Operation of zebrafish elastase (Elastase) ELISA detection kit

Operation of zebrafish elastase (Elastase) ELISA detection kitSample pretreatment methods and precautions after receiving the reagent kit, for reference:

1、 Sample pre-processing methods

The processing methods for different sample types are different, and it is necessary to strictly follow the instructions of the reagent kit. The following are the key points for handling common sample types:

1. Serum/plasma

• Collection: Collect blood using EDTA or heparin anticoagulant tubes without detection indicators to avoid hemolysis.
• Centrifuge: 2000-3000 × g, centrifuge at 4 ℃ for 10-15 minutes to separate serum/plasma.
• Storage: After packaging, freeze at -80 ℃ (to avoid repeated freeze-thaw cycles), and store at -20 ℃ for a short period of time (testing should be done as soon as possible).

2. Tissue homogenate

• Material: Fresh tissue, washed with physiological saline and then drained of water.
• Homogenization: Add pre cooled PBS or lysis buffer (containing protease inhibitor) at a weight (g): volume (mL) ratio of 1:9-10, homogenize and let stand on ice for 30 minutes.
• Centrifuge: 12000 × g, centrifuge at 4 ℃ for 10-15 minutes, and take the supernatant.
• Save: Same as serum/plasma.

3. Cell lysate

• Collect cells: Wash the cells 2-3 times with PBS, centrifuge and discard the supernatant.
• Cracking: Add RIPA lysis buffer (containing protease inhibitor), sonicate or repeatedly freeze thaw for cracking.
• Centrifuge: 12000 × g, centrifuge at 4 ℃ for 20 minutes, and take the supernatant.
• Save: Same as above.

4. Fecal/other biological samples

• Preparation: The feces need to be homogenized and then added to a solvent (such as chloroform methanol mixture) to extract lipids. After centrifugation, the organic phase is taken and dried before reconstitution.
• Storage: Avoid oxidation, it is recommended to store at -80 ℃ after packaging.

Key precautions:

• Avoid lipid contamination (such as removing solvent residues when using chloroform extraction).
• The sample may contain endogenous binding proteins, and it is necessary to ensure sufficient lysis.
• Avoid repeated freeze-thaw cycles (recommended for packaging and storage).

2、 Handling after receiving the reagent kit

1. Check the integrity of the reagent kit:

   • Confirm that the reagents, calibrators, quality control products, instructions, etc. are complete.
   • Check if the reagent bottle is sealed properly, without any leakage or delamination.

2. Storage conditions:

   • Unopened reagent kit: Store according to the instructions (usually refrigerated at 2-8 ℃).
   • Opened reagents: short-term use can be stored at 2-8 ℃, long-term storage needs to be -20 ℃ (for example, enzyme labeled Flat noodles need to be sealed in aluminum foil bags to avoid light).

3. Packaging and usage:

   • Calibration and quality control products are recommended to be packaged and frozen to avoid repeated freeze-thaw cycles.
   • Before using the reagent, it needs to be warmed up to room temperature (15-30 minutes) to avoid sudden temperature changes affecting the reaction.

4. Calibration and Quality Control:

   • When creating a standard curve, it is necessary to strictly dilute the calibration sample according to the concentration gradient.
   • The quality control product results should be within the allowable range, otherwise operational or reagent issues need to be investigated.

3、 Common problems and solutions

1. Low or high OD values after sample processing:

   • Check if the sample has degraded (such as oxidation caused by improper storage).
   • Confirm if the dilution factor of the sample is appropriate (if it exceeds the linear range, it needs to be adjusted).

2. Abnormal color rendering (such as high background and uneven color):

   • Check if there is excessive or insufficient addition of termination solution.
   • Confirm if the incubation time and temperature meet the requirements.

3. The calibration curve is not ideal:

   • Check if the standard solution is correctly reconstituted and if there is contamination.
   • Confirm the wavelength setting of the ELISA reader (usually 450nm ± 10nm).

4. Reagent failure:

   • If the reagent kit is stored improperly (such as repeated freezing and thawing or high temperature), it may cause the reagent to fail. It is recommended to contact the supplier for replacement.

4、 Other suggestions

• Pre experiment optimization of sample processing steps before the experiment (such as adjusting lysis conditions for different tissues).
• Strictly avoid cross contamination (use special pipette gun head and Flat noodles).
• If the results are abnormal, it is recommended to retest or contact the technical support of the reagent kit supplier.

Operation of zebrafish elastase (Elastase) ELISA detection kit
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