Interleukin-7 ELISA KIT

Interleukin-7 ELISA KIT

Interleukin-7 ELISA KITIt is a commonly used detection tool in scientific research, used to quantitatively determine the content of interleukin-7 in biological samples. Interleukin-7 is a 25 kDa soluble secreted protein encoded by the IL-7 gene, belonging to the hematopoietic factor family and playing a critical role in lymphocyte differentiation, proliferation, and survival. This kit can assist in the research of related disease mechanisms and drug development.

1. Kit components

• Enzyme-linked immunosorbent assay (ELISA) plate96 or 48 wells, pre coated with anti-IL-7 antibody as the carrier for the reaction.

• reference standardUsually it is freeze-dried and comes in 2 bottles. Before use, standard diluent should be used for reconstitution and diluted into different concentration gradients, such as 0, 15.6, 31.2, 62.5, 125, 250, 500, 1000 pg/mL, etc., for plotting the standard curve.

• Sample xi release solutionUsed to dilute samples with excessively high concentrations to ensure their concentration is within the effective detection range of the reagent kit.

• Enzyme labeled antibodyHRP labeled anti-IL-7 antibody, which is a concentrated solution, needs to be diluted with enzyme labeled antibody diluent according to the requirements when used.

• Enzyme linked antibody diluentSpecially used for diluting enzyme-linked antibodies.

• substrate solutionAs a TMB substrate, it can react with HRP in enzyme-linked antibodies, resulting in a color change.

• detergentGenerally, it is a concentrated solution that needs to be diluted with distilled water or deionized water in a specific ratio when used to wash the enzyme-linked immunosorbent assay (ELISA) plate, remove unbound substances, and effectively reduce background interference.

• Stop SolutionIt is mostly a strong acid solution (such as sulfuric acid) used to terminate the color reaction of the substrate, stabilize the color of the reaction solution, and facilitate subsequent readings.

• Sealing filmUsed to cover the enzyme-linked plate during the incubation process to prevent liquid evaporation and external contamination.

• User ManualDetailed introduction of the usage method, precautions, performance indicators, etc. of the reagent kit.

2. Sample requirements

• serumAfter collecting blood, leave it at room temperature for 2 hours or overnight at 4 ℃, then centrifuge at 1000 × g for 20 minutes, carefully aspirate the supernatant for later use. Blood collection should use disposable equipment that is free of pyrogens and endotoxins.

• plasmaEthylene diamine tetraacetic acid (EDTA), heparin, and other anticoagulants can be used. Within 30 minutes after sample collection, centrifuge at 1000 × g for 15 minutes at 2-8 ℃ and collect the supernatant.

• Cell culture supernatantCentrifuge the cell culture supernatant at 1000 × g for 20 minutes to remove cell debris and impurities, and then take the supernatant for detection.

• tissue homogenateRinse the tissue with pre cooled phosphate buffered saline (PBS, 0.01M, pH=7.4) to remove residual blood. After cutting the tissue, add PBS in a ratio of approximately 1:9 (w/v) and grind thoroughly on ice. Centrifuge at 5000 × g for 5-10 minutes and collect the supernatant for detection.

• If the sample is tested in the short term after collection, it can be stored at 4 ℃; If long-term storage is required, it should be frozen at -20 ℃ or -80 ℃, and repeated freezing and thawing should be avoided. Hemolytic samples are generally not suitable for testing.

3. operating procedure

• preparationRemove the reagent kit from the refrigerator and allow it to equilibrate to room temperature 30 minutes in advance. At the same time, dilute the concentrated washing solution with distilled water or deionized water according to the required ratio for later use.

• sample additionSet blank holes, standard holes, and sample holes. Add sample xi release solution to the blank well, different concentrations of standard solution to the standard well, and processed sample to the sample well. The amount of sample added to each well is usually 100 μ L. Gently mix and incubate at 37 ℃ for 90 minutes.

• washAfter incubation, discard the liquid in the wells, fill each well with washing solution, soak for 1-2 minutes, shake dry, repeat washing 5 times, and ensure that the bottom is cleaned of unbound substances.

• Add enzyme labeled antibodyAdd 100 μ L of diluted enzyme-linked immunosorbent assay working solution to each well and incubate at 37 ℃ for 60 minutes.

• Wash againRepeat the above washing steps.

• color developmentAdd 90 μ L of substrate solution to each well, gently mix well, and develop color at 37 ℃ in the dark for 15-30 minutes (depending on the actual color development situation).

• Terminate reactionAdd 50 μ L of termination solution to each well, and the color of the solution will change from blue to yellow.

• readingMeasure the absorbance (OD value) of each well at a wavelength of 450nm using an enzyme-linked immunosorbent assay reader within 15 minutes after adding the termination solution.

• calculation resultDraw a standard curve with standard concentration as the horizontal axis and OD value as the vertical axis. Find the corresponding concentration from the standard curve based on the OD value of the sample. If the sample has been diluted, multiply it by the corresponding dilution factor to obtain the actual concentration.

4. performance metrics

• Testing ScopeDifferent reagent kits have differences, and the common detection range is between 15.6-1000 pg/mL, which can meet the detection needs of IL-7 content in various samples.

• sensitivityGenerally, it can be as low as 7.8 pg/mL or even lower, and can detect trace amounts of IL-7 in the sample.

• specificityIt has high specificity for IL-7 and almost no cross reactivity with other cytokines and other substances.

• repetitivenessThe intra plate coefficient of variation is generally less than 10%, and the inter plate coefficient of variation is less than 15%, ensuring the stability and reliability of the test results.