuria
N
Espinós
Francisco
F
D000818:Animals; D002751:Chlortetracycline / Q000032:analysis; D004350:Drug Residues; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005399:Fishes; D057230:Limit of Detection; D010118:Oxytetracycline / Q000032:analysis*; D013752:Tetracycline / Q000032:analysis / Q000737:chemistry; D013754:Tetracyclines / Q000032:analysis*
D013754:Tetracyclines; D013752:Tetracycline; D002751:Chlortetracycline; D010118:Oxytetracycline