Dog FT3 ELISA Kit

Sample Collection and Storage

The following are general guidelines for sample collection and storage.
Sodium azide must not be used as a preservative during all sample collection and storage.

1. Cell Culture Supernatant:
Centrifuge at 4000 rpm for 20 minutes to remove cell particles and aggregates.
Store the supernatant below -20°C and avoid repeated freezing and thawing.

2. Serum:
Use pyrogen- and endotoxin-free tubes, avoiding any cell stimulation during the procedure.
Centrifuge at 4000 rpm for 20 minutes, carefully separate the serum, and store below -20°C.
Avoid repeated freezing and thawing.

3. Plasma:
Use heparin, EDTA, or sodium citrate as an anticoagulant.
Centrifuge at 4000 rpm for 20 minutes, remove the supernatant, and store the plasma below -20°C to avoid repeated freezing and thawing.

After sample collection, if testing cannot be completed in one go, please aliquot and freeze the sample in a single-use aliquot to avoid repeated freezing and thawing.
Thaw at room temperature before use to ensure that the sample is evenly and fully thawed.

4. Tissue homogenization:
Rinse the tissue with pre-chilled PBS (0.01 M, pH=7.4) to remove residual blood.
Weigh and mince the tissue.
Add the minced tissue to the corresponding volume of PBS (generally a 1:9 weight-to-volume ratio, for example, 1 g of tissue sample corresponds to 9 mL of PBS.
The specific volume can be adjusted appropriately according to experimental needs and recorded.
It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice.
To further lyse tissue cells, the homogenate can be ultrasonically disrupted or repeatedly frozen and thawed.
Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes, and collect the supernatant for testing.

5. Cell Extract:
Gently wash adherent cells with cold PBS, then digest with trypsin and collect the cells by centrifugation at 1000 × g for 5 minutes.
Suspension cells can be collected directly by centrifugation.
Wash the collected cells three times with cold PBS.
Resuspend in 150-200 μL of PBS per 1 × 106 cells and disrupt the cells by repeated freeze-thaw cycles (if the cell count is very low, reduce the volume of PBS).
Centrifuge the extract at 1500 × g for 10 minutes, and collect the supernatant for testing.

6. Other biological fluids:
Centrifuge at 1000 × g for 20 minutes to remove impurities and cell debris.
Collect the supernatant for analysis.

1. Before use, warm all components for at least 60 minutes to ensure they are fully warmed to room temperature.

2. Concentrated Wash Solution:
Crystals may form when the concentrated wash solution is removed from the refrigerator.
This is normal.
Heat in a water bath to completely dissolve the crystals.
Dilute the concentrated wash solution with distilled water at a ratio of 1:20: add 1 part concentrated wash solution to 19 parts distilled water.

3. Substrate:
Before use, thoroughly mix substrate solutions A and B at a 1:1 volume ratio.
Use within 15 minutes of mixing.

4. Procedure:
Bring all reagents and components to room temperature.
Duplicate wells are recommended for standards, controls, and samples.

1. Prepare the working solutions of all components of the kit as described in the previous instructions.

2. Remove the required strips from the aluminum foil pouch.
Seal the remaining strips in a ziplock bag and return them to the refrigerator.
Set up standard wells, blank wells, and sample wells.
Add 50 μL of the standard of varying concentrations to each standard well, leave the blank wells untouched, and add 50 μL of the sample to be tested to the sample wells.

3. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antigen to the standard and sample wells, except for the blank wells.

4. Cover the reaction plate with a sealing film and incubate at 37°C in a waterbath or incubator for 60 min.

5. Remove the sealing film, discard the liquid, and pat dry on absorbent paper.
Fill each well with wash solution, let it sit for 20 seconds, then shake off the wash solution and pat dry on absorbent paper.
Repeat this process five times.
If using an automated plate washer, wash the plate according to the machine's operating procedures.
Adding a 30-second soaking period can improve assay accuracy.
After washing, pat the plate dry on a clean, lint-free tissue before adding substrate.

6. Thoroughly mix substrates A and B at a 1:1 volume ratio.
Add 100 μL of the substrate mixture to all wells.
Cover the plate with sealing film and incubate at 37°C in a water bath or incubator for 15 minutes.

7. Add 50 μL of stop solution to all wells and read the absorbance (OD) of each well on a microplate reader.

Calculation of Results

1. Using the standard concentration as the horizontal axis and the corresponding absorbance (OD value) as the vertical axis, use computer software and four-parameter logistic curve fitting (4-PL) to create a standard curve equation.
Calculate the sample concentration using the sample absorbance (OD value) using this equation.

2. If the sample is diluted, the concentration measured using the above method must be multiplied by the dilution factor to obtain the final sample concentration.

Standard Curve

The concentrations of the standards are: 32, 16, 8, 4, 2, 0 pmol/L.

1. Avoid foaming when mixing protein solutions.

2. When adding calibrators and samples, change pipette tips for each calibrator concentration and sample. Common components should be added using a cantilever to avoid cross-contamination.

3. Appropriate incubation times and sufficient washing steps are essential to ensure the accuracy of experimental results.

4. When using an automated plate washer, adding a 30-second soaking step can improve detection accuracy.

5. The substrate solution is a colorless liquid. If it turns blue during storage, it has expired and should not be used.

6. The stop solution should be added in the same order as the substrate solution. After adding the stop solution, the blue substrate product will instantly turn yellow.

7. During the experiment, any remaining strips should be immediately returned to the ziplock bag and sealed (stored dry at low temperature).

8. All liquid components should be shaken thoroughly before use, and incubation should be carried out strictly according to the time, sample volume and sample addition sequence indicated in the instructions.

9. Components of different batches of this reagent must not be mixed.