Interleukin-6 receptor (soluble) ELISA KIT

Interleukin-6 receptor (soluble) ELISA KIT

Interleukin-6 receptor (soluble) ELISA KITSpecially designed for quantitative detection of soluble interleukin-6 receptor content in biological samples, it has significant implications for immune and inflammatory research in the field of scientific research.

1. Detection PrincipleBased on double antibody sandwich enzyme-linked immunosorbent assay. Enzyme linked immunosorbent assay (ELISA) plates are pre coated with specific antibodies targeting soluble interleukin-6 receptors. After the sample is added, the soluble interleukin-6 receptor binds to the solid-phase antibody. Subsequently, enzyme labeled anti-soluble interleukin-6 receptor antibodies were added to form a "solid-phase antibody soluble interleukin-6 receptor enzyme labeled antibody" complex. Wash to remove unbound substances, add substrate solution (such as TMB), and under enzymatic catalysis, the substrate will develop color. The color depth is proportional to the content of the receptor in the sample. Measure the absorbance at 450nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader and compare it with the standard curve to determine the concentration of soluble interleukin-6 receptor in the sample.

2. Kit components

• Enzyme-linked immunosorbent assay (ELISA) plateThere are 96 or 48 well specifications, pre coated with antibodies as reaction carriers.

• reference standardUsually, it is a freeze-dried product that needs to be dissolved in a standard dilution solution and diluted into a series of concentration gradients, such as 0, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 pg/mL, for drawing standard curves.

• Sample xi release solutionUsed to dilute samples with excessively high concentrations to adapt the sample concentration to the detection range of the reagent kit.

• Enzyme labeled antibodyHRP labeled anti soluble interleukin-6 receptor 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.

• detergentGenerally, it is a concentrated solution that is diluted with distilled water or deionized water in proportion and used for washing enzyme-linked immunosorbent assay plates to remove unbound substances and reduce background interference.

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

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

• User ManualDetailed introduction of usage methods, precautions, performance indicators, etc.

3. Sample requirements

• serumAfter collecting blood, leave it at room temperature for 2 hours or overnight at 4 ℃, centrifuge at 1000 × g for 20 minutes to obtain the supernatant, and use disposable equipment without pyrogen or endotoxin for blood collection.

• plasmaAnticoagulants such as EDTA and heparin can be used. Within 30 minutes after sample collection, centrifuge at 1000 × g at 2-8 ℃ for 15 minutes to obtain the supernatant.

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

• tissue homogenateWash the tissue with pre cooled PBS (0.01M, pH=7.4) to remove residual blood. Cut the tissue and add PBS at a ratio of 1:9 (w/v). Grind thoroughly on ice and centrifuge at 5000 × g for 5-10 minutes to obtain the supernatant.

• If tested in the short term after sample collection, it can be stored at 4 ℃; Long term storage requires freezing at -20 ℃ or -80 ℃, and avoiding repeated freeze-thaw cycles. Hemolytic samples are generally not suitable for testing.

4. operating procedure

• preparationRemove the reagent kit from the refrigerator, equilibrate to room temperature (about 30 minutes), and dilute and concentrate the washing solution.

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

• washDiscard the liquid in the holes, fill each hole with detergent, soak for 1-2 minutes, then shake dry. Repeat washing 5 times to ensure that unbound substances are thoroughly removed.

• 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.

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

• 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 sample OD value. If the sample has been diluted, multiply it by the corresponding dilution factor to obtain the actual concentration.

5. performance metrics

• Testing ScopeCommonly ranging from 15.6-1000 pg/mL, it meets various sample testing needs.

• sensitivityIt can be as low as 7.8 pg/mL or even lower and can detect trace amounts of soluble interleukin-6 receptor.

• specificityHighly specific to this receptor, with almost no cross reactivity with other cytokine receptors.

• repetitivenessThe intra plate coefficient of variation is generally less than 10%, and the inter plate coefficient of variation is less than 15%, ensuring stable and reliable testing results.