1. Principle
This test kit is based on the competitive enzyme immunoassay. The coupling antigens is pre-coated on the micro-well stripes. The enrofloxacin residue in the sample and coupling antigens pre-coated on the micro-well stripes compete for the anti-enrofloxacin 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 content of enrofloxacin residue in it. This value is compared to the standard curve and the content of the corresponding Enrofloxacin residue is subsequently obtained.
2. Technical specifications
Sensitivity : 0.25 ppb
Detection limit:
Tissue........................................................................................... 0.5 ppb
Serum........................................................................................... 0.5 ppb
Honey............................................................................................. 1 ppb
Cross-reaction rate:
Enrofloxacin..................................................................................... 100%
Ciprofloxacin................................................................................... 1.25%
Norfloxacin..................................................................................... 1.65%
Other Xacin medicines..................................................................... <0.1%
Recovery rate:
Tissue.......................................................................................... 85±15%
Serum.......................................................................................... 80±15%
Honey.......................................................................................... 75±15%
3. Components
1） Micro-well strips: 12 strips with 8 removable wells each
2） 6 × standard solution (1 mL each): 0 ppb, 0.25 ppb, 0.75 ppb, 2.25 ppb, 6.75 ppb and 20.25 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
4. Materials required but not provided
1) Equipments: microplate reader, printer, homogenizer, nitrogen-drying device, oscillator, centrifuge, measuring pipets, and balance with 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, dichloromethane, N-hexane, acetonitrile, Na2HPO4·12H2O, NaH2PO4·2H2O and heparin sodium ( for blood samples).
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 checked to 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:
SolutionⅠ (0.1 M NaOH): dissolve 0.4 g NaOH in the deionized water to 100 mL.
SolutionⅡ (acetonitrile-0.1 M NaOH solution): Vacetonitrile: VNaOH = 84:16.
SolutionⅢ (0.02 M PB buffer, pH 7.2): dissolve 5.16 g Na2HPO4·12H2O and 0.87 g of NaH2PO4·2H2O in the deionized water to 1 L.
Solution Ⅳ: V N-hexane: V dichloromethane = 2:8
Solution Ⅴ: dilute the 2× concentrated redissolving solution with deionized water at 1:1(1 mL 2×concentrated redissolving solution+1 mL deionized water), used for sample redissolving.
5.1 High-detection-limit samples
5.1.1 Animal tissues (meat, liver of pork and chicken, shrimp, fish, etc)
1） Weigh 2.0 ± 0.05 g of the homogenized tissue into 50 mL centrifugal tube.
2） Add 8 mL of the acetonitrile-0.1M NaOH solution, shake upside and down for 5 min, centrifuge at above 4000 r/min at 15 ℃ for 10 min.
3） Take 2 mL of the supernatant, add 2 mL 0.02 M PB buffer and 6 mL Solution Ⅳ, mix for 2 min, and centrifuge at above 4000 r/min at 15 ℃ for 5 min.
4） Discard the N-hexane phase(upper layer) and take the organic phase (lower layer ) into a dry container (clear and bright without impurity). Blow to dry with nitrogen at 50 ℃.
5） Dissolve the dry residues in 0.5 mL of the diluted redissolving solution, add 1 mL N-hexane, mix for 30 seconds, centrifuge at above 4000 r/min at 15℃ for 5min;
6） Discard the whole upper layers, take 50 µL of the lower layer for further analysis.
Fold of dilution of the sample: 2
5.1.2 Chicken blood
1） Collect the chicken blood into a centrifugal tube with the addition of the heparin sodium at (20 -30 U/mL blood) (we recommend to rinse the blood-collecting syringe with the heparin sodium). Place the blood sample for at least 1 h at room temperature. After the separation of plasma, centrifuge at above 4000 r/min at 15 ℃ for 10 min, and take 1 mL of the plasma.
2） Add 4 mL acetonitrile(100%), shake upside down for 5 min, and centrifuge at above 4000 r/min at 15℃ for 10 min.
3） Transfer the supernatant into another centrifugal tube, add 2 mL of 0.02 M PB buffer, and mix thoroughly;
4） Add 5 mL dichloromethane, mix thoroughly for 5 min, and centrifuge at 4000 r/min at 15℃ for 10 min. Discard the upper layer , and transfer organic phase (the lower) into a drying bottle (clear and bright without impurity). Then blow to dry completely with the nitrogen at 50 ℃.
5） Dissolve the dry residues in 1 mL of the diluted redissolving solution, add 1 mL N-hexane, mix for 30 seconds and centrifuge at above 4000 r/min at 15 ℃ for 5 min.
6） Discard impurity of the upper and middle layers, take 100 µL of the lower, and add 100 µL of the diluted redissolving solution. Mix for 30 seconds.
7） Take 50 µL for further analysis.
Fold of dilution of the sample:2
5.2 Honey
1） Take 1 g honey, add 2 mL 0.02 M PB buffer, dissolve it completely.
2） Add 8 mL dichlormethane, vortex for 5 min and centrifuge at 4000 r/min for 5 min.
3） Remove the upper layer, and take the organic phase (lower layer) into a dry container. Blow to dry with nitrogen or air at 50 ℃.
4） Dissolve the dry residues in 1mL of the diluted redissolving solution, and dilute it at 1:3 (1 mL sample solution+3 mL of the diluted redissolving solution). Mix for 30 seconds.
5） Take 50 µL for further analysis.
Fold of dilution of the sample: 4