1 Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Fluoroquinolones in the pork, chicken, fish, shrimp and honey, etc. The Fluoroquinolones in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Fluoroquinolones 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 Fluoroquinolones in the sample. This value is compared to the standard curve and the Fluoroquinolones concentration is subsequently obtained.
2 Technical specifications
Sensitivity: 1 ppb
Cross-reactions
Enrofloxacin.................................................................................... 100%
Ciprofloxacin................................................................................ 92.88％
ofloxacin..................................................................................... 98.86％
Oxolinic Acid.............................................................................. 116.86％
Danofloxacin.............................................................................. 102.63％
Norfloxacin................................................................................. 148.65％
Lomefloxacin................................................................................ 86.24％
Pefloxacin.................................................................................. 156.60％
Enoxacin...................................................................................... 98.97％
Flumequine................................................................................... 96.73％
Marbofloxacin............................................................................. 110.63％
Amifloxacin.................................................................................. 98.86％
Nadifloxacin................................................................................. 56.81％
Recovery rate
Tissue(chichen, liver, pork, shrimp, fish)......................................... 80±15%
Serum.......................................................................................... 80±15%
Honey.......................................................................................... 75±15%
Detection limit
Enrofloxacin.................................................................................... 2 ppb
Ciprofloxacin....................................................................... approx 2 ppb
ofloxacin............................................................................. approx 2 ppb
Danofloxacin....................................................................... approx 2 ppb
Norfloxacin.......................................................................... approx 1 ppb
Lomefloxacin....................................................................... approx 2 ppb
Pefloxacin........................................................................... approx 1 ppb
Enoxacin............................................................................. approx 2 ppb
OxolinicAcid........................................................................ approx 2 ppb
Flumequine.......................................................................... approx 2 ppb
Marbofloxacin...................................................................... approx 2 ppb
Amifloxacin......................................................................... approx 2 ppb
Nadifloxacin........................................................................ approx 4 ppb
3 Components
1) Micro-well strips: 12 strips with 8 removable wells each
2) 6× standard solution (1 mL each): 0 ppb, 1 ppb, 3 ppb, 9 ppb, 27 ppb, 81 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, homogeniser, 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: HCl(approx 36.5%), Dichloromethane, acetonitrile, N-hexane, Na2HPO4·12H2O, NaH2PO4·2H2O, heparin sodium (specific for blood samples).
5 Sample pre-treatment
Instructions (The following points must be dealt with before the pre-treatment)
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) 0.1 M HCl solution: dissolve 860 µL HCl(36.5%) in deionized water to 100 mL.
2) CH3CN - 0.1 M HCl solution: ⅤCH3CN :ⅤHCl =84:16.
3) 0.05 M PB buffer (pH=7.2): dissolve 12.9 g Na2HPO4·12H2O and 2.175 g NaH2PO4·2H2O in deionized water to 1 L.
4) 0.02 M PB buffer: dissolve 5.16 g Na2HPO4·12H2O and 0.87 g NaH2PO4·2H2O in deionized water to 1 L.
5) N-hexane- Dichloromethane solution: ⅤN-hexane :ⅤDichloromethane = 2：8
6) The 2× concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL condentrated redissolving solution + 1 mL deionized water), The resulting solution will be used for the treated sample redissolving.
5.1 High-detection-limit samples
a. Animal tissue (chiken/liver, pork/liver, shrimp, fish.etc)
1. Weigh 2.0 ± 0.05 g of the homogenized sample into 50 mL centrifugal tube.
2. Add 4 mL of the CH3CN-0.1 M HCl solution, shake upside down thoroughly for 5 min, then add 4 mL N-hexane- Dichloromethane solution, and shake for 10 min. centrifuge at above 4000 r/min at 15 ℃ for 10 min.
3. Take 2 mL of the clear organic phase into a dry container. Blow to dry completely with nitrogen at 56℃.
4. Dissolve the dry residues in 1 mL of the diluted redissolving solution, add 1 mL N-hexane, mix for 2 min and centrifuge at above 4000 r/min at 15 ℃ for 5 min.
5. Discard upper layer and take 50 µL solution for further analysis.
Fold of dilution of the sample: 2
b. Chicken blood
1 Collect the chicken blood into centrifugal tube with the addition of the heparin sodium at (20-30 units heparin sodium) / (1 mL of blood), (Recommend to rinse the blood-collecting syringe with the heparin sodium). Place the blood sample for at least 1 h at room temperature (20-25 ℃). After the separation of plasma, centrifuge at above 4000 r/min at 15 ℃ for 10 min, and take 1 mL plasma.
2 Add 4 mL of the anhydrous acetonitrile (100%), mix thoroughly by shaking upside down for 10 min, and centrifuge at above 4000 r/min at 15 ℃ for 10 min;
3 Transfer the supernatant into a new centrifugal tube, add 2 mL 0.02 M PB buffer, mix evenly;
4 Add 5 mL dichloromethane, mix thoroughly for 10 min, centrifuge at 4000 r/min at 15 ℃ for 10 min. Discard the upper layer, transfer the organic phase (lower layer) into a drying bottle (clear - bright without impurity). Blow to dry with nitrogen or completely by rotary evaporation at 50 ℃
5 Dissolve the dry residues in 1 mL of the diluted redissolving solution, add 1 mL of N-hexane, mix for 2 min , centrifuge at above 4000 r/min at 15 ℃ for 5 min;
6 Gently remove the upper and middle layers of impurity, take 100 µL of the lower layer, add 100 µL of the diluted redissolving solution, mix properly;
7 Take 50 µL for further analysis.
Fold of dilution of the sample: two
5.2 Low-detection-limit samples
Animal tissue (chiken, pork, liver, shrimp, fish.etc)
1. Weigh 2.0 ± 0.05 g of the homogenized sample, add 8 mL 0.02 M PB buffer, mix thoroughly by shaking upside down for 10 min, centrifuge at above 4000 r/min at 15 ℃ for 10 min (if the supernatant is too turbid, take 2-4 mL, centrifuge again).
2. Take