Human Interleukin-6 (IL-6) ELISA Kit

(Product No.: F01310)

Principle

This experiment uses the double antibody sandwich ABC-ELISA method. Anti-human IL-6 monoclonal antibody is coated on the enzyme-labeled plate. IL-6 in the standard and sample binds to the monoclonal antibody. Biotinylated anti-human IL-6 is added, forming an immune complex attached to the plate. Horseradish peroxidase-labeled Streptavidin binds to the biotin. Substrate working solution is added to develop a blue color, followed by the stop solution. The OD value is measured at 450nm. The concentration of IL-6 is proportional to the OD value, and the concentration of IL-6 in the specimen can be determined by plotting a standard curve.

Kit Components (Store at 2-8°C)

Preparation of Reagents and Collection of Blood Samples

1. Dilute the 10× sample buffer 1:10 with distilled water (e.g., 1ml concentrated diluent + 9ml distilled water).

2. Collect specimens: serum, plasma (EDTA, citrate, heparin anticoagulated), cell culture supernatant, tissue homogenate, etc. Test as soon as possible; store at 2-8°C for 48 hours; for longer storage, freeze at -20°C or -70°C and avoid repeated freeze-thaw cycles.

3. Preparation of standard solution: Take 8 1.5ml centrifuge tubes. Add 900ul of sample buffer to the first tube, and 500ul to the second to eighth tubes. Add 100ul of 10ng/ml standard solution to the first tube, mix thoroughly on a vortex mixer, then pipette 500ul and transfer to the second tube. Repeat this serial dilution. Discard 500ul from the seventh tube. The eighth tube is the blank control.

4. Wash buffer: Dilute 1:20 with distilled water (e.g., 1ml concentrated wash buffer + 19ml distilled water).

Assay Procedure

1. Sample addition: Add 100ul of standard or test sample to each well. Mix the plate thoroughly and incubate at 37°C for 40 minutes.

2. Washing: Wash the plate thoroughly with wash buffer 4-6 times, and blot dry on filter paper.

3. Add 50ul of distilled water and 50ul of the first antibody working solution to each well (blank adds 100ul water). Mix the plate thoroughly and incubate at 37°C for 20 minutes.

4. Washing: Same as before.

5. Add 100ul of enzyme-labeled antibody working solution to each well. Incubate the plate at 37°C for 10 minutes.

6. Washing: Same as before.

7. Add 100ul of substrate working solution to each well. Incubate in the dark at 37°C for 15 minutes.

8. Add 100ul of stop solution to each well and mix.

9. Measure the absorbance at 450nm with a microplate reader within 30 minutes.

Result Calculation and Interpretation

1. It is recommended to subtract the blank value from all OD values before calculation. If the blank OD is below 0.1, direct calculation is acceptable.

2. Using the standard concentrations of 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 0 pg/ml as the x-axis and the OD values as the y-axis, plot the standard curve using software with a four-parameter polynomial fit.

3. Calculate the corresponding IL-6 concentration based on the sample OD value. The software can be requested from our company via email.

Kit Performance

1. Sensitivity: The minimum detectable concentration of IL-6 is less than 8pg/ml.

2. Specificity: Can detect both recombinant and natural human IL-6. No cross-reactivity with other human cytokines.

3. Repeatability: Intra- and inter-assay coefficients of variation are both less than 10%.

Precautions

1. It is recommended to run duplicates for both standard and test samples. A standard curve should be included in each assay.

2. The washing process is critical. Inadequate washing may lead to precision errors and falsely elevated OD values.

3. During the assay, all reagents should be brought to room temperature. After opening the strip, reseal the remaining strips to keep them dry.

4. This kit uses a highly sensitive TMB solution. If the color development is too strong, precipitation may occur, which is normal and does not affect result interpretation.

5. The kit components listed in the manual are for a 96T kit. For a 48T kit, the amounts should be halved!

6. This kit should be stored at 4°C. For research use only, not for clinical diagnosis!

The above instructions are for reference only! Please refer to the manual inside the kit for actual instructions!