Methods

The new time-resolved fluorescence based label technology (Fig. 1.; click to enlarge) employing intrinsically fluorescent lanthanide chelates provides a very high sensitivity and a stable chemistry suitable for simple automation (3). According to the technology a delayed fluorescent signal originating from the lanthanide chelate labelled component is measured once the background fluorescence has declined. This results in a high signal-to-noise -ratio because the background fluorescence present in the prompt fluorescence in avoided in the measurement. The fluorescent signal can be measured directly from the solid-phase surface without any signal enhancement step (4).

In the present project the power of time-resolved fluoroimmunoassay (TR-FIA) technology is combined with a novel assay format using the one-step all-in-one -well concept (Fig. 2.; click to enlarge) (5). This enables the development of fast veterinary drug residue immunoassays in which all assays are performed in microtitration wells that contain all the assay specific reagents in dry form. An analyser developed for rapid assays in the clinical field will be used in the project and the immunoassay design allows both non-competitive and competitive assays to be carried out.
The competitive assay format contains an analyte specific antibody immobilised onto the surface of the well and the labelled competing analyte is dried in a small volume on top of the insulating layer. The one-step all-in-one dry reagent immunoassay concept is extremely simple for the end-user because only the addition of a common assay solution and the sample is needed. The same system can be extended to also cover assays for other analytes of interest in the field.

Confirmatory methods have already been developed to support the results of the screening assays (Fig. 3.; click to enlarge. Revised EU criteria for the applicability of analytical methods maintains the current position of the European Commission in: a) expressing a preference for the use of mass spectrometric methods in the confirmation of compounds with an MRL and b) an obligation to use of mass spectrometric methods in the Reference analysis of compounds listed in Annexe IV of Council Regulation 2377/90. LC-MS(/MS) techniques have been used in the development of new methods (6) and in the improvement of existing chemical procedures which detect residues of the drugs included in the project.

The results of the project are going to be presented in Technology Transfer Events. If you are interested in attending these training events, send an e-mail to mika.tuomola@utu.fi and we will keep you informed about the forthcoming meetings.

References

3. Lövgren, T., Meriö, L., Mitrunen, K., Mäkinen, M-L., Mäkelä, M., Blomberg, K., Palenius, T., Pettersson, K. 1996. One-step all-in-one dry reagent immunoassays with fluorescent europium chelate label and time-resolved fluorometry. Clin. Chem. 42, 1196-1201
4. Lövgren, T., Heinonen, P., Lehtinen, P., Hakala, H., Heinola, J., Harju, R., Takalo, H., Mukkala, V-M., Schmid, R., Lönnberg, H., Pettersson, K., Iitiä, A. 1997. Sensitive bioaffinity assays with individual microparticles using time-resolved fluorometry. Clin. Chem. 43, 1937-1943
5. Tuomola, M., Cooper, K.M., Lahdenperä, S., Baxter, G.A., Elliott, C.T., Kennedy, D.G., Lövgren, T. 2002. A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle. Analyst 127, 83-86
6. Yakkundi, S., Cannavan, A., Elliott, C.T., Lovgren, T., Kennedy, D.G. 2001. Development and validation of for the confirmation of nicarbazin in chicken liver and eggs using LC-electrospray MS-MS according to the revised EU criteria for veterinary drug residue analysis. Analyst 126, 1985-1989