1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of AOZ in the sample. The coupling antigens are pre-coated on the micro-well stripes. The AOZ in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-AOZ 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 AOZ in it. This value is compared to the standard curve and the AOZ concentration is subsequently obtained.
2. Technical specifications
Sensitivity: 0.02 ppb
Detection limit
Tissue(Chicken, duck, porcine meat, beef and mutton).................. 0.04 ppb
Liver(Chicken, duck, porcine meat, beef and mutton)..................... 0.04 ppb
Honey, milk, intestine, urine.......................................................... 0.04 ppb
Fish, shrimp(some interference)..................................................... 0.1 ppb
Recovery rate
Tissue, liver.................................................................................. 90±10%
Honey, milk, intestine.................................................................... 75±15%
Cross-reaction rate
AOZ................................................................................................ 100%
AHD............................................................................................. <0.01%
AMOZ.......................................................................................... <0.01%
SEM............................................................................................ <0.01%
3. Components
1） Micro-well strips: 12 strips with 8 removable wells each
2） 6× standard solution (1 mL each): 0 ppb, 0.02 ppb, 0.06 ppb, 0.18 ppb, 0.54 ppb and 1.62 ppb
3） Enzyme conjugate (7 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 (C7H5NO3)(10 mL)........................................ white cap
4. Materials required but not provided
1） Equipments: microplate reader, printer, homogenizer, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (a reciprocal sensibility 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 (approx 36.5%), K2HPO4·3H2O(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 diluted with deionized water at 1:1(1mL 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 deionized water to 100 mL.
6) 1 M NaOH: dissolve 4 g NaOH in deionized water to 100 mL.
5.1 Samples preparation
a) milk
1. Take 5 mL sample into centrifuge tube; add C and D solution, 250 µL each.
2. Mix thoroughly, use vortex, centrifuge at above 4000 r/min at 2-10 ℃ for 10 min with centrifuge of constant temperatures, if centrifuge of constant temperature is not available, chill sample temperature to approx 8 ℃, then centrifuge.
3. Continue as described in step (1-7, b).
b) Tisssue, Honey, Intestine, Liver, Urine
1. Weigh 1± 0.05 g of the homogenized sample (tissue, intestine or liver), or 1 mL urine, or put 1.1 mL of the centrifuged supernatant (equivalent to 1mL milk sample) in a plastic tube, add 4 mL of the deionized water, 0.5 mL 1 M HCI and 100 µL 2-Nitrobenzaldehyde (C7H5NO3) to each tube, shake properly.
2. Incubate at 37 ℃ over night ( approx 16 hours) or incubate at 56℃ by water bath(2 hours).
3. Add 5 mL 0.1 M K2HPO4, 0.4 mL 1 M NaOH and 5 mL ethyl acetate to each tube, shake strongly for 5 min.
4. Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
5. Transfer 2.5 mL of the ethyl acetate layer (upper layer) into a new centrifugal tube and evaporate to dryness by nitrogen or air at 50 ℃.
6. Dissolve the dry residues in 1 mL N-hexane, 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 1 M NaOH and 10 mL ethyl acetate to each tube, shake vigorously for 5 min.
5. Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
6. Transfer 5 mL ethyl acetate into a clean centrifuge tube and blow to dryness by nitrogen or air at 50 ℃.
7. Dissolve the dry residues in 1 mL N-hexane, add 1 mL of the diluted redissolving solution, mix properly, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min.
8. Take 50 µL of the lower layer for the analysis.
Fold of dilution of the sample: 2