1. Principle

The BIOKAU^®β-agonists test kit is based on the competitive enzyme immunoassay for the detection of β-agonists in the urine, pork and other samples. The coupling antigen is pre-coated on the micro-well stripes. The β-agonists in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-β-agonists 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 β-agonists in the sample. This value is compared to the standard curve and the β-agonists residues is subsequently obtained.

2. Technical specifications

Sensitivity: 0.3 ppb

Detection limit:

Tissue.......................................................................................... 0.3 ppb

Urine, serum................................................................................. 0.3 ppb

Feed............................................................................................. 30 ppb

Recovery rate:

Urine, serum................................................................................. 90±10%

Tissue.......................................................................................... 80±10%

Feed............................................................................................ 80±15%

Cross-reaction rate:

Clenbuterol...................................................................................... 100%

Salbutamol...................................................................................... 120%

Bromchlorbuterol............................................................................. 130%

Brombuterol...................................................................................... 90%

Mabuterol.......................................................................................... 70%

Clenpenterol...................................................................................... 65%

Mapenterol........................................................................................ 55%

Tebutalin........................................................................................... 42%

Carbuterol......................................................................................... 35%

Clenproperol..................................................................................... 15%

Cimaterol........................................................................................... 12%

Dobutamine...................................................................................... <3%

Ritodrine........................................................................................... <1%

Ractopamine.................................................................................. <0.5%

Precision:

For the intra-assay precision, CV<8%

For the inter-assay precision, CV<10%

3. Components

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

2）   6× standard solution (1 mL each): 0 ppb, 0.3ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb, 25 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

8）   20× concentrated washing buffer (40 mL)..................................... white cap

9）   2× concentrated redissolving solution (50 mL)..................... transparent 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, 100-1000 µL, and multi-channel 250 µL;

3)      Reagents: Acetonitrile (CH3CN), NaOH, ethyl acetate, HCI (approx 36.5%), N-hexane, isopropanol(C₃H₈O)

5. Sample pre-treatment

Solution preparation before sample pre-treatment:

1)      1 M HCI: dissolve 8.6 mL HCI (approx 36.5%) in deionized water to 100 mL.

2)      1 M NaOH: dissolve 4 g NaOH in deionized water to 100 mL.

3)      Dillute 2× concentrated redissolving solution with deionized water at 1:1(1× concentrated redissolving solution, used for sample redissolving) and 1:3(0.5× concentrated redissolving solution, used for extraction of tissue sample)