1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Nitrofuran (SEM) in chicken, fish, shrimp, milk, honey, whole egg, etc. The coupling antigens are pre-coated on the micro-well stripes. The SEM in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-SEM 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 SEM in it. This value is compared to the standard curve and the SEM concentration is subsequently obtained.
2. Technical specifications
Sensitivity: 0.1 ppb
Detection limit
Tissue(Chicken, duck, porcine meat, beef and mutton)···················· 0.2 ppb
Liver(Chicken, duck, porcine meat, beef and mutton)······················· 0.2 ppb
Milk, intestine, honey····································································· 0.2 ppb
Fish, shrimp (some interference) ··················································· 0.3 ppb
Recovery rate
Tissue, liver·················································································· 90±15%
Honey, milk, intestine···································································· 80±15%
Cross-reaction rate
SEM ······························································································ 100%
AHD····························································································· ﹤0.1%
AMOZ··························································································· ﹤0.1%
AOZ······························································································ ﹤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, 2.7 ppb 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） 2× concentrated redissolving solution (50 mL).................. transparent cap
10） 2-Nitrobenzaldehyde(C7H5NO3) solution (10 mL)........................... white cap
4. Materials required but not provided
1） Equipments: microplate reader, printer, honogenizer, nitrogen-drying device, vortex, centrifuge, measuring pipets, 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(approx 36.5%), K2HPO4·3H2O, K2Fe(CN)5NO·3H2O and ZnSO4·7H2O(only 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 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) the 2× concentrated redissolving solution is diluted 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 by vortex; centrifuge at above 4000r/min at 4-12 ℃ 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 to 7, b).
b) Tissue, intestine, liver, honey
1. weigh 1± 0.05 g of the homogenized sample (tissue, honey, intestine, liver), or 1.1 mL the supernatant of centrifugal milk(equivalent to 1 mL of milk sample), add 4 mL of the deionized water, 0.5 mL 1 M HCI and 100 µL 2-Nitrobenzaldehyde solution to each well, 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 10 mL ethyl acetate to each tube, shake vigorously for 5 min.
4. Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
5. Transfer 5 mL ethyl acetate into a clean centrifuge tube and blow to dryness by nitrogen or air at 50 ℃.
6. 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.
7. Take 50 µL of the lower layer for the 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. &