Tissue, acquatic product, honey

Nitrofuran (AOZ) ELISA Test Kit

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of AOZ (3-anmino-2-oxazolidinone) in the meat, chicken, fish, shrimp and honey. 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 AOZ concentration in the sample. This value is compared to the standard curve and the content of the corresponding AOZ is subsequently obtained.

2. Technical specifications

Sensitivity : 0.05ppb

Detection limit:

Tissue and honey.......................................................................... 0.1 ppb

Shrimp,fish(some interference)....................................................... 0.2 ppb

Recovery rate:

Shrimp and fish............................................................................ 90±10%

Honey ,chicken meat /liver............................................................. 75±15%

Cross-reaction rate:

AOZ................................................................................................. 100%

AHD............................................................................................... <0.1%

AMOZ............................................................................................. <0.1%

SEM............................................................................................... <0.1%

3. Components

1.    Micro-well strips: 12 strips with 8 removable wells each

2.     6× standard solution (1 mL each): 0 ppb, 0.05 ppb, 0.15 ppb, 0.45 ppb, 1.35 ppb and 4.05 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)( 15.1 mg)...................................... white 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 to 200 µL and 100 to 1000 µL, and multi-channel 250 µL.

3) Reagents: methanol, NaOH, ethyl acetate, N-hexane, HCI(approx 36.5%), K₂HPO₄·3H₂O, 2-Nitrobenzaldehyde(C₇H₅NO₃), K₂Fe(CN)₅NO·3H₂O and ZnSO₄·7H₂O

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. 10 mM 2-Nitrobenzaldehyde solution: dissolve 15.1 mg 2-Nitrobenzaldehyde in 10 mL methanol.

2. The 2×concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL 2× concentrated redissolving solution + 1 mL deionized water), used for sample redissolving.

3. C solution (for milk sample): dissolve 12.5 g K₂Fe(CN)₅ NO·3H₂O in deionized water to 100 mL.

4. D solution(for milk sample): dissolve 29.8 g ZnSO₄.7H₂O in deionized water to 100 mL.

5. 0.1 M K₂HPO₄ :dissolve 22.8 g K₂HPO₄·3H₂O in deionized water to 1 L.

6. 1 M HCl: dissolve 8.6 mL HCI(approx 36.5%) in water to 100 mL.

7. 1 M NaOH: dissolve 4 g NaOH in wate