The interleukin-5 receptor (soluble) ELISA KIT adopts double antibody sandwich ELISA technology to accurately quantify the receptor in serum and other samples. It is easy to operate and helps in research fields such as immunology.
Interleukin-5 receptor (soluble) ELISA KIT
Interleukin-5 receptor (soluble) ELISA KITIt is a tool used for quantitative detection of soluble interleukin-5 receptor content in biological samples, playing an important role in scientific research and helping to gain a deeper understanding of physiological and pathological processes such as immune regulation and inflammatory response. Here are several aspects to introduce to you:
Detection PrincipleUsing double antibody sandwich enzyme-linked immunosorbent assay (ELISA) technology. The enzyme-linked immunosorbent assay (ELISA) plate of the kit is pre coated with specific antibodies against soluble interleukin-5 receptor. After adding the sample, the soluble interleukin-5 receptor in the sample specifically binds to the solid-phase antibody. Next, enzyme labeled anti-soluble interleukin-5 receptor antibodies are added, which form a "solid-phase antibody soluble interleukin-5 receptor enzyme-linked antibody" complex with the receptor already bound to the solid-phase antibody. After thorough washing to remove unbound substances, the substrate solution (such as 3,3 ', 5,5' - tetramethylbenzidine, or TMB) is added. Under the catalysis of enzymes in enzyme-linked antibodies, the substrate undergoes a color reaction, and the depth of color is proportional to the content of soluble interleukin-5 receptors 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 a standard curve, the concentration of the receptor in the sample can be calculated.
Kit components
Enzyme-linked immunosorbent assay (ELISA) plateThere are specifications of 96 or 48 wells, which have been pre coated with anti soluble interleukin-5 receptor antibodies and serve as carriers for the reaction.
reference standardUsually it is freeze-dried and comes with 2 bottles. Before use, standard dilution solution 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., 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 effective detection range of the reagent kit.
Enzyme labeled antibodyHRP (horseradish peroxidase) labeled anti soluble interleukin-5 receptor antibody, which is a concentrated solution, needs to be diluted with enzyme labeled antibody diluent as required 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 certain proportion when used to wash the enzyme-linked immunosorbent assay (ELISA) plate, remove unbound substances, and 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 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 ℃, then 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.
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 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
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.
