Human CCL5/RANTES ELISA Kit

Description

Accurate measurement of human CCL5, also known as RANTES, requires an assay that can quantify low concentrations across commonly used biological sample types while supporting consistent plate-based workflows. The Human CCL5/RANTES ELISA Kit is designed for the quantitative determination of human CCL5/RANTES concentrations in serum, plasma and cell culture supernatants.

This quantitative sandwich ELISA uses a microplate pre-coated with a monoclonal antibody specific for human CCL5. Bound CCL5 is detected with a biotin-linked detection antibody, followed by streptavidin-HRP and TMB color development. The resulting color intensity is proportional to the amount of human CCL5/RANTES present in the sample.

The assay provides a sensitivity of 0.53 pg/mL and a standard range of 15.63–1,000 pg/mL. Absorbance is measured at 450 nm using a standard microplate reader, with wavelength correction at 570 nm or 630 nm when available.

For research use only. Not for use in diagnostic procedures.

Quick Details

Specifications

Features

• Quantitative measurement of human CCL5/RANTES concentrations.

• Quantitative sandwich ELISA assay format.

• Monoclonal anti-human CCL5 capture antibody pre-coated on the microplate.

• Biotin-linked CCL5 detection antibody with streptavidin-HRP detection.

• HRP-TMB colorimetric readout at 450 nm.

• Suitable for human serum, plasma and cell culture supernatants.

• Compatible with EDTA, citrate and heparin plasma.

• 0.53 pg/mL stated sensitivity.

• 15.63–1,000 pg/mL standard range.

• Available in 48T and 96T kit sizes.

Applications

The Human CCL5/RANTES ELISA Kit is intended for quantitative CCL5 measurement in research samples. Supported applications include:

• Quantification of human CCL5/RANTES in serum samples.

• Measurement of human RANTES concentrations in EDTA, citrate or heparin plasma.

• Detection of secreted CCL5 in cell culture supernatants.

• Human chemokine profiling studies.

• Research involving CCL5-associated inflammatory responses.

• Studies of leukocyte recruitment to inflammatory sites.

• Research involving the chemotactic activity of CCL5 toward T cells, eosinophils and basophils.

• Studies involving cytokine-associated natural killer cell activation.

• Research examining CCL5 released by CD8-positive T cells.

This product is intended exclusively for research use. It is not intended for disease diagnosis, patient screening or therapeutic decision-making.

Principle of the Assay

This assay employs a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human CCL5 is pre-coated onto the microplate.

1. Standards and samples are added to the coated wells.

2. A biotin-linked detection antibody specific for human CCL5 is added.

3. Human CCL5 present in the sample is bound between the immobilized capture antibody and the detection antibody.

4. Unbound substances are removed by washing.

5. Streptavidin-HRP is added and binds to the biotin-linked detection antibody.

6. After another wash, TMB substrate is added.

7. Color develops in proportion to the amount of CCL5 bound in the well.

8. Stop Solution is added, and absorbance is measured at 450 nm.

Standard Curve

A standard curve must be generated during each assay. The standard series described in the product manual includes the following concentrations:

The standard curve data supplied in the manual are provided for demonstration only. A new standard curve must be run with each assay.

Sample Collection and Preparation

Cell Culture Supernatants

Remove particulates by centrifugation. Test freshly prepared samples immediately or divide them into aliquots and store at or below -20°C. Avoid repeated freeze-thaw cycles.

Cell culture supernatants may be added at 100 µL per well. Samples with high CCL5/RANTES concentrations may be diluted using the corresponding cell culture medium.

Serum

Collect blood using a serum separator tube and allow it to clot for 30 minutes. Centrifuge for 10 minutes at 1,000 × g. Remove the serum and test it immediately or divide it into aliquots and store at or below -20°C.

Normal serum samples require a 100-fold dilution. The suggested dilution is:

5 µL serum sample + 495 µL 1X Assay Buffer

Plasma

Collect plasma using EDTA, citrate or heparin as the anticoagulant. Centrifuge at 1,000 × g within 30 minutes of collection. Test freshly prepared plasma immediately or divide it into aliquots and store at or below -20°C.

Normal plasma samples require a 100-fold dilution. The suggested dilution is:

5 µL plasma sample + 495 µL 1X Assay Buffer

General Sample Handling

• Clarify samples containing visible precipitates before testing.

• Do not use grossly hemolyzed or lipemic specimens.

• Samples tested within 24 hours may be stored at 2–8°C.

• For longer storage, aliquot samples and store at -20°C.

• Avoid repeated freeze-thaw cycles.

• Dilution with 1X Assay Buffer may be necessary for samples with high CCL5/RANTES concentrations.

Assay Workflow

1. Bring all kit reagents and samples to room temperature before use.

2. Prepare the standards, samples, detection antibody, streptavidin-HRP, Assay Buffer and Washing Buffer.

3. Wash the required microplate wells once using 300 µL of 1X Washing Buffer per well.

4. Add 100 µL of each two-fold diluted standard to the standard wells.

5. Add 100 µL of Assay Buffer or culture medium to the blank well.

6. For serum or plasma, add 80 µL of 1X Assay Buffer and 20 µL of prediluted sample per well.

7. For cell culture supernatants, add 100 µL of sample per well.

8. Add 50 µL of diluted detection antibody to each well.

9. Seal the plate and incubate for 2 hours at 25°C ± 3°C on a microplate shaker at 300 rpm.

10. Wash the wells six times using 300 µL of 1X Washing Buffer per wash.

11. Add 100 µL of diluted streptavidin-HRP to each well.

12. Incubate for 45 minutes at 25°C ± 3°C on a microplate shaker at 300 rpm.

13. Wash the wells six times.

14. Add 100 µL of TMB substrate to each well.

15. Incubate for 5–30 minutes at room temperature while protecting the plate from light.

16. Add 100 µL of Stop Solution to each well.

17. Measure optical density within 30 minutes at 450 nm, with correction at 570 nm or 630 nm when available.

Kit Components

Components from reagent kits with different batch numbers must not be used interchangeably.

Additional Equipment Required

• Microplate reader capable of measuring absorbance at 450 nm.

• Optional wavelength correction at 570 nm or 630 nm.

• Single-channel and multichannel micropipettes with disposable tips.

• 50–300 µL adjustable multichannel micropipette.

• Multichannel micropipette reservoir.

• Polypropylene tubes for standard and sample dilution.

• Deionized or distilled water.

• Laboratory vessels required for reagent preparation.

• Microplate shaker capable of operating at 300 rpm.

Storage and Shipping

• Unopened kit: Store at 2–8°C and refer to the product label for the expiration date.

• Opened microplate: Return unused strips to the aluminum foil bag, reseal the bag and store at 2–8°C for approximately one month.

• Reconstituted standard: Store at -20°C for approximately one month and discard after single-use reconstitution.

• Other opened components: Store at 2–8°C for approximately one month.

• Prepared 1X Washing Buffer: Store at 2–25°C for up to 30 days.

• Prepared 1X Assay Buffer: Store at 2–8°C for up to 30 days.

• Shipping condition: Transport at approximately 4°C with blue ice.

Documents

Human CCL5/RANTES ELISA Kit User Manual

Frequently Asked Questions

For research use only. Not for use in diagnostic procedures.