The interleukin-6 ELISA KIT uses a double antibody sandwich ELISA method to accurately quantify interleukin-6 in serum and other samples. It is easy to operate and provides key data for research in immunology, inflammation, and other fields.
Interleukin-6 ELISA KIT
Interleukin-6 ELISA KITIt is a tool for quantitative detection of interleukin-6 content in biological samples, widely used in scientific research fields such as immunology and inflammation. Here is an introduction for you:
Detection PrincipleAdopting double antibody sandwich ELISA technology. The enzyme-linked immunosorbent assay (ELISA) plate in the kit has been pre coated with anti-IL-6 specific antibodies. After adding the sample, IL-6 in the sample specifically binds to the solid-phase antibody, forming an antigen antibody complex. Subsequently, enzyme labeled anti-IL-6 antibodies are added, which further bind to IL-6 already bound to the solid-phase antibody, forming a sandwich structure of "solid-phase antibody IL-6 enzyme labeled antibody". After the washing step, remove unbound substances and then add substrate solution (such as TMB). Under the catalysis of enzymes, the substrate undergoes a color reaction, and the depth of color is proportional to the content of IL-6 in the sample. By measuring the absorbance at a specific wavelength (usually 450nm) using an enzyme-linked immunosorbent assay (ELISA) reader and comparing it with the standard curve, the concentration of IL-6 in the sample can be calculated.
Kit components
Enzyme-linked immunosorbent assay (ELISA) plateThere are 96 or 48 well specifications, pre coated with anti-IL-6 antibody, which is the carrier for the reaction.
reference standardGenerally, it is freeze-dried and usually comes in 2 bottles. Before use, it is necessary to dissolve the standard dilution solution and dilute it into different concentration gradients, such as 0, 15.6, 31.2, 62.5, 125, 250, 500, 1000 pg/mL, etc., to draw the standard curve.
Sample xi release solutionUsed to dilute test samples with excessively high concentrations, ensuring that the sample concentration is within the detection range of the reagent kit.
Enzyme labeled antibodyHRP labeled anti-IL-6 antibody, which is a concentrated solution, needs to be diluted with enzyme-linked antibody diluent as required.
Enzyme linked antibody diluentSpecially used for diluting enzyme-linked antibodies.
substrate solutionLike TMB substrate, it reacts with HRP in enzyme-linked antibodies to produce a color change.
detergentMost of them are concentrated solutions, which need to be diluted with distilled water or deionized water in proportion when used to wash the enzyme-linked plate, remove unbound substances, and reduce background interference.
Stop SolutionGenerally, it is 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 reading.
Sealing filmCover the enzyme-linked immunosorbent assay (ELISA) 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.
Sample requirements
serumAfter collecting blood, leave it at room temperature for 2 hours or overnight at 4 ℃, centrifuge at 1000 × g for 20 minutes, and take the supernatant for later use. Blood collection should use disposable equipment that is free of pyrogens and endotoxins.
plasmaAnticoagulants such as ethylenediaminetetraacetic acid (EDTA) and heparin can be used. Within 30 minutes after sample collection, centrifuge at 1000 × g at 2-8 ℃ for 15 minutes 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 at a ratio of 1:9 (w/v) and grind thoroughly on ice. Centrifuge at 5000 × g for 5-10 minutes and collect the supernatant for detection.
If tested in the short term after sample 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.
operating procedure
preparationTake out the reagent kit from the refrigerator and equilibrate it 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.
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-6 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-6 in the sample.
specificityIt has high specificity for IL-6 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.
