1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Tylosin in the sample. The coupling antigens are pre-coated on the micro-well stripes. The Tylosin in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Tylosin antibody. 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 Tylosin in it. This value is compared to the standard curve and the Tylosin concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 1.5 ppb

Detection limit

Meat, liver........................................................................................ 3 ppb

Honey........................................................................................... 1.5 ppb

Recovery rate

Meat............................................................................................. 90±15%

Liver............................................................................................. 80±10%

Honey.......................................................................................... 85±15%

3. Components

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

2）   6× standard solution (1 mL each): 0 ppb, 1.5 ppb, 4.5 ppb, 13.5p pb ,40.5 ppb,121.5 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 (450 nm / 630 nm), homogenizer, oscillator, votex, centrifuge, and 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: ethanenitrile (CH₃CN), Methanol, deionized water, 0.1 M HCI, NaOH, CHCl₃

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:

1)      0.1 M NaOH: dissolve 0.4 g NaOH in deionized water to 100 mL.

2)      Sample extracting solution: mix 84 mL acetonitrile and 16 mL 0.1 M HCl, then add 18 mL methanol, mix evenly.

3)      the 2× concentrated redissolving solution is mixed with the deionized water at 1:1 (1 mL concentrated redissolving solution + 1 mL deionized water)

5.1 Liver and meat sample

1.      Homogenize the sample.

2.      Take 2± 0.05 g of the homogenized sample into 50 mL tube, add 8 mL of Sample extracting solution, shake properly with oscillator for 5 min. 

3.      Centrifuge at above 4000 r/min at room temperature (20 - 25 ℃) for 10 min.

4.      Take 2 mL supernatant, add 1 mL of 0.1 M NaOH, mix properly, add 3 mL CHCl₃ to extract, shake with oscillator for 5 min.

5.      Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min, remove the upper layer, take the lower, blow to dry with nitrogen.

6.      Dissolve the dry residues in 1 mL of the diluted redissolving solution.

7.      Take 50 µL for analysis.

Fold of dilution of the sample: 2        Detection limit :3 ppb

5.2 Honey

1.      Weigh 1± 0.05 g sample into 50 mL centrifuge tube, add 2 mL deionized water, vortex for 2 min to dissolve, then add 10 mL CHCl₃, shake upside and down for 5 min.

2.      Centrifuge at above 4000 r/min at room temperature (20 – 25 ℃) for 10 min, remove the upper layer, take organic phase (the lower), blow to dry with nitrogen.

3.      Dissolve the dry residue in 1 mL of the diluted redissolving solution.

4.      Take 50 µL for analysis.

Fold of dilution of the sample: 1    Detection limit:1.5 ppb