Colistin ELISA Test Kit High Reproducibility High Sensitivity

Be sure samples are properly stored. In general, samples should be refrigerated at 2-4°C for no more than 1-2 days.

Freeze samples to a minimum of -20°C if they need to be stored for a longer period. Frozen samples can be thawed at room temperature (20 – 25C / 68 – 77F) or in a refrigerator before use.

1.Preparation of 1X Colistin Extraction Buffer Ⅰ:

Mix 1 volume of 10X Colistin Extraction Buffer Ⅰ with 9 volumes of distilled water.

2.Preparation of 1X Colistin Extraction Buffer Ⅱ for Meat Extraction Mix 1 volume of 6X Colistin Extraction Buffer Ⅱ with 5 volumes of distilled water.

3. Preparation of 3X Colistin Extraction Buffer Ⅱ for Feed and Shrimp Extraction Mix 3 volume of 6X Colistin Extraction Buffer Ⅱ with 3 volumes of distilled water.

Meat(beef, pork and chicken)

1. Remove fat from meat , homogenize a reasonable amount of sample with a suitable mixer.

2. Weigh out 1 g of the homogenized meat sample,add 3.5 mL 1X Colistin Extraction Buffer Ⅰ and 500 uL 1X Colistin Extraction Buffer Ⅱ,add 3mL n-hexane vortex or shake for 3 minutes.

3. Centrifuge for 10 minutes at 4,000 x g at room temperature (20 – 25C / 68 – 77F). 4. Aspirate and discard the n-hexane layer, then transfer 500 uL of supernatant to a new tube containing 200 uL of 10X Colistin Extraction Buffer,200 uL of 100% ethanol and 100 uL of Colistin Balance Buffer.

5. Vortex or shake for 30 seconds at maximum speed.

6. Use 75 uL per well in the ELISA test.

Note: Dilution factor:10 Meat Powder

1. Homogenize a reasonable amount of sample with a suitable mixer.

2. Weigh out 0.5 g of the homogenized sample, add 3.5 mL 1X Colistin Extraction Buffer Ⅰ and 500 uL 1X Colistin Extraction Buffer Ⅱ,add 3mL n-hexane vortex or shake for 3 minutes.  SAMPLE PREPARATIO

N3. Centrifuge for 10 minutes at 4,000 x g at room temperature (20 – 25C / 68 – 77F).

4. Aspirate and discard the n-hexane layer, then transfer 500 uL of supernatant to a new tube containing 200 uL of 10X Colistin Extraction Buffer,200 uL of 100% ethanol and 100 uL of Colistin Balance Buffer.

5. Vortex or shake for 30 seconds at maximum speed.

6. Use 75 uL per well in the ELISA test.

Note: Dilution factor: 16. Feed

1. Homogenize a reasonable amount of sample with a suitable mixer.

2. Weigh out 0.5 g of the homogenized sample, add 2 mL of 0.1M HCl and 2 mL of n-hexane, and mix by vortexing at high speed for 3 minutes.

3. Centrifuge for 10 minutes at 4,000 x g at room temperature (20 – 25C / 68 – 77F). 4. Aspirate and discard the n-hexane layer, then transfer 200uL of supernatant to a clean centrifuge tube, add 150uL of 1×Colistin Extraction Buffer Ⅰ, and shake at high speed for 30 seconds.

5. Use 75 uL per well in the ELISA test.

Note: Dilution factor:8.75 Fish/Shrimp

1. Homogenize a reasonable amount of sample with a suitable mixer.

2. Weigh out 1.0 g of the homogenized meat sample, add 3.5 mL 1X Colistin Extraction Buffer Ⅰ and 500 uL 3X Colistin Extraction Buffer Ⅱ,add 3mL n-hexane vortex or shake for 3 minutes.

3. Centrifuge for 10 minutes at 4,000 x g at room temperature (20 – 25C / 68 – 77F). 4. Aspirate and discard the n-hexane layer, then transfer 200 uL of supernatant to a new tube containing 300uL distilled water, 200 uL of 10X Colistin Extraction Buffer, 200 uL of 100% ethanol and 100 uL of Colistin Balance Buffer.

5. Vortex or shake for 30 seconds at maximum speed.

6. Use 75 uL per well in the ELISA test.

Note: Dilution factor:25