1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Diazepam in the feed, urine, liver and meat. The coupling antigen is pre-coated on the micro-well stripes. The Diazepam in the sample and the coupling antigen pre-coated on the micro-well stripes compete for the anti-Diazepam 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 Diazepam in it. This value is compared to the standard curve and the Diazepam concentration is subsequently obtained.
2. Technical specifications
Sensitivity: 0.1 ppb
Detection limit
Tissue............................................................................................. 1 ppb
Urine............................................................................................... 1 ppb
Feed............................................................................................. 10 ppb
Recovery rate
Urine........................................................................................... 80 ±10%
Feed........................................................................................... 75 ±10%
Tissue(meat, liver)........................................................................ 85 ±10%
Cross-reactions rate
Diazepam........................................................................................ 100%
Nitrazepam....................................................................................... 7.6%
Oxazepam........................................................................................ 8.8%
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 (12 mL)............................................................. red cap
4） Antibody working solution (7 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, votex, centrifuge, measuring pipets, balance (a sensibility reciprocal of 0.01 g).
2) Micropipettors: single-channel 20 to 100 µL and 200 to 1000 µL, and multi-channel 250 µL.
3) Reagents: N-hexane, NaOH,Dichloromethane(CH2Cl2), CH3CN.
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) 1 M NaOH: dissolve 4 g NaOH in 100 mL deionized water.
2) The 2×concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL concentrated redissolving solution + 1 mL deionized water), used for the treated sample redissolving.
3) N-hexane-CH2Cl2 solution: ⅤN-hexane:ⅤCH2Cl2 = 5:3.
5.1 Animal tissues (meat ,liver)
1. Take the sample, homogenize at 10000 r/min for 1 min,
2. Weigh 2 ± 0.05 g of the homogenized sample, put into 50 mL centrifugal tube, add 5 mL CH3CN,1 mL 2 M NaOH, shake properly for 10 min, centrifuge at above 4000 r/min at 10 ℃ for 10 min,
3. Take 3 mL supernatant(upper layer) into a new centrifugal tube, add 200 µL 2 M NaOH, 6 mL N-hexane-CH2Cl2 solution, shake for 10 min, and centrifuge at above 4000 r/min at 20-25 ℃ for 5 min,
4. Static for 5-10 min, transfer all supernatant into a new centrifugal tube, blow to dry with nitrogen,
5. Dilute residues in 1 mL of the diluted redissolving solution,
6. Take sample solution, dilute at 1:9 ( 50 µL sample solution + 450 µL diluted redissolving solution),
7. Take 50 µL for further analysis.
Fold of dilution of the sample: 10 Detection limit: 1 ppb
5.2 Urine
1. Put 1 mL clear sample into 50 mL centrifuge tube, add 4 mL 0.1 M NaOH, shake properly for 2-5 min,
2. Transfer 1 mL liquid into another centerifugal tube, add 10 mL N-hexane, shake for 5 min, and centrifuge at above 4000 r/min at 20-25 ℃ for 5 min,
3. Transfer 5 mL supernatant into a new centrifugal tube, blow to dry with nitrogen,
4. Dilute with 1 mL of the diluted redissolving solution,
5. Take 50 µL for further analysis.
Fold of dilution of the sample: 10 Detection limit: 1ppb
5.3 Feed
1. Put 1.0 ± 0.05 g feed into 50 mL centrifugal tube, add 6 mL deionized water and 1 mL 1 M NaOH, vortex for 1 min, add 6 mL N-hexane-CH2Cl2 solution(5:3), shake properly for 10 min, centrifuge at above 4000 r/min at room temperature for 5 min,
2. Take 3 mL supernatant(upper layer), blow to dry with nitrogen at 50 ℃,
3. Dilute with 1 mL of the diluted redissolving solution .
4. Dilution: at 1:49(10 µL sample + 490 µL the diluted redissolving solution ).
5. Take 50 µL for further analysis
Fold of dilution of the sample: 100 Detection limit: 10 ppb
Notice: We recommend the standard 3 as cut off because of some interference.