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1. Product Description

Clenbuterol belongs to the group of ß-agonists. It has been known that ß-agonists are suitable for use as performance improvers within the field of livestock production; in particular, the meat/fat ratio in fattened animals can be improved or the growth accelerated. However, such compounds have not been authorized as fattening adjuvants up to now.This ELISA KIT is a competitive enzyme immunoassay for the quantitative analysis of clenbuterol in muscle, liver, urine.

2. Test principle

The basis of the test is the antigen-antibody reaction. The microtiter wells are coated with sheep anti- clenbuterol antibodies. Free clenbuterol enzyme conjugate compete for the clenbuterol antibody binding sites. At the same time, the anti- clenbuterol antibodies are also bound by the immobilized capture antibodies. Any unbound enzyme conjugate is then removed in a washing step. Substrate/chromogen is added to the wells and incubated. Bound enzyme conjugate converts the chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm; the absorption is inversely proportional to the clenbuterol concentration in the sample.

3. Kit Contents

Each kit contains sufficient materials for 96 measurements (including standard analyses). Each test kit contains:

1 x Microtiter plate with 96 wells

Clenbuterolstandard solutions(1ml)：0、0.25ppb、0.75ppb、2.25ppb、6.75ppb、20.25ppb

16 × HRP-Conjugated Antibody................................................................................0.9 ml

HRP-Conjugated Antibody Dilution buffer................................................................15 ml

TMB Buffer A............................................................................................................. 7 ml

TMB Buffer B........................................................................................................... 7 ml

Stop Buffer............................................................................................................ 7 ml

10 × Washing Buffer...................................................................................................50 ml

4. Required Materials Not Provided With the Kit

(1) Equipment:

- Microtiter plate reader (450 nm)

- Vortex mixer

- 20, 100 and 1000 μl pipettes

- Multi-channel pipette: 50-300μl

- Incubator

- Centrifuge

- Balance

- Vibrator

- Volumetric flask: 100ml，500ml，1000ml

(2) Reagents:

- Trichloroacetic acid

- NaOH

- Tert-butyl ether

- Concentrated HCl

- Tris

- Methanol

- Acetonitrile

- Ethyl acetate

- H₃PO₄

(3) Solution preparation

- 3% Trichloroacetic acid solution: Mix 3.0g Trichloroacetic acid and deionized water to 200ml.

- 1 M HCl solution: Mix 8.6ml concentrated HCl and water to 100ml.

- 1 M NaOH solution: Mix 4.0 g NaOH and water to 100ml.

- 0.05 M HCl solution: Mix 0.43 ml concentrated HCl and water to 100ml.

- 0.09 M Tris solution: Mix 1.09 g Tris and water to 100ml.

- Washing buffer: Mix 1 volume 10× Washing Buffer with 9 volume of distilled water.

- HRP-Conjugated antibody working buffer: Mix 1 volume 16×HRP-Conjugated antibody with 15

volume of HRP-Conjugated Antibody Dilution buffer.

5. Sample Preparation

(1) Urine

- Detect urine sample directly. If the urine was turbid, please centrifuge.

Note: Dilution factor:1.

(2) Liver, kidney, meat or tissue with low fat content

− Homogenize 2g of sample with a mixer and transfer it into a centrifugal vial. Add 10ml 3%

Trichloroacetic acid solution. Mix for 10min.

− Centrifuge: 5 min / 3000 g or at a higher speed.

− Keep 5ml upper layer. Use 1 M NaOH adjust the pH to 9-11(about 1.5ml 1M NaOH )

− Add 10ml Tert-butyl ether. Mix.

− Centrifuge: 5 min / 3000 g or at a higher speed.

− Use 5ml upper layer, and evaporate by nitrogen-blow.

− Use 0.5ml 0.05M HCl to dissolve the residue. Add 0.5 ml 0.09M Tris. Use 30μl in the assay.

Note: Dilution factor:1.

(3)Meat and tissue with high fat content

− Homogenize 3g of sample with a mixer and transfer it into a centrifugal vial. Add 7.5ml 3%

Trichloroacetic acid solution. Mix for 10min.

− Centrifuge: 5 min / 3000 g or at a higher speed.

− Keep 5ml upper layer. Use 1 M NaOH adjust the pH to 9-11(about 0.7ml 1M NaOH )

− Centrifuge: 5 min / 3000 g or at a higher speed.

− Use 5ml upper layer, and evaporate by nitrogen-blow.

− Use 0.5ml 0.05M HCl to dissolve the residue. Add 0.5 ml 0.09M Tris. Use 30μl in the assay.

Note: Dilution factor:1.

6. ELISA Testing protocol

(1) Insert a sufficient number of wells into the microwell holder for all standards and samples to be run in duplicate. Record standard and sample positions.

(2) Add 30μl standard solution or sample solution. Then add 100μl of HRP-conjucated antibody buffer and incubate for 30 min at room temperature (about 26 °C).

(3) Pour the liquid out of the wells and tap the microwell holder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. Fill all the wells with 250 μl washing buffer and pour out the liquid again. Repeat four more times.

(4) Add 100 μl of TMB Buffer (mix TMB buffer A 1:1 with TMB buffer B) to the bottom of each well. Incubate for 15 min at 37℃ in the dark.

(5) Add 50 μl of the stop solution to each well. Mix gently by shaking the plate manually and measure the absorbance at 450 nm. Read within 30 minutes after addition of stop solution.

7. Results

A standard curve can be constructed by plotting the mean relative absorbance (%) obtained from each refernce standard against its concentration in ng/ml on a logarithmic curve.

Use the mean relative absorbance values for each sample to determine the corresponding concentration of the tested drug in ng/ml from the standard curve. A special program with Excel functionality, Triphil ELISA test results. Please contact your local distributor or [email protected]

In order to obtain the clenbuterol concentration in ppb actually contained in a sample, the concentration read from the calibration curve must be further multiplied by the corresponding dilution factor.

8. Detection limit and Recovery rate

(1) Kit sensitivity: 0.25ppb

(2) Detection limit：

Urine…………………………………………………………………………………1ppb

Tissue (liver, kidney or meat) …………………………………………………………1ppb

(3) Recovery rate：

Urine……………………………………………………………………………100±15%

Tissue (liver, kidney or meat) …………………………………………………75±15%

9. Specificity

The specificity of the ELISA Kit test was determined by analyzing the cross-reactivities to corresponding substances.

Clenbuterol ………………………………………………………………………100%

Terbutalin ……………………………………………………………………… ≤2%

Salbutamol ………………………………………………………………………≤4%

Isoproterenol ………………………………………………………………………≤2%

Adrenalin ………………………………………………………………………<2%

Noradrenalin …………………………………………………………………<2%

10. Warnings and Precautions

--Make sure plates and reagents are brought up to room temperature. Keep the kit components out of the kit box for a least 1hour before starting the assay.

-- Shake up the reagent before using it.

--Carefully label each reagent to make sure the reagents are not intermixed. Kits with different expiration dates might generate different range of OD readings.

--In general, a value of less than 0.6 in zero standard reading may indicate certain degrees of deterioration of reagents.

--Incubate at 37℃, too high or too low temperature may generate different range of OD readings. And avoid sunlight beaming directly.

--The stop buffer is 1M H₂SO₄, Please caution.

--The TMB buffer becoming blue before use is indicate the deterioration of reagent

--Always refrigerate plates in sealed bags at drying condition without light.

--Store the kit at 2-8℃, the shelf life is 12 months when the kit is properly stored.