1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Clenbuterol in the sample. The coupling antigen is pre-coated on the micro-well stripes. The Clenbuterol in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Clenbuterol 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 Clenbuterol in the sample. This value is compared to the standard curve and the Clenbuterol residues is subsequently obtained.
2. Technical specifications
Sensitivity: 0.1 ppb
Incubation Temperature: 25℃
Incubation Time: 30min—15min
Detection limit
Urine............................................................................................ 0.1 ppb
Tissue (method one)..................................................................... 0.4 ppb
Tissue (method two)...................................................................... 0.1 ppb
Feed............................................................................................... 1 ppb
Recovery rate
Urine............................................................................................ 95±22%
Tissue.......................................................................................... 75±20%
Feed............................................................................................ 80±20%
Cross-reaction rate
Clenbuterol...................................................................................... 100%
Terbutalin.......................................................................................... <1%
Mabuterol ........................................................................................ <1%
Brombuterol...................................................................................... <1%
Salbutamal....................................................................................... < 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
8） 20X Sample extract solution (50 mL)…………………………………. blue 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 30～300 μl;
3) Reagents: Acetonitrile (CH3CN), NaOH, ethyl acetate, methanol, n-Hexane, HCI (approx 36.5%), Na2 SO4(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.
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(for tissue samples): dissolve 0.4 g NaOH in deionized water to 100 mL.
3) CH3CN-0.1 M HCI solution(for tissue samples): ⅤCH3CN :ⅤHCI =84:16
4) Sample extract solution: 1 part 20X Sample extract solution + 19 parts deionized water, mix evenly for tissue sample extract.
5.1 Urine
Take 20 µL clear urine, directly detect it (If urine is muddy, must filter or centrifuge at 4000 r/min for 10 min, then take clear urine). Store at frozen environment if don’t use.
Fold of dilution of sample: 1
5.2 Tissue (liver, pork, etc.)
Method one
1. Weigh 2±0.05 g homogeneous sample, put it into a 50ml centrifuge tube, add 6 ml sample extract solution, shake thoroughly for 2 min, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.(Tips: If the fat content is higher in the sample, can firstly put it in the water bath at 85 ℃ for 10 min after shaking, then centrifuge)
2. Take 20 µL clear supernatant for analysis.
Fold of dilution of sample: 4
Method two
1. Weigh 2±0.05 g homogeneous sample, put it into a 50ml centrifuge tube, add 6 mL CH3CN-0.1 M HCI solution, shake for 2 min, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
2. Take 3 mL of clear supernatant, add 2 mL 0.1 M NaOH and 6 mL ethyl acetate, shake for 1 min, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min. Take all supernatant (almost is clear), blow to dry with nitrogen or air at 50～60 ℃.
3. Add 1 mL deionized water, mix for 30s, redissolve residues properly.
4. Take 20 µL for analysis.
Fold of dilution of sample: 1
5.3 Feed
1. Grind sample, weigh 1.0±0.05 g, put it into a 50ml centrifuge tube, add 10 mL methanol, then add 5 g Na2 SO4, shake thoroughly for 2 min, centrifuge at above 4000 r/min at 15℃ for 10 min
2. Take 1 ml supernatant (must be clear), blow to dry with nitrogen or air at 50～60℃. Add 1 ml deionized water to dissolve the dry residue, then add 1 mL N-hexane. Mix properly for 30 seconds, centrifuge at above 4000 r/min at room temperature 15 ℃ for 5 min. Remove up-layer organic phase.
3. Take down-layer phase 20 µL for analysis.
Fold of dilution of sample: 10