Cor1p.a5final.doc

CORTICOSTEROID EIA
A microtiter plate based enzyme immunoassay
for the screening of milk, urine, tissue and feed samples on the presence
of Corticosteroids
Dexamethasone,
Betamethasone,
Flumethasone,
Triamcinolone,
Prednisolone.
BRIEF INFORMATION
11. LITERATURE
The CORTICOSTEROID-EIA is a competitive enzyme immunoassay for the screening of milk, urine, tissue (liver and muscle) and feed samples on the presence of corticosteroids like 1. Council of the European Communities, Council Directive 86/469/EEC of 16 Septem- dexamethasone, betamethasone, flumethasone, prednisolone 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 2. Brunn. H and Georgii, S. Identification and quantification of dexamethasone and in total 40 samples can be analyzed. The EIA kit contains all the reagents, including standards related xenobiotic corticosteroids in cattle urine with ELISA and HPLC/ELISA. to perform the test. Chemicals for the preparation of tissue and feed extracts are not included. Archiv für Lebensmittel Hygiene 45 (4), 1994, 96. 3. Council of the European Communities; Council Directive 1441/95 of June 26, 1995. After homogenisation and defatting, (diluted) milk can be applied directly to the test. The detection limits in milk are 0.15 ng/ml for dexamethasone, betamethasone and 4. P. Stouten, W. Haasnoot, G. Cazemier, P. Berende and H. Keukens; flumethasone, 0.45 ng/ml for triamcinolone and 0.75 ng/ml for prednisolone.(4) Immunochemical detection of corticosteroids in milk, liver, kidney and muscle. Proceedings Euro-Residue III 1996 ed. N. Haagsma, A. Ruiter, p. 902. Urine diluted in a buffer, can be applied directly to the test or after on HPLC-fractionation (2). The detection limits in urine range from 5-20 ng/ml (depending of the corticosteroid 12. ORDERING INFORMATION
For ordering the Corticosteroid EIA kit, please use cat. code 5081COR1p [3]. For feed samples, two extraction procedures are described: Procedure I- A simple extraction procedure using a mixture of water and acetonitrile only. Procedure II - A more extended extraction procedure including a three phases extraction step. The total time required to perform the test, including preparation (with Procedure I) of up to 40 samples, is about 3-4 hours (including a 1 hour EIA incubation step). Using Procedure II the total time will be about 7-8 hours for screening up to 40 samples. The detection limits of the test in feed samples are about: Tissue samples, liver or muscle, can be applied to the test after a three phase extraction and cleaning procedure. The detection limits are: (bovine) liver 1 ng/g, (calf) muscle 0,2 ng/g. 1. INTRODUCTION
Synthetic corticosteroids, are widely used in live-stock breeding. After application of The amount of dexamethasone and cross-reacting compounds in the tissue sample are such corticosteroids a delay time of 48 hours for milk and 72 hours for meat is expressed as dexamethasone equivalent concentration (ng/g). The dexamethasone prescribed. Recently the E.U. established MRL's for dexamethasone in milk, equivalents (ng/g of tissue) corresponding to the % maximal OD of each sample can be read muscle/kidney and liver of 0.3; 0.5 and 2.5 µg/kg, repectively [3]. Corticosteroids are directly from the calibration curve (no dilution applied). 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 "POSITIVE" SAMPLES FOUND WITH THIS EIA HAVE TO BE CONFIRMED ON THE PRESENCE OF CORTICOSTEROIDS BY A MORE SPECIFIC METHOD With this EIA kit is possible to screen milk, urine, tissue and feed samples in a short time for the presence of corticosteroids like dexamethasone, betamethasone, flumethasone and other cross-reacting corticosteroids. 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- 2. 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 feed extract) and enzyme labelled dexamethasone compete for the specific antibody binding sites (competitive enzyme After an incubation time of one hour, 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 the chromogen into a coloured product. The substrate reaction is stopped by Figure 1: Example of a calibration curve
the addition of sulphuric acid. The colour intensity is measured photometrically at 450 nm and is inversely proportional to the steroid concentration in the standard solution or 3. SPECIFICITY AND SENSITIVITY
10. INTERPRETATION OF RESULTS
The dexamethasone EIA utilizes antibodies raised in rabbits against protein conjugated Substract the mean OD value of the wells A1 and A2 from the individual OD of the dexamethasone. As shown in figure 1, the calibration curve is linear between 0.125 to 4 wells containing the standards and the samples. The OD values of the six standards and the samples (mean values of the duplicates) are divided by the mean OD value of the zero standard (wells B1 and B2) and multiplied by 100. The zero standard is thus equal to 100% (maximal OD) and the other OD values are quoted in percentages of the ------------------------------------------------- X 100 = % maximal OD The values (% maximal OD) calculated for the standards are plotted (on the linear Y- axis) versus the dexamethasone equivalent concentration (ng/ml) on a logarithmic X- axis. The calibration curve should be linear in the 0.125 - 4 ng/ml range. The amount of dexamethasone and cross-reacting compounds in the milk sample are expressed as dexamethasone equivalent concentration (ng/g). The dexamethasone equivalents (ng/g of milk) corresponding to the % maximal OD of each sample can be read directly from the calibration curve (no dilution applied). Cross-reactivities were determined in the competitive EIA and calculated at 50% inhibition. REMARK: Although not tested, the antibodies will probably show high cross-reactivities
Using the direct method (7.2.1), the dexamethasone equivalents have to be multiplied with dexamethasone-21 and betamethasone-21 derivatives. Using HPLC-fractionation, (7.2.2) the multiplication factor depends on the amount of urine injected into the HPLC and on the dilution applied after fractionation. 4. HANDLING AND STORAGE
- Store the kit at + 2EC to + 8EC in a dark place. The amount of dexamethasone and cross-reacting compounds in the feed samples are - After the expiry date (see kit label) has passed, it is no longer possible to accept any expressed as dexamethasone equivalent concentration (ng/g). The dexamethasone equivalents (ng/g of feed) corresponding to the % maximal OD of each sample can be - It is advised to unpack the sealed microtiter plate, reconstitute or dilute the kit components, immediately before use and after equilibration of kit to room temperature. Using Procedure I for sample preparation, these dexamethasone equivalents have to be multiplied by 500 (0.002 g of feed/ml extract). - After the lyophilized kit components have been reconstituted, the antibody components are only guaranteed for 4 weeks (stored at + 2EC to + 8EC in the dark). Using Procedure II for sample preparation, these dexamethasone equivalents have to be It is advised to aliquot the conjugate and store at -20EC for prolonged storage. multiplied by 100 (0.01 g of feed/ml final extract). - Any direct action of light on the chromogen solution should be avoided. Using Procedure I for sample preparation: Feed samples are "positive" if the dexamethasone equivalent is > 100 ng/g.
If the following phenomena are observed, this may indicate a degradation of the reagents: A blue colouring of the chromogen solution before putting it into the wells, Using Procedure II for sample preparation: A weak or absent colour reaction of the first standard (0-standard) (E450nm < 0.6). Feed samples are "positive" if the dexamethasone equivalent is > 20 ng/g.
5. KIT CONTENTS
Reconstitute the vial of lyophilized antibodies with 3 ml dilution buffer, mix thoroughly and The reagents included in the kit are sufficient to carry out at least 96 analyses keep in the dark until use. Store the vial immediately after use in the dark at +2EC to +8EC. (including standard analyses). Each standard and sample is analyzed in duplicate. 9. ASSAY PROCEDURE
1 sealed dry microtiter plate (12 strips, 8 wells each), coated with antibodies to rabbit IgG, ready to use. Do not wash! In EIA's, between each immunological incubation step, unbound components have to be removed efficiently. This is achieved by apppropiate rinsing. It should be clear that each 6 vials containing (4, 2, 1, 0.5, 0.25, 0.125 ng/ml) standard solutions of rinsing procedure must be carried out with care to guarantee inter- and intra-assay results. Basicly manual rinsing or rinsing with automatic plate washing equipment can be done as 1 vial containing lyophilized conjugate (peroxidase conjugated to dexamethasone, 1 vial containing lyophilized antibodies (anti-dexamethasone; gold cap), 1. Empty the content of each well by turning the microtiter plate upside down followed by a 1 vial containing the Substrate solution, ready to use (12 ml), 2. Fill all the wells to the rim (300 µl) with washing solution. 3. This rinsing cycle (1 and 2) should be carried out 3 times. 1 vial containing dilution buffer pH 7, ready to use (20 ml, white cap), 4. Turn the plate upside down and empty the wells by a firm short vertical movement. 5. Place the inverted plate on absorbent paper towels and tap the plate firmly to remove 1 vial containing 10 times concentrated rinsing buffer (60 ml), 6. Take care that none of the wells dry out before the next reagent is dispensed. 1 vial containing stop solution, ready to use (15 ml, red cap). Rinsing with automatic microtiter plate washing equipment When using automatic plate washing equipment, check that all wells can be aspirated 6. PRECAUTIONS
completely, that the washing solution in correctly dispensed reaching the rim of each well during each rinsing cycle. The washer should be programmed to execute three rinsing cycles. The stop solution contains 0.5 M sulphuric acid. Do not allow the reagent to get Assay Protocol
Avoid contact of all biological materials with skin and mucous membranes. Prepare samples according to section 7 and prepare reagents according to section 8. All standards and samples should be simultaneously tested in duplicate. Microtiter plate is ready for use, do not wash! Do not eat, drink, smoke, store or prepare foods, or apply cosmetics within the 1. Pipet 100 µl of dilution buffer in duplicate (well A1, A2). 2. Pipet 50 µl of dilution buffer in duplicate (well B1, B2). TMB is toxic by inhalation, in contact with skin and if swallowed; observe care 3. Pipet 50 µl of standard solution in duplicate (well C1, C2 to, H1, H2). 4. Pipet 50 µl of each sample solution in duplicate to the remaining wells of the microtiter plate Do not use components past expiration date and do not intermix components from 5. Pipet 25 µl of conjugate (DEX-HRPO) to all wells, except wells A1 and A2. 6. Pipet 25 µl antibody solution to all wells, except wells A1 and A2. 7. Seal the microtiter plate and shake the plate for 1 min. Each well is ultimately used as an optical cuvette. Therefore, do not touch the 8. Incubate for 1 hour in the dark at room temperature (20-25EC). undersurface of the wells, prevent damage and dirt. 9. Discard the solution from the microtiter plate and wash 3 times with rinsing buffer. 10. Pipet 100 µl substrate solution into each well. Optimal results will be obtained by strict adherence to this protocol. Careful 11. Incubate 30 min. at room temperature (25 EC) in the dark. pipetting and washing throughout this procedure are necessary to maintain 12. Add 100 µl stop solution to each well. 13. Read the OD values immediately at 450 nm. 7. SAMPLE PREPARATION
To 2 g of the minced muscle or tissue samples, 10 ml of a mixture of acetonitrile/water (7:3; v/v) are added. After mixing (Vortex) for 1 min, the Milk samples are analysed directly after defattening (centrifugation for 10 min at samples are shaked (head over head) for 1 h. After centrifugation (15 min at 2.000 rpm at 4EC). Of the (defatted) milk, 50 µL were pipetted in the microtite plate. 10.000 rpm at 4EC), 2.5 ml of the supernatant is pipetted in a test tube and 4 ml of hexane and 1 ml of dichloromethane are added. After mixing (Vortex) for 1 min and centrifugation (5 min at 2.000 rpm), 1 ml of the middle phase (corresponding to 0.2 g of sample) is evaporated to dry at 50EC under a stream of nitrogen and the residue is dissolved in 200 µL To 100 µl of urine, 4.9 ml of buffer (Phosphate Buffer/Tween*) are added. Phosphate Buffer/Tween*(1 g of sample/ml) of which 50 µL were pipetted in To 1 ml of urine, 6 ml of a 50 mM Sodiumacetate buffer (pH 4.8) and 16 µl Helix pomatia juice are added. The mixture is incubated for 2 hr at 37EC. Bring the mixture to room temperature and extract (by shaking or 10 min) with successively one portion of 10 ml and two portions of 5 ml ethylacetate. Separate the organic layer by centrifugation (8 min. 3000 rpm and 4EC). Collect the organic layers and evaporate at 50EC (Rotavapor). Dissolve the residue in 500 µl of methanol/water (55/45 v/v) and filter the solution (0.45 µ filter). Inject 100 µl in an HPLC (see method described by Brunn. and Georgii 2). HPLC fractions (each 260 µl) are collected and to each fraction, 4.74 ml of Phosphate 8. PREPARATION OF REAGENTS
Before starting the test, equilibrate reagents to room temperature (20-25 EC). Any reagent not used should be put back into storage immediately at 2-8EC. To 1 g of the grinded feed sample 3 ml water and 7 ml acetonitrile (Merck 3) are added. After shaking for 15 min and 5 min waiting, 100 µl of the clear liquid is pipetted into a tube and 4.9 ml of Phosphate Buffer/Tween* is added Return unused strips into plastic bag, seal bag with dessicant and store at 2-8 EC for use in subsequent assays. Retain also the strip-holder. Of the clear water/acetonitrile extract obtained with procedure I, 2.5 ml are pipetted into a 10 ml tube. 1 ml Dichlormethane (Merck 6050) and 4 ml hexane (Merck 4367) are added. Shake for 1 min and allow the 3 phases to separate. The rinsing buffer is delivered 10 times concentrated. Prepare dilutions freshly before Take 100 µl of the middle phase and evaporate to dryness. Add 1 ml use. Per strip 40 ml of diluted rinsing buffer is used (4 ml concentrated rinsing buffer + Phosphate Buffer/Tween* to the residu and mix on vortex [0.01 g feed/ml The substrate solution (ready to use) precipitates at 4EC. Take care that this vial is at room temperature (keep in the dark) and mix the content before pipetting into the Reconstitute the vial of lyophilized conjugate (DEX-HRPO) with 3 ml dilution buffer, mix thoroughly and keep in the dark until use. Store the vial immediately after use in the dark at +2EC to +8EC. For prolonged storage aliquot and refrigerate at -20 C.

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