Community Research and Development Information Service - CORDIS

Screening by chemiluminescence immunoassay

Synthetic corticosteroids, are widely used in live-stock breeding. After application of such corticosteroids a delay time of 48 hours for milk and 72 hours for meat is prescribed. Corticosteroids are found in cattle feed and are suspected to be used as growth-promotors. In all European Community countries however, the use of growth promoters has been banned since 1986. During this project we have developed an immunoassay for a number of corticosteroids. This CORTICOSTEROID-EIA is a competitive enzyme immunoassay for the screening of urine samples on the presence of corticosteroids like dexamethasone, betamethasone, flumethasone and triamcinolone. This EIA system uses antibodies raised against protein conjugated dexamethasone. With this EIA-kit 96 analyses can be performed. Samples and standards are measured in duplicate, which means that in total 40 samples can be analysed. The EIA kit contains all the reagents, including standards to perform the test. Chemicals for the SPE and preparation of the urine samples are not included.

This test makes no distinction between the different corticosteroids and can therefore only be used as a screening method. For the confirmation of the presence and the identification of the specific steroid, more specific analytical methods (HPLC or GC-MS) are recommended.

PRINCIPLE OF THE DEXAMETHASONE EIA:
The microtiter plate based EIA kit consists of 12 strips, each 8 wells, precoated with sheep antibodies to rabbit IgG. In one incubation step, specific antibodies (rabbit anti-dexamethasone), enzyme labelled dexamethasone (enzyme conjugate) and dexamethasone standards or samples are added to the precoated wells. The specific antibodies are bound by the immobilised anti-rabbit antibodies and at the same time dexamethasone (in the standard solution or in the sample) and enzyme labelled dexamethasone compete for the specific antibody binding sites (competitive enzyme immunoassay).

After an incubation time of one hour at 37C, the non-bound (enzyme labelled) reagents are removed in a washing step. The amount of bound enzyme conjugate is visualized by the addition of substrate chromogen (tetramethylbenzidine, TMB). Bound enzyme transforms in 30 minutes the chromogen substrate into a coloured product. The substrate reaction is stopped by the addition of sulphuric acid. The colour intensity is measured photometrical at 450 nm and is inversely proportional to the steroid concentration in the standard solution or the sample. Total time to perform the assay for 25 samples, including sample preparation is one day.

SPECIFICITY AND SENSITIVITY:
The dexamethasone EIA utilizes antibodies raised in rabbits against protein conjugated dexamethasone. The calibration curve is linear between 0.125 to 2 ng/ml of dexamethasone.

Cross-reactions:
- Flumethasone 179%;
- Dexamethasone 100%;
- Betamethasone 55%;
- Triamcinolone 49%;
- Methylprednisolone 8%;
Cross-reactivities were determined in the competitive EIA and calculated at 50% inhibition.

A sample preparation method with glucuronidase treatment and solid phase extraction (SPE)to minimise the matrix effect of urine samples is included.

A sample preparation method was also developed for the analysis of corticosteroids in meat using an organic extracion and SPE. This has been validated in LCMS-MS but is also applicable in the EIA.

The EIA can be implemented in a slaughtering house environment using urine samples or pre-urines obtained from the slaughtered animals. A simple laboratory instrumentation is necessary to perform the sample preparations and the EIA which contains easy and ready to use reagents. Some laboratory skills are however requested.

Related information

Contact

Piet VAN WICHEN, (Manager R&D)
Tel.: +31-26-3630364
Fax: +31-26-3645111
E-mail
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