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New β-Agonist combo ELISA Diagnostic Kit for urine

Introduction

Ractopamine, Clenbuterol and Salbutamol are three main kinds of β-Agonist, which are used

for the treatment of asthma in human and animal.

It has been shown that using β-Agonist to feed farm animals at high dose can shift the flow of

nutrients from adipose tissue toward muscle tissue. Due to the popularity of improved lean / fat

meat ratio and the enhanced production efficiency, β-Agonist is illegally used in livestock

production. Recently, several cases of intoxication have been reported regarding abuse of

Ractopamine / Clenbuterol / Salbutamol or other kinds of β-Agonist.

The fast screening method, developed for the detection of β-Agonist in biological samples by

enzyme immunoassay, allows analyzing large number of samples in short time（60 min）.

Therefore, the FLOGEN New β-Agonist combo ELISA Diagnostic Kit has been used in testing

the drug residue generally.

Principle

The FLOGEN quantitative β-Agonist ELISA diagnostic kit is based on

the immunological reaction between specific antigen and antibody. It

relies on the competition of enzyme conjugate and Ractopamine /

Clenbuterol / Salbutamol residue in the sample to bind to the

Ractopamine / Clenbuterol / Salbutamol specific antibody immobilized

on the microtiter wells. In the test procedure, the sample and the enzyme

conjugate are added into the antibody-immobilized microtiter plate well.

The drug in the sample will compete with the enzyme conjugate to bind

to the limited amount of antibody. When a sufficient amount of drug is

present, the drug will saturate the antibody. Thus the enzyme reaction

will not proceed and only light color will be shown indicating a positive

result. If there is no drug in the sample, it will generate deep color due to

the enzyme reaction with the substrate.

Product Content

1. Microtiter well plate：8 microwells / strip, 12 strips / plate.

2. Clenbuterol standard：each bottle for 0 ppb, 0.25 ppb, 0.5 ppb, 1 ppb, 2 ppb, 4ppb 1.0

mL / bt.（Ready to use）

3. 10X Concentrate Washing Solution：1 bts / kit, 50 mL / bt.（Dilute 10X before use）

4. 150X Concentrate Enzyme Conjugate：1 bts / kit, 0.1 mL / bt.（Dilute 150X before use）

5. Enzyme Conjugate Dilution Solution：1 bts / kit, 50 mL / bt.（Ready to use）

6. Substrate Solution（TMB）：1 bt, 12 mL / bt.（Ready to use）

7. Stop Solution：1 bt, 13 mL / bt.（Ready to use）

Instructions

A. Precaution

1. Take out the samples, Microtiter well plate, Clenbuterol standard 6 bottles, 10X Concentrate

Washing Solution, 150X Concentrate Enzyme Conjugate, Enzyme Conjugate Dilution Solution,

Substrate Solution（TMB）and Stop Solution from the refrigerator for 40 minutes before the

reaction and make sure the whole kit is back to the room temperature.

2. Dilute the 10X Concentrate Washing Solution with 10 folds of double distilled water. The 1X

Washing Solution is now ready for the plate washing.

3. Dilute the 150X Concentrate Enzyme Conjugate with 150 folds of Enzyme Conjugate Dilution

Solution. The diluted conjugate must be used immediately. After reconstitution, the enzyme

conjugate is stable at -20 for 1℃℃℃℃ week.

4. Take out the required amount of microtiter well strips. The remaining strips are put back to the

aluminum bag, and stored in the 2~8 refrigerator.℃

B. Required materials but not provided

- Microtiter plate spectrophotometer

- Centrifuge Machine

- Variable 50 µL , 100 µL , 1000 µL – micropipettes

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C. Sample preparation：：：：

� Urine

1. Centrifuge urine samples with 1700g for 10 minutes.

2. Supernatant is now ready for application to the ELISA microtiter plate.

Sensitivity: 0.25 ppb

Dilution Factor: 1

D. Operation procedure：

1. Add 30 µL of the CB standard solution（0, 0.25, 0.5, 1, 2, 4 ppb）to the appropriate microtiter

wells.

2. Add 30 µL of the urine samples to the appropriate microtiter wells.

3. Add 120 µL of the diluted enzyme conjugate solution to each well, mix gently by knocking the

edge of the plate and incubate for 30 min at room temperature（19~25℃）in the dark.

4. Dump the reaction solution out of the microtiter wells.

5. Add washing buffer to fill the microtiter wells and dump it out.

6. Repeat above procedure for 3 times.

7. Pat it dry after the final wash.

8. Add 100 µL of the substrate solution to each microtiter wells and incubate for 20 minutes at

room temperature in the dark.

9. Add stop solution 100 µL to each microtiter wells.

10. Measure the absorbance at 450 nm（ref 630 or 650 nm）.

E. Test result interpretation：

1. Divide the absorbance value of each standard and sample by the absorbance of the Maximum

Binding（B0）（absorbance of 0 ppb standard） and multiply by 100, the Maximum Binding is

thus made equal to 100% and the absorbance values are quoted in percentage：

absorbance standard（（（（or sample））））/ absorbance（（（（0 ppb standard））））* 100 = B/B0(%)

2. Enter the B/B0 values calculated for each standard in a semi-logarithmic system of coordinates

against the standard clenbuterol concentration, draw the standard curve.（Fig.1）Take the B/B0

values for each sample and interpolate the corresponding concentration from the calibration

curve.

For example：Divide the absorbance value of each sample by the absorbance of 0 ppb standard

and multiply by 100 = X%, than the concentration = e[(48.045-X)/15.467]

ppb.（Fig.1）

F. Detection limit

The lowest detection limit of the FLOGEN β-agonist kit is around 0. ppb. The OD value of this

concentration is significantly different from the negative standard.

G. Standard curve y = -15.467Ln(x) + 48.045R2 = 0.997102040

60801000.01 0.1 1 10 100

Fig.1：Calibration Curve for FLOGEN New β-Agonist combo Kit

H. Reproducibility

The precision of this test has been determined from the result of six different experiments. Figure

2 shown the coefficient of variation（CV%）obtained from the absorption values of the standards

are plotted against the corresponding clenbuterol concentrations. The coefficients of variation are

lower than 10% in the whole range of testing concentration which a high reproducibility of the

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results is ensured.

0.01.02.03.04.05.06.00 0.25 0.5 1 2 4STD Conc.

CV(%

)

Fig.2：Interassay Precision Profile for FLOGEN New β-Agonist combo Kit

I. Cross-reactivity

The specificity of β-Agonists ELISA kits has been determined by analyzing the cross-reactivity of the

corresponding substances.

Compound 85% Cross-Reactivity（%）

Clenbuterol 100

Salbutamol 80

Ractopamine 85

Bromobuterol 95

Cimaterol 115

Mabuterol 60

Tulobuterol 65

Zilpaterol 10

Phenethylamine A 160

Mapenterol 50

Terbutaline 20

After the caculation from the above, 1.00 ppb Clenbuterol is adequate for 1.25 ppb Salbutamol，1.25

ppb Ractopamine，1.05 ppb Bromobuterol，2.22 ppb Mabuterol，1.05 ppb Cimaterol，5.00 ppb

Zilpaterol，2.00 ppb Tulobuterol，0.87 ppb Phenethylamine A，2.00 ppb Mapenterol，5.00 ppb

Terbutaline, 2 ppb Mapenterol, 5 ppb Terbutaline .

J. Sensitivity

Sample Sensitivity (ppb)

Urine 0.25

K. Recovery rate

Sample（spike CB in ppb） Recovery rate（%）

Urine 60 ~ 140

L. Q & A

1. Low OD value after adding the stop solution.

i. Forget to add the enzyme conjugate.

Ans: Please follow the correct procedures in the package insert.

ii. The kit is not wholly back to the room temperature.

Ans: It should take enough time to allow the kit back to the room temperature.

iii. Run with low room temperature（< 19℃）

Ans: If the room temperature is lower than 19 , please increase the incuba℃ tion period at step 3

of Operation procedure .

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iv. Wash the plate using the washing buffer not from the kit provided.

Ans: Please use the provided washing buffer to wash the plate.

v. The kit is out of date.

Ans: Please use the kit still within Exp. Date.

vi. If waiting too long to read the result after adding the stop solution.

Ans: Read within 20 minutes after addition of stop solution.

2. The standard curve is different from the curve shown on the QC report.

i. The position for the incubation is interfered by the current of the air conditioner.

Ans: Make sure that the incubation condition is stable.

ii. The operation time takes too long.

Ans: Please follow the procedure.

iii. Contamination has occurred to the intra microtiter wells.

Ans: Be careful not to spill when loading the samples.

iv. The volume of standard or enzyme conjugate is not shown equal in each well.

Ans: Please use the calibrated pipetman.

v. Load the sample or reagent with incorrect operation.

Ans: The tip should not contact with the microtiter wells.

vi. There is something wrong with washing procedure.（ex. The pipeline is plugged, and dose not go

through to next procedure after washing the microtiter wells）.

Ans: Please monitor the washing procedure and watch on washing machine momently, and go

through next procedure right the way after washing the microtiter wells.

3. High OD value

I. Room temperature is higher .（> 25 ℃）

Ans: If the room temperature is higher than 25 , please decrease the incubation time at step 3 of ℃Operation procedure.

II. Contamination occurred in the TMB.

Ans: Do not use the TMB solution if it shows a little blue.

III. Reaction time is too long.

Ans: Please use the right reaction time.

IV. The reagent is contaminated by enzyme conjugate.

Ans: Every time throw away the used tip, the contamination could be avoided. The used container

should be washed carefully, especially for those with enzyme conjugate and substrate.（Immerse in

bleach first, then wash thoroughly）.

4. Negative control OD values are lower than positive control OD values.

i. Unequal mixing of reagents is happened in microtiter wells.

Ans: Please knock the edge of the plate gently to mix the reagents well after the addition each

reagents.

ii. Incomplete washing of plates.

Ans: Please refer to 2-vi.

iii. Unequal addition of samples or enzyme conjugate.

Ans: Please refer to 2-iv.

5. RPM / RCF conversion :

RCF(g) = 1.118 × γ × (RPM / 1000)2

Where

RPM is rotating speed measured in revolutions per minutes. γ is the rotational radius measured in millimeters (mm).

Centrifuge condition of β-Agonist combo ELISA Diagnostic Kit should be performed in 1700g,

or the maximum rotation speed (3000 ~ 3500 rpm).