Rat T3 ELISA Kit

Sample Collection, Preparation, and Storage

1. Serum: After placing whole blood samples at room temperature for 2 hours or at 4°C overnight, centrifuge at 1000×g for 20 minutes. Remove the supernatant for testing. Blood collection tubes should be disposable, pyrogen-free, and endotoxin-free. Store at -20°C or -80°C and avoid repeated freezing and thawing.

2. Plasma: Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing. EDTA-Na₂ is recommended as an anticoagulant. Avoid using samples with hemolysis or hyperlipidemia. Store at -20°C or -80°C and avoid repeated freezing and thawing. 3. Tissue Homogenization: Take an appropriate amount of tissue and wash it in pre-chilled PBS (0.01M, pH 7.0-7.2) to remove blood (lysed red blood cells in the homogenate will affect the measurement results). After weighing, mince the tissue and mix it with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio; the specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS). Pour the mixture into a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or freeze-thawed repeatedly (keep the sonication in an ice bath and repeat the freeze-thaw cycle twice). Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes. Remove the supernatant for analysis. (The tissue homogenate should also be assayed for protein concentration to obtain a more accurate concentration of the test substance per milligram of protein.) 4. Cell Culture Supernatant: Centrifuge the cell supernatant at 1000 × g for 20 minutes to remove impurities and cell debris. Remove the supernatant for testing and store at -20°C or -80°C, but avoid repeated freezing and thawing.

5. Urine: Collect the first morning urine (midstream) or 24-hour urine collection. Centrifuge at 2000×g for 15 minutes, collect the supernatant, and store the sample at -20°C. Avoid repeated freezing and thawing.

6. Saliva: Collect the sample using a saliva sample collection tube, then centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing, or aliquot and store at -20°C. Avoid repeated freezing and thawing.

7. Other biological samples: Centrifuge at 1000×g for 20 minutes, collect the supernatant, and store at -20°C. Avoid repeated freezing and thawing.

Precautions

1. The sample should be clear and transparent, and suspended matter should be removed by centrifugation. Hemolysis of the sample will affect the results, so hemolyzed samples should not be used.

2. If the sample is tested within 1 week after collection, it can be stored at 4°C. If testing cannot be done promptly, please aliquot the sample into a single-use amount and freeze at -20°C (for testing within 1 month) or -80°C (for testing within 3-6 months). Avoid repeated freeze-thaw cycles. Please bring the sample to room temperature before the experiment.

3. If the concentration of the test substance in your sample is higher than the highest value of the standard, please dilute it appropriately based on the actual situation (it is recommended to conduct a preliminary experiment to determine the dilution factor).

Sample Dilution Principles

If your test sample needs to be diluted, the general dilution principles are as follows:

1. 50-fold dilution: One-step dilution. Add 5 μL of sample to 245 μL of standard and sample diluent for a 50-fold dilution.

2. 100-fold dilution: One-step dilution. Add 5 μL of sample to 495 μL of standard and sample diluent for a 100-fold dilution.

3. 1000-fold dilution: Two-step dilution. Add 5 μL of sample to 95 μL of Standard & Sample Diluent for a 20-fold dilution. Then add 5 μL of the 20-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution, for a total dilution of 1,000. 4. 100,000-fold dilution: Three-step dilution. Add 5 μL of sample to 195 μL of Standard & Sample Diluent for a 40-fold dilution. Then, add 5 μL of the 40-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution. Finally, add 5 μL of the 2,000-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution, for a total dilution of 100,000-fold. 5. For each dilution step, use at least 3 μL of sample volume, and do not exceed a 100-fold dilution. Excessively small sample volumes can lead to greater errors during mixing. Ensure thorough mixing at each dilution step to avoid foaming. 6. For very high dilution ratios, dilute with PBS first, and then use the Standard & Sample Diluent provided in the kit as the final step.

Preparation Before Assay

1. Remove the test kit from the refrigerator 30 minutes in advance and equilibrate to room temperature.

2. Dilute 25 μg of concentrated wash buffer to 1 μg of working buffer with double-distilled water. Return any unused portion to 4°C.

3. Standard: Add 1.0 mL of Universal Diluent for Standards & Samples to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Then gently mix (concentration: 10,000 pg/mL). Subsequently, serially dilute the standard to 10,000 pg/mL, 5,000 pg/mL, 2,500 pg/mL, 1,250 pg/mL, 625 pg/mL, 312.5 pg/mL, and 156.25 pg/mL. The standard diluent (0 pg/mL) serves as a blank well. Prepare the required amount of standard solution and set aside. It is recommended to add the prepared standard solution to the sample within 15 minutes; it is not recommended to leave it for an extended period.

4. Biotin Conjugate Working Solution (1x): Centrifuge before opening the bottle. Dilute with Biotin Conjugate Diluent immediately before use. Prepare the solution based on the pre-calculated total volume required for each experiment (50 μL per well). Add 0.1-0.2 mL more than the actual volume. For example, prepare 10 μL of biotin conjugate with 990 μL of Biotin Conjugate Diluent, mix gently, and prepare within one hour of use.

5. Streptomycin-Horseradish Peroxidase Conjugate Working Solution (1x): Centrifuge before opening the bottle. Dilute with Enzyme Conjugate Diluent immediately before use. Prepare the solution based on the pre-calculated total volume required for each experiment (100 μL per well). Add 0.1-0.2 mL more than the actual volume. Prepare a solution of 10 μL of enzyme conjugate to 990 μL of enzyme conjugate diluent, mix gently, and prepare within one hour of use.

6. TMB Substrate - Use a pipette to aspirate the required volume of solution. Do not return any remaining solution to the reagent bottle.

Preparation

1. Before use, equilibrate all materials and prepared reagents to room temperature. Before use, mix all reagents thoroughly, taking care not to create any foam.

2. The user should calculate the number of samples likely to be used in the entire assay. Please reserve sufficient sample in advance.

3. Estimate the concentrations before measurement. If these values are not within the standard curve range, the user must determine the optimal sample dilution for their specific assay.

Procedure

1. Before beginning the assay, equilibrate all reagents to room temperature and prepare all reagents in advance. When diluting reagents or samples, mix thoroughly and avoid foaming. If the sample concentration is too high, dilute with sample diluent to bring the sample within the test range of the kit. 2. Sample Addition: Set up separate wells for standards and samples to be tested. Add 50 μL of the standard or sample to be tested, being careful not to create bubbles. Add the sample to the bottom of the wells of the ELISA plate, minimizing contact with the well walls. Next, add 50 μL of biotin conjugate (1 x) to each well and gently shake to mix. Cover the plate or film and incubate at 37°C for 1 hour. 3. To ensure the validity of the experimental results, use a fresh standard solution for each experiment. 4. After the 1-hour incubation, discard the liquid in the wells, spin dry, and wash the plate three times, adding 200 μL of wash solution (1 x) to each well, soaking for 1-2 minutes each time, and spin dry. 5. Then, add 100 μL of streptomycin-HRP (1 x) to each well and gently shake to mix. Cover the plate or cover the plate with film and incubate at 37°C for 1 hour. 6. Discard the liquid in the wells, spin dry, and wash the plate 5 times. Add 200 μL of wash solution (1 x) to each well, soaking for 1-2 minutes each time, and spin dry. 7. Add 90 μL of TMB colorimetric reagent to each well and develop the color at 37°C in the dark for 20 minutes (the time can be shortened or extended depending on the actual color development, but should not exceed 30 minutes). 8. Add 50 μL of stop solution to each well to terminate the reaction (the blue color will immediately turn yellow). The order of adding the stop solution should be as similar as possible to the order of adding the substrate solution. To ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time expires. 9. Measure the optical density (OD) of each well sequentially using a microplate reader at 450 nm. Perform assays within 5 minutes after adding the stop solution. 10. *Samples may require dilution. Please refer to the Sample Handling section. Calculation of Results 1. The OD values of the competition standards and samples can be directly substituted into the calculations. If replicate wells are used, the average value should be used for calculations. 2. For ease of calculation, although concentration is the independent variable and OD value is the dependent variable, the graphs use the OD value of the standard as the horizontal axis (X-axis) and the concentration of the standard as the vertical axis (Y-axis). To ensure intuitive visualization of the results, the graphs present raw data rather than logarithmic values. The OD values of the standard curve may vary due to differences in experimental conditions (such as operator, pipetting technique, plate washing technique, and temperature). The provided standard curve is for reference only; experimenters should establish their own standard curve based on their own experiments. The OD value of the used sample can be used to calculate the sample concentration on the standard curve, and then multiplied by the dilution factor to obtain the actual concentration of the sample. It is recommended to use professional curve drawing software, such as Curve Expert.

Sample type

Recovery rate range

Average recovery

Serum (n=5)

89-103%

96%

Sample type

1:2

1:4

1:8

1:16

Serum (n=5)

90-99%

95-106%

82-101%

93-105%

EDTA Plasma (n=5)

92-104%

82-96%

95-102%

87-98%

heparin plasma (n=5)

87-98%

89-103%

82-95%

86-99%

Chinese name

96T

Storage conditions

ELISA plate (detachable)

12 strips×8 wells

Lyophilized Standard

2

4°C/-20°C

Standard & Sample Dilution

20 mL

4°C/-20°C

Biotin conjugate (100×)

60 μL

4°C/-20°C

Biotin conjugate diluent

10 mL

4°C/-20°C

Concentrated HRP enzyme conjugate (100×)

120 μL

4°C/-20°C

Enzyme conjugate diluent

12 mL

4°C/-20°C

Concentrated washing solution (25×)

20 mL

4°C/-20°C

Chromogenic substrate solution (TMB)

10 mL

4°C/-20°C (protect from light)

Reaction stop solution

6 mL

4°C/-20°C

Sealing film

2

Normal temperature