1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Melamine in the sample. The coupling antigen is pre-coated on the micro-well stripes. The Melamine in the sample and the coupling antigens pre-coated on the micro-well stripes compete for anti-Melamine antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the testing sample has a negative correlation with the Melamine concentration in the sample. This value is compared to the standard curve and the Melamine concentration is subsequently obtained.
2. Technical specifications
Sensitivity : 5 ppb
Detection limit:
Milk................................................................................................. 5 ppb
Tissue(chicken, pork, duck, fish, shrimp and liver)............................ 10 ppb
Feed............................................................................................ 500 ppb
Egg............................................................................................. 100 ppb
Recovery rate:
Milk.............................................................................................. 90±15%
Tissue.......................................................................................... 85±10%
Feed............................................................................................ 85±10%
Egg.............................................................................................. 80±10%
Cross-reaction rate:
Melamine......................................................................................... 100%
Cyanuric acid..................................................................................... 60%
Triazine.............................................................................................. <1%
Diamino atrazine................................................................................ <1%
3. Components
1） Micro-well strips: 12 strips with 8 removable wells each
2） 6× standard solution (1 mL each): 0 ppb,15 ppb, 45 ppb, 135 ppb, 405 ppb, 1215 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)......................................................... white cap
7） Stop solution (7 mL).................................................................. yellow cap
8） 20× concentrated washing buffer (40 mL)..................................... white cap
9） 5× 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 reciprocal sensibility of 0.01 g).
2) Micropipettors: single-channel 20~200 µL, 100~1000 µL; and multi-channel 250 µL;
3) Reagents: HCl, NaOH, Acetonitrile(CH3CN), N-hexane.
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 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 5×concentrated redissolving solution is diluted with deionized water at 1:4 (1 mL concentrated redissolving solution + 4 mL deionized water)
2) 1 M HCl: dissolve 8.6 mL HCl(approx 36.5%) in the deionized water to 100 mL.
3) 0.1 M NaOH solution: dissolve 0.4 g NaOH in the deionized water to 100 mL.
4) 1 M NaOH solution: dissolve 4 g NaOH in the deionized water to 100 mL.
5) Acetonitrile-0.1 M NaOH solution: Mix 84 mL Acetonitrile with 16 mL 0.1 M NaOH solution.
5.1 Feed
1 Take 2±0.05 g homogenized sample; Add 2 mL 1 M HCl and 16 mL deionized water.
2 Mix for 3 minutes, and then vortex for 5 minutes.
3 Centrifuge at above 4000 r/min at 15 ℃ for 10 min. Take 10 mL supernatant and adjust pH to 6-8 with 1 M NaOH.
4 Centrifuge at above 4000 r/min at 15 ℃ for 15 min. Take the supernatant(if it’s still not clear, should centrifuge at more speed).
5 Dilute the supernatant with the diluted redissolving solution at 1:9(100µL supernatant + 900µL diluted redissolving solution, mix for 30 s).
6 Take 50 µL for analysis.
Fold of dilution of sample :100
5.2 milk
1 Take 2 mL milk sample(without fat) to tube;
2 Add 8 mL Acetonitrile-0.1 M NaOH solution, mix throughly for 5 min. Centrifuge at above 4000 r/min at 15 ℃ for 10 min.
3 Take 2 mL supernatant and blow to dry by nitrogen at 60 ℃.
4 Dissolve the dry residue in 1 mL N-hexane, then add 1mL of the diluted redissolving solution. Mix properly for 30 seconds, centrifuge and remove the N-hexane layer(top layer)
5 Take 50 µL for analysis
Fold of dilution of sample :5
5.3 Tissue(meat, liver, chicken, shrimp, fish, duck)
1 Take 2±0.05 g homogenized sample to 50 mL tube.
2 Add 8 mL Acetonitrile-0.1 M NaOH solution and mix for 5 min, Centrifuge at above 4000 r/min at 15 ℃ for 10 min. Take 2 mL supernatant and blow to dry by nitrogen at 60 ℃.
3 Dissolve the dry residue in 1 mL N-hexane, then add 1mL of the diluted redissolving solution. Mix properly for 30 seconds, centrifuge and remove the N-hexane layer(top layer)
4 Take 50 µL for analysis
Fold of dilution of sample :2
5.4 Egg
1. Take 2±0.05 g homogenized sample(Egg white, egg yolk or whole egg) into 50 mLcentrifugal, add 8mL Acetonitrile-0.1 M NaOH solution, mix evenly for 5min.
2. Centrifuge at above 4000 r/min at 15 ℃ for 10 min, transfer 1 mL supernatant into a new centrifugal tube and evaporate to dryness by nitrogen at 60 ℃.
3. Dissolve the dry residues in 1 mL N-hexane, add 1mL of the diluted redissolving solution, mix properly for 30s, centrifuge and remove the N-hexane layer(top layer).
4. Dilute sample with the diluted redissolving solution at 1:3 (50 µL sample + 150µL diluted redissolving solution, mix for 30 s).
5. Take 50 µL for further analysis.
Fold of dilution of sample :20