1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Benzyl penicillin in the chicken, pork, duck, honey, milk, fish, shrimp and egg, etc. The antigens conjugated Benzyl penicillin is pre-coated on the micro-well stripes, Benzyl penicillin in the sample and the conjugated antigens pre-coated on the micro-well stripes compete for the anti-Benzyl penicillin 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 Benzyl penicillin concentration in the sample. This value is compared to the standard curve and concentration of Benzyl penicillin in the sample is subsequently obtained.
2. Technical specifications
Sensitivity : 0.05 ppb
Detection limit:
Chicken, duck, pork, liver, fish, shrimp................................................ 1 ppb
Honey, milk....................................................................................... 1 ppb
Recovery rate:
Chicken, duck, pork,liver, fish, shrimp.............................................. 85±10%
Honey, milk.................................................................................... 70±10%
Cross-reaction rate:
Benzyl penicillin................................................................................. 100%
Ampicillin........................................................................................... 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, 0.05 ppb, 0.15 ppb, 0.45 ppb, 1.35 ppb and 4.05 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, homogeniser, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (a reciprocal sensibility of 0.01 g)
2) Micropipettors: single-channel 20 to 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, ZnSO4·7H2O, HCI(36%), 2M H2SO4
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 2×concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL 2× concentrated redissolving solution+1 mL deionized water), ued for sample redissolving
2 0.1 M NaOH(for second milk sample preparement and tissue): dissolve 0.4 g NaOH in deionized water to 100 mL
3 CH3CN-0.1 M NaOH solution: ⅤCH3CN :ⅤNaOH =84:16 (84 mL CH3CN + 16 mL 0.1 M NaOH). (for second milk sample preparement and tissue)
4 C solution(for first milk sample preparement)/ 0.36M Na2Fe(CN)5·NO·2H2O: dissolve 10.7 g Na2Fe(CN)5·NO·2H2O in deionized water to 100 mL .
5 D solution(for first milk sample preparement)/1 M ZnSO4: dissolve 28.8 g ZnSO4·7H2O in deionized water to 100 mL.
6 Acidic Acetonitrile (CH3CN) (for honey sample): add 150 µL 2 M H2SO4 into 100 mL Acetonitrile (CH3CN), mix properly.
7 1M HCI (for honey sample): dissolve 41.7 mL HCI(36%) in deionized water to 500 mL.
8 1M NaOH(for honey sample) : dissolve 4 g NaOH in deionized water to 100 mL.
5.1 Tissue (Chicken, duck, pork or pork liver, fish, shrimp)
1 Homogenize the sample
2 Take 2± 0.05 g of the homogenized sample into 50 mL tube, add 8 mL of CH3CN-0.1 M NaOH solution, mix it with vortex for 2 min, shake for 5 min , centrifuge at above 4000 r/min at room temperature(20-25 ℃) for 10 min.
3 Take 1 mL of the supernatant, blow to dry by nitrogen or air at 56 ℃.
4 Dissolve the dry residues in 1 mL N-Hexane, add 1 mL of the diluted redissolving solution, shake vigorously for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min.
5 Remove N-Hexane phase (upper layer), take the lower to dilute at 1:4 ( 50 µL sample + 200 µL the diluted redissolving solution). Mix for 30 seconds.
6 Take 50 µL for analysis.
Fold of dilution of the sample: 20
5.2 Honey
1 Put 4.0 ± 0.05 g honey into centrifuge tube , add 0.5 mL 1 M NaOH, mix properly, then place it for 20 min.
2 Add 0.5 mL 1 M HCI, mix properly(pH=3), add 7 mL of the Acidic Acetonitrile (CH3CN) (pH=4), shake vigorously for 5 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 10 min.
3 Taker 3 mL of the supernatant (upper layer) and evaporate to dryness by nitrogen at 56 ℃.
4 Dissolve the dry residue in 1 mL of the diluted redissolving solution.
5 Dilute with the diluted redissolving solution at 1:19(20 µL sample+380 µL diluted redissolving solution). Mix for 30 seconds.
6 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 centrifuge 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 the supernatant(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 Take 2 mL milk (removed fat) into centrifuge tube.
2 Add 8 mL of the CH3CN-0.1 M NaOH solution, shake vigorously for 5 min, centrifuge at above 4000 r/min at 15 ℃ for 10 min, take 1 mL supernatant(upper layer) and evaporate to dryness by nitrogen at 56 ℃.
3 Dissolve the dry residues in 1 mL N-Hexane, add 1 mL of the diluted redissolving solution, mix properly for 30 seconds, centrifuge and remove N-hexane layer.
4 Dilute the lower layer at 1:3 (50 µL sample + 150 µL the diluted redissolving solution).
5 Take 50 µL for analysis.
Fold of dilution of the sample: 20