1. Principle

The test kit is based on the competitive enzyme immunoassay for the detection of Ractopamine in the sample. The conjugated antigens is pre-coated on the micro-well stripes. The Ractopamine in the sample and the coupling antigens pre-coated on the micro-well stripes compete for anti-Ractopamine 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 Ractopamine concentration in the sample. The value is compared to the standard curve and the Ractopamine concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 0.05 ppb

Incubator temperature:  25℃

Incubator time:  30min～15min

Detection limit

Urine.......................................................................................... 0.05 ppb

Tissue……………………………………………………………………… 0.2 ppb

Meat........................................................................................... 0.05 ppb

Liver............................................................................................. 0.1 ppb

Feed............................................................................................ 0.5 ppb

Cross-reactions

Ractopamine................................................................................... 100%

Dobutamine........................................................................................ 1%

Salbutamol..................................................................................... <0.1%

Clenbuterol..................................................................................... <0.1%

Recovery rate

Urine............................................................................................ 95±18%

Tissue.......................................................................................... 75±21%

Feed............................................................................................ 80±25%

3. Components

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

2）   6× standard solution (1 mL each): 0 ppb, 0.05 ppb, 0.15ppb, 0.45 ppb, 1.35 ppb, 4.05 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, oscillator, centrifuge, measuring pipets, balance ( a reciprocal sensibility of 0.01 g)

2)        Micropipettors: single-channel 20-200 µL and 100-1000 µL, and multi-channel 30-300 µL;

₃₎        Reagents: N-hexane, Acetonitrile (CH₃CN), Methanol, Na₂SO₄ (100%)

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.

Samples preparation

5.1 Urine

Take 20 µL clear urine, directly detect it (If urine is muddy, must filter or centrifuge at 4000 r/min for 5 min, then take clear urine). Store at frozen environment if don’t use.

Fold of dilution of sample: 1              

5.2 Tissue (Method No.1)

1.      Weigh 2±0.05 g homogenized tissue sample into 50ml centrifuge tube, add 6 mL deionized water, shake thoroughly for 2 min, centrifuge at 4000 r/min at room temperature (20-25 ℃) for 10 min (If the fat content is high in tissue sample, after shaking, put it in 85℃ water bath for 10min, then centrifuge.).

2.      Take 20 µL clear supernatant for analysis.

Fold of dilution of sample: 4               

5.3 Tissue (Method No.2)

1.  Weigh 2±0.05 g homogenized tissue sample into 50ml centrifuge tube, add 8 mL CH₃CN solution, shake thoroughly for 2 min, centrifuge at 4000 r/min at 15 ℃ for 10 min.

2.  Take 5 mL supernatant, blow to dry with nitrogen or air at 50-60 ℃.

Meat sample

Add 1ml deionized water, mix for 30s, take 20 µL for analysis.

Fold of dilution of sample: 1

Liver sample

Add 2ml N-hexane shake to dissolve, then add 1ml deionized water, mix for 30s, centrifuge at 4000 r/min at 15 ℃ for 5 min, move the up-layer; take 50µl down-layer, mix with 50µl deionized water evenly; take 20 µL for analysis.

Fold of dilution of sample: 2

5.4 Feed

1.      Grind sample, weigh 1.0±0.05 g, add 10 mL Methanol, then add 5g Na₂SO₄ (100%), shake with oscillator for 2 min, centrifuge at above 4000 r/min at 15 ℃ for 10 min.

2.      Take 1 ml supernatant, blow to dry with nitrogen or air at 50-60 ℃, use 1ml deionized water to dissolve the dry residue, then add 1 ml N-hexane, mix 30s; centrifuge at above 4000 r/min at 15℃ for 5 min, remove the up-layer organic phase.

3.      Take 20 µL down-layer for analysis.

Fold of dilution of sample: 10