1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Aminohydantion (AHD) in the sample. The coupling antigens are pre-coated on the micro-well stripes. The Aminohydantion (AHD) in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-AHD 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 AHD in it. This value is compared to the standard curve and the AHD concentration is subsequently obtained.
2. Technical specifications
Sensitivity: 0.05 ppb
Detection limit
Tissue(Chicken, duck, porcine meat, beef and mutton)···················· 0.1 ppb
Liver(Chicken, duck, porcine meat, beef and mutton)······················· 0.1 ppb
Milk, intestine, honey····································································· 0.1 ppb
Fish, shrimp (some interference) ·················································· 0.15ppb
Recovery rate
Tissue, liver·················································································· 90±15%
Honey, milk, intestine···································································· 80±15%
Cross-reaction rate
AHD································································································ 100%
AMOZ··························································································· ﹤0.1%
AOZ······························································································ ﹤0.1%
SEM····························································································· ﹤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, 0.15ppb, 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
10) 2-Nitrobenzaldehyde (10 mL)······················································· white cap
4. Materials required but not provided
1） Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex, centrifuge, measuring pipets, and 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: NaOH, ethyl acetate, n-Hexane, HCI (approx36.5%), K2HPO4 (for all samples), K2Fe(CN)5NO·3H2O and ZnSO4·7H2O(for milk sample)
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 absorbing 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) the 2×concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL concentrated redissolving solution + 1 mL deionized water), used for sample redissolving.
2) C solution (for milk sample): dissolve 12.5 g K2Fe(CN)5NO·3H2O in deionized water to 100 mL.
3) D solution (for milk sample): dissolve 29.8 g ZnSO4·7H2O in deionized water to 100 mL.
4) 0.1 M K2HPO4: dissolve 22.8 g K2HPO4·3H2O in deionized water to 1 L.
5) 1 M HCl: dissolve 8.6 mL HCI (approx 36.5%) in water to 100 mL.
6) 1 M NaOH: dissolve 4 g NaOH in water to 100 mL.
5.1 Samples preparation
a) milk
1. Put 5 mL milk into centrifuge tube, add C and D solution, 250 μL each.
2. Mix thoroughly, use vortex, centrifuge at above 4000 r/min at 4-12 ℃ for 10 min, if centrifuge of constant temperature is not avaible, chill sample to approx 8 ℃, then centrifuge.
3. Continue as described in step (1-7,b).
b) Tissue, intestine, liver, honey
1. Weigh 1± 0.05 g of the homogenized sample (Tissue, intestine, liver or honey), or put 1.1 mL supernatant of centrifuged milk (equivalent to 1 mL milk sample) into a plastic tube, add 4 mL of the deionized water, 0.5 mL 1 M HCI and 100 μL 2-Nitrobenzaldehyde to the tube, shake properly.
2. Incubate at 37 ℃ over night ( approx 16 h) or incubate at 56℃ in water bath(2 hours).
3. Add 5 mL 0.1 M K2HPO4, 0.4 mL 1 M NaOH and 5mL ethyl acetate, shake vigorously for 5 min.
4. Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
5. Transfer 2.5 mL ethyl acetate (upper layer) into a new centrifugal tube and evaporate to dry by nitrogen or air at 50 ℃.
6. Dissolve the dry residue in 1 mL N-hexane, then add 1mL of the diluted redissolving solution. Mix properly for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
7. Take 50 µL of the lower for analysis.
Fold of dilution of the sample:2
c) Special samples(Fried cooked foods, Non-fried cooked food)
1. weigh 1± 0.05 g of the homogenized sample in 50 mL tube.
Fried cooked foods: Add 4.5 mL Methanol and 0.5 mL distilled water, shake strongly for 3 min. Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min, Discard all solutions. then add 5 mL Acetonitrile and 5 mL N-hexane, shake strongly for 3 min. Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min, Discard all solutions.
Non-fried cooked foods: Add 5 mL distilled water and 5 mL N-hexane, shake strongly for 3 min, Discard all solutions.
2. Add 4 mL distilled water, 0.5 mL 1 M HCl and 100µL 2-Nitrobenzaldehyde(C7H5NO3) solution to the tube, shake properly.
3. Incubate at 37 ℃ over night ( approx 16 hours) or incubate at 56℃ by water bath(2 hours) .
4. Add 5 mL 0.1 M K2HPO4, 0.4 mL