Interleukin-6 receptor ELISA KIT

Interleukin-6 receptor ELISA KIT

Interleukin-6 receptor ELISA KITIt is a tool used in scientific research to quantitatively detect the content of interleukin-6 receptor in biological samples. IL-6R plays a crucial role in immune regulation and inflammatory response processes, and detecting its content is of great significance for related research.

1. Detection PrincipleThe reagent kit often uses double antibody sandwich enzyme-linked immunosorbent assay. Pre coated with specific antibodies against IL-6R on an enzyme-linked immunosorbent assay (ELISA) plate. After adding the sample, IL-6R in the sample specifically binds to solid-phase antibodies. Next, enzyme labeled anti-IL-6R antibody is added, which forms a "solid-phase antibody IL-6R enzyme labeled antibody" complex with IL-6R already bound to the solid-phase antibody. After thorough washing to remove unbound substances, substrate solution (such as TMB) is added. Under the catalysis of enzymes, the substrate undergoes a color reaction, and the color intensity is proportional to the content of IL-6R in the sample. Measure the absorbance at a specific wavelength (usually 450nm) using an enzyme-linked immunosorbent assay (ELISA) reader, compare it with the standard curve, and calculate the concentration of IL-6R in the sample.

2. Composition of reagent kit

• Enzyme-linked immunosorbent assay (ELISA) plate96 or 48 wells, pre coated with anti-IL-6R antibody, serve as carriers for the reaction to occur.

• reference standardGenerally, it is freeze-dried and 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, 31.2, 62.5, 125, 250, 500, 1000 pg/mL, etc., for drawing 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-6R antibody, which is a concentrated solution, needs to be diluted with enzyme labeled antibody diluent as required when used.

• Enzyme labeled antibody xi release solutionSpecially used for diluting enzyme-linked antibodies.

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

• detergentUsually a concentrated solution, it needs 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.

• Termination liquidIt 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 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.

3. 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.

4. 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 the 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.

5. performance metrics

• Testing ScopeDifferent reagent kits have differences, and the common detection range is 31.2-2000 pg/mL, which can meet the detection needs of IL-6R content in various samples.

• sensitivityGenerally, it can be as low as 15.6 pg/mL or even lower, and can detect trace amounts of IL-6R in the sample.

• specificityIt has high specificity for IL-6R and almost no cross reactivity with other cytokine receptors 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.

6. Precautions

• Strictly follow the instructions and do not mix reagents from different batches of reagent kits.

• Use disposable suction tips during operation to prevent cross contamination, and ensure precise sample addition.

• Adequate washing is the key to ensuring accurate results, as washing the plate without washing the bottom can easily lead to false positive results.

• The substrate solution is sensitive to light and needs to be stored and used in the dark. The color reaction should be carried out under dark conditions.

• Terminating liquid is corrosive, please take precautions during operation to avoid contact with skin and eyes.

• It is recommended to perform double well testing on both standard samples and samples to improve the accuracy of the results. This kit is only for scientific research purposes and cannot be used for clinical diagnosis.