1. Principle

The test kit is based on the competitive enzyme immunoassay for the detection of Salbutamol in the sample. The coupling antigen is pre-coated on the micro-well stripes. The Salbutamol in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Salbutamol antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with the Salbutamol in it. This value is compared to the standard curve and the Salbutamol residues is subsequently obtained.

2. Technical specifications

Sensitivity: 0.1ppb

Detection limit

Tissue(First method)..................................................................... 0.4 ppb

Tissue(Second method)................................................................ 0.1 ppb

Urine, serum................................................................................. 0.1 ppb

Feed............................................................................................... 1 ppb

Recovery rate

Urine, serum................................................................................. 95±10%

Tissue.......................................................................................... 85±15%

Feed............................................................................................ 85±15%

Cross-reaction rate

Salbutamol...................................................................................... 100%

Terbutalin......................................................................................... < 1%

Clenbuterol...................................................................................... < 1%

Ractopamine.................................................................................... < 1%

3. Components

1）   Micro-well strips: 12 strips with 8 removable wells each

2）   6× standard solution (1 mL each): 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb

3）   Enzyme conjugate (7 mL)............................................................... red cap

4）   Antibody working solution (10 mL)................................................ blue cap

5）   Substrate A solution (7 mL)......................................................... white cap

6）   Substrate B solution (7 mL)......................................................... black cap

7）   Stop solution (7 mL).................................................................. yellow cap

4. Materials required but not provided

1)      Equipments: microplate reader, printer, homogenizer, nitrogen-drying device, vortex, oscillator, centrifuge, measuring pipets, balance (a sensibility reciprocal of 0.01 g)

2)      Micropipettors: single-channel 20-200 µL and 100-1000 µL, and multi-channel 250 µL;

3)      Reagents: Acetonitrile (CH₃CN), NaOH, ethyl acetate, N-hexane, HCI (approx 36.5%), Methanol, Na₂SO₄

5. Sample pre-treatment

Instructions (The following points must be dealt with before the pre-treatment)

1)      Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

2)      Before the experiment, each experimental utensil must be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results.

Solution preparation before sample pre-treatment:

1)      0.1 M HCI: dissolve 0.86 mL HCI (approx 36.5%) in deionized water to 100 mL.

2)      0.1 M NaOH: dissolve 0.4 g NaOH in deionized water to 100 mL.

3)      CH₃CN-0.1 M HCl solution:  V _CH3CN:V _HCl=84:16

5.1 Samples preparation

a) Urine and serum

Take 20 µL clear urine or serum, directly detect it (If urine and serum are muddy, must filter or centrifuge at 4000 r/min at 15 ℃ for 10 min, then take clear urine and serum). Store at frozen if don’t use.

Fold of dilution of sample: 1

b) First method of tissue(liver, pork, etc.)

1.      Weigh 2 ± 0.05 g sample, add 6 mL deionized water, mix properly for 2 min, Then centrifuge at 4000 r/min at room temperature (20-25 ℃) for 10 min(If the fat is too much, shake for 10 min in water bath at 85 ℃).

2.      Take 20 µL for analysis.

Fold of dilution of sample: 4

Second method of tissue(liver, pork, etc.)

1.         Weigh 2 ± 0.05 g sample, add 6 mL CH₃CN-0.1 M HCl solution, mix properly for 2 min, Then centrifuge at 4000 r/min at room temperature (20-25 ℃) for 10 min.

2.         Take 3 mL supernatant, add 2 mL 0.1 M NaOH and 6 mL ethyl acetate. Mix properly for 1 min, Then centrifuge at 4000 r/min at room temperature (20-25 ℃) for 10 min. Blow to dryness with nitrogen or air at 50℃.

3.         Add 1 mL deionized water, mix for 30 seconds.

4.         Take 20 µL for analysis.

Fold of dilution of sample: 1

c)  Feed

1.      Take 1 ± 0.05 g grinded sample, add 10 mL Methanol and then 5 g Na₂SO₄, shake for 2 min with oscillator; Centrifuge at 4000 r/min for 10 min at 15 ℃

2.      Take 1 mL supernatant, Blow to dryness with nitrogen or air at 56℃. Resolve the dry residue with 1 mL deionized water, then add 1 mL N-hexane, mix for 30 seconds. Centrifuge at 4000 r/min for 5 min at 15 ℃

3.      Take 20 µL for further analysis.

Fold of dilution of sample: 10

6. ELISA procedures

1.      Bring test kit to the room temperature (20-25 ℃) for at least 30 min, note that each reagent must be shaken evenly before use; put the required micro-well strips into plate frames. Re-sealed the unused microplate, stored at 2-8 ℃, not frozen;

2.      Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate; record their positions;

3.      Add 20 µL of the sample or standard solution to separate duplicate wells, then add 50µL/well of the Enzyme conjugate, and then add antibody working solution, 80 µL/well, seal the microplate with the cover membrane, and incubate at 25℃ for 30 min;

4.      Pour the liquid out of microwell, wash the microplate with the deionized water at 250 µL/well for 4-5 times. Each time for 15-30 sec, flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips). .

5.      Coloration: add 50 µL of substrate A solution and 50 µL B solution into each well. Mix gently by shaking the plate manually, and incubate at 25 ℃ for 15 min at dark for coloration;

6.      Determination: add 50 µL stop solution into each well. Mix gently by shaking the plate manually. Set the wavelength of the microplate reader at 450 nm to determine the OD value of every well. (Recommend to read the OD value at the dual-wavelength 450/630 nm within 5 min).

8. Result judgment

There are two methods to judge the results; the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with Salbutamol concentration in the sample.

8.1  Qualitative determination

The concentration range (ng/mL) can be obtained from comparing the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sample Ⅰ is 0.313, and that of the sample Ⅱ is 1.032, the OD value of standard solutions is: 1.892 for 0 ppb, 1.501 for 0.1 ppb, 1.175 for 0.3 ppb, 0.751 for 0.9 ppb, 0.421 for 2.7 ppb ,0.198 for 8.1 ppb, accordingly the concentration range of the sample Ⅰ is 2.7 to 8.1 ppb, and that of the sample Ⅱ is 0.3 to 0.9 ppb.

8.2    Quantitative determination

The mean values of the absorbance values obtained for the average OD value (B) of the sample and the standard solution divided by the OD value (B0) of the first standard solution (0 standard) and subsequently multiplied by 100%, that is

B—the average (double wells) OD value of the sample or the standard solution

B0—the average OD value of the 0ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the Clenbuterol standard solutions (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the sample from the standard curve by incorporating its absorption percentage into the standard curve. The resulting value is subsequently multiplied by the corresponding dilution fold, finally obtaining the Salbutamol concentration in the sample.

Using the professional analyzing software of this kit will be more convenient for the accurate and rapid analysis of a large amount of samples. (Please contact us for this software)