LSY-10069 Thiamphenicol ELISA Test Kit for Food safety ISO certificated 96 wells/kit

Thiamphenicol ELISA Test Kit

Catalog No. LSY-10069

1. Principle

This test kit is based on the indirect competitive enzyme immunoassay for the detection of Thiamphenicol in samples. The coupling antigens are pre-coated on the micro-well stripes. The Thiamphenicol in the sample and the conjugate antigens pre-coated on the micro-well stripes compete for the anti- Thiamphenicol antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value has a negative correlation with the Thiamphenicol concentration in the sample. This value is compared to the standard curve and the Thiamphenicol concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 0.1ppb

Incubator temperature: 25℃

Incubator time: 30min～15min

Detection limit:

Tissue (Method 1) 1ppb

Tissue (Method 2) 0.2ppb

Recovery rate:

Tissue 90 ±30%

Cross-reaction rate:

Thiamphenicol 100%

Florfenicol 105%

theChloramphenicol ＜1%

3. Components

4. Materials required but not provided

• Equipments: microplate reader, homogenizer, printer, nitrogen-drying device, oscillator, centrifuge, measuring pipets, balance( a reciprocal sensibility of 0.01 g), incubator.
• Micropipettors: single-channel 20-200µL and 100-1000 µL, and multi-channel 30～300 μl;
• Reagents: NaOH, Acetonitrile, ethyl acetate, Na₂SO₄

5. Sample pre-treatment

Instructions

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

• Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents.
• Before the experiment, each experimental utensil 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:

Solution 1 Extracting A solution

1 part of 20X extracting A solution + 19 parts of deionized water

Solution 2 Extracting B solution

1 part of 2X extracting B solution + 1 part of deionized water

Solution 3 Acetonitrile-ethyl acetate mixed solution

V _Acetonitrile:V _{ethyl acetate} = 4 : 1

Solution 4 1M NaOH solution

Take 4g solid NaOH, add deionized water to make the volume to 100 ml

Solution 5 0.01M NaOH solution

Take 0.04g solid NaOH, add deionized water to make the volume to 100 ml

Solution 6 Sample redissolving solution:

Dilute the 20× concentrated redissolving solution with deionized water at 1:19 (1 part 2× concentrated redissolving solution +19 parts of deionized water).

Samples preparation

a) Tissue (method 1)

• Weight 2 ± 0.05 g of the homogenized tissue sample into a 50ml centrifuge tube, add 3ml Extracting A solution, vortex for 3min;
• Then add 600µl 1M NaOH solution and 2.4ml Extracting B solution, vortex for 3min;centrifuge at above 4000 r/min at 25℃ for 5 min;
• Take 200µl up-layer liquid, add 300µl Sample redissolving solution, mix it evenly;
• Take 50 µL of above mixed solution for analysis.

Fold of dilution of the sample: 10

b) Tissue (method 2)

• Weight 2 ± 0.05 g of the homogenized tissue sample into a 50ml centrifuge tube;
• Add 2ml 0.01M NaOH solution and 6ml Acetonitrile-ethyl acetate mixed solution, then add 2g Na₂SO₄, vortex for 3min; centrifuge at above 4000 r/min at 25℃ for 5 min;
• Take 4ml of organic phase into a drying container and blow dry with nitrogen at 56°C;
• Add 1 mL of the sample redissolving solution to dissolve the dry residues, then add 1 mL N-hexane, mix for 10 seconds; centrifuge at above 4000 r/min at 25℃ for 5 min.
• Remove up-layer organic phase, take 50 µL of the down-layer liquid for analysis.

Fold of dilution of the sample: 1