Investigation of dissociation enhanced lanthanide fluoroimmunoassay as an alternative screening test for veterinary drug residues

Abstract

Both the number of drugs used in veterinary medicine and the diversity of procedures employed to detect their residues are ever increasing. Many laboratories that carry out such testing have employed a variety of immunoassays to serve as rapid screening tests. Owing to the high sensitivity, good reproducibility and availability of multi-analyte assays dissociation enhanced time-resolved fluorescence immunoassay (dissociation enhanced lanthanide fluoroimmunoassay, DELFIA) kits have become a market leader in the human clinical field. No commercial DELFIA kits are presently available for veterinary drug analysis.

A DELFIA method was developed for the quantitative analysis of residues of medroxyprogesterone (MP) in bovine bile. Commercially available DELFIA reagents were used in a microtitre assay format. Within- and between-assay s_r values (relative standard deviation) were 4.2–9.9 and 5.1–8.5%, respectively. Detection limits were calculated (mean + 3 s of a known negative population) as being 0.52 and 4.91 µg l^–1 for male and female animals, respectively.

A second study was carried out to optimize the labelling of haptens with a range of lanthanide metals. Purification of the hapten–lanthanide conjugates produced was achieved using fast protein liquid chromatography. Sensitive standard curves were produced for nortestosterone and diethylstilbestrol (curve mid-points of 30 and 40 pg, respectively). By using two different lanthanide labels (Eu and Tb) the simultaneous measurement of these compounds in a single assay system was achieved. Use of a third lanthanide label (Sm) yielded a product of low signal intensity and sensitivity.