1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Ampicillin in the sample. The antigens conjugated Ampicillin is pre-coated on the micro-well stripes, Ampicillin in the sample and the conjugated antigens pre-coated on the micro-well stripes compete for the anti-Ampicillin 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 Ampicillin concentration in the sample. This value is compared to the standard curve and concentration of Ampicillin in the sample is subsequently obtained.
2. Technical specifications
Sensitivity : 0.1 ppb
Detection limit:
Chicken, duck, pork,liver, fish, shrimp................................................. 2 ppb
Honey, milk....................................................................................... 2 ppb
Because of some interference in honey and milk, the detection limit is 4 ppb.
Recovery rate:
Chicken, duck, pork, liver, fish, shrimp.......................................... 75%±10%
Honey, milk................................................................................. 70%±10%
Cross-reaction rate:
Ampicillin........................................................................................... 100%
Benzyl penicillin.................................................................................. 0.8%
Cloxacillin........................................................................................... 0.2%
Dicloxacillin........................................................................................ 0.1%
Amoxicillin.......................................................................................... 0.1%
Ceftiofu.............................................................................................. 0.1%
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, 2.7ppb and 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） 5× concentrated redissolving solution (50 mL)..................... transparent cap
4. Materials required but not provided
1) Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (a reciprocal sensibility of 0.01 g)
2) Micropipettors: single-channel 20-200 µL and 100 to 1000 µL, and multi-channel 250 µL
3) Reagents: NaOH, Acetonitrile(CH3CN), N-Hexane, deionized water, Na2Fe(CN)5·NO·2H2O and ZnSO4
5. Sample pre-treatment
Instructions
The following points must be dealt with before the pre-treatment of any kind of sample:
1） Only the disposable tips can be used for the experiments and the tips must be changed when used for different reagents.
2） Before the experiment, each experimental equipment 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 5×concentrated redissolving solution is mixed with deionized water at 1:4 (1 mL 5× concentrated redissolving solution+ 4 mL deionized water), ued for sample redissolving.
2 0.1 M NaOH : dissolve 0.4 g NaOH in deionized water to 100 mL.
3 CH3CN-0.1M NaOH solution: ⅤCH3CN:ⅤNaOH =84:16 (84 mL CH3CN + 16 mL 0.1 M NaOH).
4 C solution(for milk sample): dissolve 10.7 g Na2Fe(CN)5·NO·2H2O in deionized water to 100 mL .
5 D solution(for milk sample): dissolve 28.8 g ZnSO4·7H2O in deionized water to 100 mL.
6 1 M NaOH solution: dissolve 4g NaOH in deionized water to 100 mL.
5.1 Tissue (Chicken, duck, pork, liver, fish or shrimp)
1 Homogenize the sample.
2 Take 2± 0.05 g of the homogenized sample into 50 mL centrifugal tube, add 2 mL 1 M NaOH solution, rock it with vortex for 2 min, add 8 mL CH3CN, shake for 10 min, centrifuge at above 4000 r/min at room temperature(25 ℃) for 10 min.
3 Take 1 mL of the supernatant, blow to dry by nitrogen or air at 50-60 ℃.
4 Dissolve the dry residues in 1 mL of the diluted redissolving solution, then dilute at 1:3 ( 50 µL sample + 150 µL the diluted redissolving solution).
6 Take 50 µL for further analysis.
Fold of dilution of the sample: 20
5.2 Honey
1 Put 1.0 ± 0.05 g honey into centrifuge tube, add 3 mL diluted redissolving solution, then static for 20 min after shaking vigorously. centrifuge at above 4000 r/min at room temperature (25 ℃) for 10 min.
2 Take upper layer, diluted 1:4 in diluted redissolving solution(20 µL sample + 80 µL the diluted redissolving solution).
3 Take 50 µL for further analysis.
Fold of dilution of the sample: 20
5.3 Milk
a) First method
1 Take 2 mL fresh milk into 5 mL centrifugal tube, add 50 µL C solution , mix properly.
2 Add 50 µL D solution ,shake for 1 min , centrifuge at 4000 r/min at 10 ℃ for 10 min.
3 Take clear liquid(upper layer), dilute with the diluted redissolving solution at 1:19 (20 µL sample+380 µL diluted redissolving solution).
4 Take 50 µL for further analysis.
Fold of dilution of the sample: 20
Note: Repeat again if centrifuged sample is muddy
b) Second method
1 Put 2 mL milk (removed fat) into centrifugal tube.
2 Add 8 mL of the CH3CN-0.1 M NaOH solution, shake vigorously for 10min, centrifuge at above 4000 r/min at 15 ℃ for 10 min, take 1 mL supernatant (upper layer), evaporate to dryness by nitrogen at 60 ℃.
3 Dissolve the dry residues in 1 mL N-Hexane, add 1 mL of the diluted redissolving solution, mix properly for 1 min, centrifuge and remove N-hexane phase.
4 Take the lower to dilute 1:3 ( 50 µL sample + 150 µL the diluted redissolving solution).
5 Take 50 µL for analysis.
Fold of dilution of the sample: 20