Interleukin-7 receptor (soluble) ELISA KIT

Interleukin-7 receptor (soluble) ELISA KIT

Interleukin-7 receptor (soluble) ELISA KIT, is a tool used for quantitative detection of soluble interleukin-7 receptor content in biological samples, widely used in fields such as immunology research.

1. Detection PrincipleAdopting the double antibody sandwich ELISA method. The microplate in the reagent kit has been pre coated with specific antibodies against soluble interleukin-7 receptor. After adding the sample, the soluble interleukin-7 receptor in the sample binds to the solid-phase antibody. Subsequently, enzyme labeled anti soluble interleukin-7 receptor antibodies are added, which will form a "solid-phase antibody soluble interleukin-7 receptor enzyme labeled antibody" complex with the receptor already bound to the solid phase. After washing to remove unbound substances, substrate solution (such as TMB) is added. Under the catalytic action of the enzyme, the substrate undergoes a color reaction, and the color intensity is proportional to the content of soluble interleukin-7 receptor 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.

2. Kit components

• Enzyme-linked immunosorbent assay (ELISA) plate96 or 48 wells, pre coated with anti soluble interleukin-7 receptor antibody, serve as carriers for the reaction.

• reference standardUsually 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., to draw the standard curve.

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

• Enzyme labeled antibodyHRP labeled anti soluble interleukin-7 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 to produce a color change.

• detergentGenerally, it is a concentrated solution that needs to be diluted with distilled water or deionized water in 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 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 ℃, 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 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, remove cell debris and impurities, and then take the supernatant.

• tissue homogenateWash the tissue with pre cooled PBS to remove residual blood, cut the tissue into pieces, and add PBS at a ratio of 1:9 (w/v). 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 stored for a long time, it needs to 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 let it equilibrate to room temperature (approximately 30 minutes). Dilute and concentrate the washing solution.

• sample additionSet blank holes, standard holes, and sample holes. 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 againSame as 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.

5. performance metrics

• Testing ScopeCommonly ranging from 31.2-2000 pg/mL, it can meet the detection needs of soluble interleukin-7 receptor content in various samples.

• sensitivityIt can be as low as 15.6 pg/mL or even lower, and can detect trace amounts of the receptor in the sample.

• specificityHas high specificity for soluble interleukin-7 receptor and no significant 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 different batches of reagent kits.

• Use disposable suction tips during operation to avoid cross contamination and ensure accurate sample addition.

• Thoroughly washing is the key to ensuring accurate results, and not washing the bottom of the plate can easily lead to false positives.

• 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.

• Termination fluid 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 and cannot be used for clinical diagnosis.