CD25 ELISA Kit

Catalog No. ABIN2472085

Product Details CD25 ELISA Kit

Target: CD25 (IL2RA) (Interleukin 2 Receptor, alpha (IL2RA))

Binding Specificity: Soluble

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 78.13-5000 pg/mL

Minimum Detection Limit: 78.13 pg/mL

Application: ELISA

Purpose: This assay employs the quantitative sandwich enzyme immunoassay technique for quantitative detection.

Sample Type: Serum, Plasma, Cell Culture Supernatant

Analytical Method: Quantitative

Specificity: SIL-2Rα / CD25, Human

Sensitivity: 2.99 pg/mL

Components: Plate, Standard, Diluent

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with correction wavelength set at 570 nm or 630 nm.
Pipettes and pipette tips.
50 μL to 300 μL adjustable multichannel micropipette with disposable tips.
Multichannel micropipette reservoir.
Beakers, flasks, cylinders necessary for preparation of reagents.
Deionized or distilled water.
Polypropylene test tubes for dilution.

Application Details

Sample Volume: 100 μL

Assay Time: 3 - 4 h

Plate: Pre-coated

Protocol: 1. Prepare all reagents and standards as directed.
2. Add 50 μL Assay Buffer to each well.
3. Add 50 μL Standard or sample per well within 15 minutes.
4. Add 50 μL Detect Antibody to each well.
5. Incubate for 2 hours at RT.
6. Aspirate and wash 6 times.
7. Add 100 μL Streptavidin-HRP to each well.
8. Incubate for 45 min at RT.
9. Aspirate and wash 6 times.
10. Add 100 μL Substrate Solution to each well. Incubate for 10 - 30 minutes at RT. Protect from light.
11. Add 100 μL Stop Solution to each well.
12. Read at 450 nm within 30 minutes. Correction 570 or 630 nm

Reagent Preparation:

If crystals form in the Buffer Concentrates, warm and gently stir them until completely dissolved. Washing Buffer (1x) Pour entire contents (50 mL) of the Washing Buffer (20x) into a clean 1000 mL graduated cylinder. Bring to final volume of 1000 mL with pure or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2 to 25 °C. Washing Buffer (1x) is stable for 30 days. Assay Buffer (1x) Pour the entire contents (5 mL) of the Assay Buffer (10x) into a clean 100 mL graduated cylinder. Bring to final volume of 50 mL with distilled water. Mix gently to avoid foaming. Store at 2 to 8 °C. Assay Buffer (1x) is stable for 30 days. Detect Antibody Mix well prior to making dilutions. Make a 1:100 dilution of the concentrated Detect Antibody solution with Assay Buffer (1x) in a clean plastic tube as needed. The diluted Detect Antibody should be used within 30 minutes after dilution. Streptavidin-HRP Mix well prior to making dilutions. Make a 1:100 dilution of the concentrated Streptavidin-HRP solution with Assay Buffer (1x) in a clean plastic tube as needed. The diluted Streptavidin-HRP should be used within 30 minutes after dilution. Sample Dilution If your samples have high sIL-2Rα content, dilute serum/plasma samples with Assay Buffer (1x). For cell culture supernates, dilute with cell culture medium. Human sIL-2Rα Standard Reconstitute Human sIL-2Rα Standard by addition of distilled water. Reconstitution volume is stated on the label of the standard vial. Swirl or mix gently to insure complete and homogeneous solubilization (concentration of reconstituted standard = 20000 pg/ mL). Allow the standard to reconstitute for 10 - 30 minutes. Mix well prior to making dilutions. Use polypropylene tubes. Human sIL-2Rα/CD25 ELISA Kit 7 For serum/plasma samples, mixing concentrated human sIL-2Rα standard (150 μL) with 150 μL of Standard Diluent creates the high standard (10000 pg/ mL). Pipette 150 μL of Standard Diluent into each tube. Use the high standard to produce a 1:1 dilution series . Mix each tube thoroughly before the next transfer. Standard Diluent serves as the zero standard (0 pg/ mL). For cell culture supernates, mixing concentrated human sIL-2Rα standard (150 μL) with 150 μL of cell culture medium creates the high standard (10000 pg/ mL). Pipette 150 μL of cell culture medium into each tube. Use the high standard to produce a 1:1 dilution series. Mix each tube thoroughly before the next transfer. Cell culture medium serves as the zero standard (0 pg/ mL).

Sample Collection: Cell
Culture Supernates:

Remove particulates by centrifugation and assay freshly prepared samples immediately or aliquot and store samples at ≤ 20 °C for later use. Avoid repeated freeze thaw cycles. Serum:

Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 10 minutes at 1000 g. Remove serum and assay freshly prepared samples immediately or aliquot and store samples at ≤ 20 °C for later use. Avoid repeated freeze thaw cycles. Plasma:

Collect plasma using EDTA, citrate or heparin as anticoagulant. Centrifuge at 1000 g within 30 minutes of collection. Assay freshly prepared samples immediately or aliquot and store samples at ≤ 20 °C for later use. Avoid repeated freeze thaw cycles. Other biological samples might be suitable for use in the assay. Cell culture supernates, serum and plasma were tested with this assay. Dilution with Assay Buffer may be needed. Note: Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. If samples are to be run within 24 hours, they may be stored at 2 to 8 °C. For longer storage, aliquot samples and store frozen at 20 °C to avoid loss of bioactive human sIL:
2Rα. Avoid repeated freeze thaw cycles

Assay Procedure:

Bring all reagents and samples to room temperature before use.
1. Prepare all reagents including microplate, samples, standards and working solution as described in the previous sections.
2. Remove excess microplate strips and return them to the foil pouch containing the desiccant pack, and reseal for further use.
3. Add 300 μL Washing Buffer (1x) per well, and allow it for about 30 seconds before aspiration. Soaking is highly recommended to obtain a good test performance. Empty wells and tap microwell strips on absorbent pad or paper towel to remove excess Washing Buffer (1x). Use the microwell strips immediately after washing. Do not allow wells to dry.
4. Add 50 μL of Assay Buffer (1x) to each well.
5. Add 50 μL of Standard or sample to each well. Ensure reagent addition is uninterrupted and completed within 15 minutes.
6. Add 50 μL of Detect Antibody to each well.
7. Seal the plate with an adhesive film. Incubate at room temperature (18 to 25 °C) for 2 hours on a microplate shaker set at 100 rpm.
8. Aspirate each well and wash by filling each well with 300 μL Washing Buffer (1x), repeat five times for a total six washes. Complete removal of liquid at each step is essential to the best performance. After the last wash, remove any remaining Washing Buffer (1x) by aspirating or decanting. Invert the plate and tap it against clean paper towels.
9. Add 100 μL of Streptavidin-HRP to each well.
10. Seal the plate with a fresh adhesive film. Incubate at room temperature (18 to 25 °C) for 45 minutes on a microplate shaker set at 100 rpm.
11. Repeat aspiration/wash as in step 8. 12. Add 100 μL of Substrate Solution to each well. Incubate for 10 - 30 minutes at room temperature. Protect from light.
13. Add 100 μL of Stop Solution to each well. The color will turn yellow. If the color in the well is green or if the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
14. Measure the optical density value within 30 minutes by microplate reader set to 450 nm. If wavelength correction is available, set to 570 nm or 630 nm. If wavelength correction is not available, subtract readings at 570 nm or 630 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Reading directly at 450 nm without correction may generate higher concentration than true value.

Calculation of Results:

Average the duplicate optical density readings for each standards and sample, then subtract the average optical density value of the zero standard. Standard Concentration as horizontal axis, optical density (OD) Value as the vertical axis, regressing the data and create a standard curve using computer software. The data may be linearized by plotting the log of the sIL-2Rα concentrations versus the log of the OD and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. Note: The finally concentration of top standard is 5000 pg/mL. If instruction in this protocol have been followed samples have been diluted by 1:1 ratio (50 μL sample + 50 μL Assay Buffer), the concentration read from the standard curve must be multiplied by the dilution factor (x2). If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Assay Precision: Intra-assay precision (precision within an assay): Three serum-based and buffer-based samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay precision (precision between assays): Three serum-based and buffer-based samples of known concentration were tested in six separate assays to assess inter-assay precision.

Spike recovery:
The spike recovery was evaluated by spiking 3 levels of human sIL-2Rα into five health human serum samples. The un-spiked serum was used as blank in these experiments. The recovery ranged from 93 % to 112 % with an overall mean recovery of 102 %.

Restrictions: For Research Use only

Handling

Buffer: Buffer contains: 0.02 % sodium azide

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Intended for research use only and are not for use in diagnostic or therapeutic procedures.
Treat all chemicals with caution because they can be potentially hazardous.
It is recommended that this product is handled only by persons who have been trained in laboratory techniques and in accordance with the principles of good laboratory practice. Wear personal protection equipment such as laboratory coat, safety glasses and gloves.
Avoid direct contact with skin or eyes. Wash immediately with water in the case of contact with skin or eyes. Avoid contact of skin or mucous membranes with kit reagents or specimens. See material safety data sheet(s) for specific advice.
Pure water or deionized water must be used for reagent preparation.
The Stop Solution provided with this kit is an acid solution. Wear personal protection equipment with caution.
Do not expose kit reagents to strong light during storage and incubation.
Rubber or disposable latex gloves should be worn while handling kit reagents or specimens.
Avoid contact of substrate solution with oxidizing agents and metal.
Avoid splashing or generation of aerosols.
Use disposable pipette tips and/or pipettes to avoid microbial or cross-contamination of reagents or specimens which may invalidate the test.
Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagent.
Exposure to acid inactivates the HRP and antibody conjugate.
Substrate solution must be warmed to room temperature prior to use.
Decontaminate and dispose specimens and all potentially contaminated materials as they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121.5 °C.
Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0 % sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite.
All reagents including microplate, samples, standards and working solution should be warmed to room temperature before use.
To obtain accurate results, using adhesive film to seal the plate during incubation is suggested.
It is recommended that all samples and standards be assayed in duplicate.
Avoid foaming when mixing or reconstituting solutions containing protein.
To avoid cross-contamination, use separate reservoirs for each reagent and change pipette tips between each standard, sample and reagent.
When using an automated plate washer, adding a 30 seconds soak period before washing step and/or rotating the plate between wash steps may improve assay precision.
When pipetting reagents, maintain a consistent order of addition from well-to-well.
Keep Substrate solution protected from direct strong light. Substrate Solution should turn to gradations of blue after a proper color development.
Read absorbance within 30 minutes after adding stop solution.
Take care not to scratch the inner surface of the microwells.

Storage: 4 °C

Storage Comment: Store kit reagents between 2 and 8 °C. Immediately after use remaining reagents should be returned to cold storage (2 to 8 °C).
Expiry of the kit and reagents is stated on labels. Expiration date of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, this reagent is not contaminated by the first handling.
Unopened Kit: Store at 2 - 8 °C (See expiration date on the label).
Opened/Reconstituted Reagents: Up to 1 month at 2 - 8 °C.
Reconstituted Standard: Up to 1 month at ≤ -20 °C in a manual defrost freezer. Avoid repeated freeze-thaw cycles.
Microplate Wells: Up to 1 month at 2 - 8 °C. Return unused strips to the foil pouch containing the desiccant pack, reseal along entire edge to maintain plate integrity. Provided this is within the expiration date of the kit

Target Details for CD25

Target: CD25 (IL2RA) (Interleukin 2 Receptor, alpha (IL2RA))

Alternative Name: IL-2Ralpha/CD25 (IL2RA Products)

Synonyms: CD25 ELISA Kit, IDDM10 ELISA Kit, IL2R ELISA Kit, TCGFR ELISA Kit, IL2RAC ELISA Kit, IL-2RA ELISA Kit, Il2r ELISA Kit, Ly-43 ELISA Kit, interleukin 2 receptor subunit alpha ELISA Kit, interleukin-2 receptor subunit alpha-like ELISA Kit, interleukin 2 receptor, alpha chain ELISA Kit, IL2RA ELISA Kit, Il2ra ELISA Kit, LOC100222957 ELISA Kit

Background: The interleukin-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, that binds and responds to a cytokine called IL-2. IL-2R is consist of three chains, an alpha chain (IL-2Rα, CD25), a beta chain (IL-2Rβ, CD122) and a gamma chain (IL-2Rγ, CD132). The α chain binds IL-2 with low affinity, the combination of β and γ together form a complex that binds IL-2 with intermediate affinity, primarily on memory T cells and NK cells.

Gene ID: 3559

NCBI Accession: NM_000417

UniProt: P01589

Pathways: JAK-STAT Signaling, Growth Factor Binding, Activated T Cell Proliferation