1. Principle

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

2. Technical specifications

Sensitivity: 0.05 ppb

Detection limit:

Chicken,duck, pork(meat or liver), shrimp, fish, milk

Tetracyclines..................................................................................... 1ppb

Minocycline...................................................................................... 1ppb

RoliTetracyclines............................................................................... 1ppb

Aureomycin...................................................................................... 1ppb

Demethylchlor Tetracyclines............................................................... 3ppb

Terramycin........................................................................................ 2ppb

Doxycycline...................................................................................... 3ppb

Honey

Tetracyclines..................................................................................... 2ppb

Minocycline...................................................................................... 2ppb

RoliTetracyclines............................................................................... 2ppb

Aureomycin...................................................................................... 2ppb

Demethylchlor Tetracyclines............................................................... 6ppb

Terramycin........................................................................................ 4ppb

Doxycycline...................................................................................... 6ppb

Cross-reaction rate:

Tetracyclines.................................................................................... 100%

Minocycline...................................................................................... 125%

RoliTetracyclines............................................................................... 110%

Aureomycin...................................................................................... 100%

Demethylchlor Tetracyclines................................................................ 35%

Terramycin......................................................................................... 58%

Doxycycline....................................................................................... 45%

Recovery rate:

Tissue, milk................................................................................... 85±10%

Honey.......................................................................................... 70±10%

3. Components

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

2）   6× standard solution (1 mL each): 0 ppb, 0.5 ppb, 1.5 ppb, 4.5 ppb, 13.5 ppb, 40.5 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）   5× concentrated redissolving solution (50 mL)..................... transparent cap

4. Materials required but not provided

1)      Equipments: microplate reader (450 nm / 630 nm), homogenizer, oscillator, centrifuge, balance (a sensibility reciprocal of 0.01 g), measuring pipets

2)      Micropipettors: single-channel 20 to 200 µL and 100 to 1000 µL, and multi-channel 250 µL;

3)      Reagents: NaOH, Trichloroacetic acid, Na₂HPO₄·12H₂O, deionized water, C₆H₈O₇·H₂O, EDTA·Na₂·2H₂O, H₂C₂O₄, CH₃OH(≥99.5%), RIDA^RC₁₈ column

5. Sample pre-treatment

Instructions

The following points must be dealt with before the pre-treatment of any kind of sample:

1) This product can detect the following tissue samples:animal tissue,poultry,and aquatic tissue,

eg:chicken, duck, beef, rabbit, fish, shrimp,etc.

2) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

3) 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)      Solution A: dilute 5× concentrated redissolving solution with deionized water at 1:4.  

2)      Solution B(3% Trichloroacetic acid, for tissue sample): dissolve 3 g Trichloroacetic acid in deionized water to 100 mL.

3)      Solution C(1% Trichloroacetic acid, for honey sample): dissolve 1 g Trichloroacetic acid in deionized water to 100 mL.

4)      Solution D(0.1M NaoH,for tissue sample): dissolve 0.4 g NaOH in deionized water to 100 mL

5)      Solution E: dissolve 27.6 g Na₂HPO₄·12H₂O, 12.9 g C₆H₈O₇·H₂O and 37.2 g EDTA·Na₂·2H₂O in deionized water to 1000 mL(adjust pH 4.0)

6)      Solution F: dissolve 1.8 g H₂C₂O₄ in CH₃OH to 1L.

5.1 Tissue(Chicken, pork, porcine liver, shrimp or fish)

1.      Take 2± 0.05 g of the homogenized sample into 50 mL centrifuge tube, add 4 mL of Solution B, shake properly with oscillator for 5 minutes. 

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

3.      Take 100 µL supernatant into another tube, add 100 µL of 0.1 M NaOH and then diluted with 800 µL Solution A, mix for 30s(If it’s not clear, Centrifuge at above 4000 r/min for 10 minutes).

4.      Take 50 µL for analysis.

Fold of dilution of the sample: 20       

5.2 Honey

1.      Weigh 1± 0.05 g homogenized sample into centrifuge tube, add 2 mL Solution C, shake properly with oscillator for 5 minutes. 

2.      Centrifuge at above 4000 r/min for 10 minutes.

3.      Take 100 µL supernate into another tube and add 1900 µL Solution A, mix for 30s

4.      Take 50 µL for analysis.

Fold of dilution of the sample: 40

5.3 Milk  

1.Take 1ml milk into centrifuge tube, add 3 mL of Solution B, shake properly with oscillator for 5 minutes. 

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

3.Take 100 µL supernate into another tube and add 400 µL Solution A, mix for 30 s.

4.Take 50 µL for analysis.

Fold of dilution of the sample: 20

5.4 Tissue(Chicken, pork, porcine liver, shrimp or fish) by RIDA^RC18 column

1.      Take 5± 0.05 g of the homogenized sample into 50ml centrifuge tube, add 25 mL of Solution E, shake properly with oscillator for 30 minutes. 

2.      Centrifuge at above 4000 r/min at 15 ℃ for 10 minutes.

3.      Take all the supernatant to a new tube, extract the precipitate with 25 mL of solution E again.

4.      Put the two supernatant together, and filtered by filter paper.

5.      Take 5 mL filter solution for purification.

Purification procedure

1.       Take 3 mL CH₃OH(100%) to wash the C₁₈ column.

2.       Take 2 mL deionized water to wash the C₁₈ column.

3.       Add the 5 mL filter solution to the C₁₈ column, 15 drop/min.

4.       Wash the column by 3 mL deionized water.

5.       Empty the water in the column.

6.       Washed by 1 mL Solution F, 15 drop/min. gather the wash sulution.