• Cytokine ELISA Protocol

Introduction

The enzyme linked immunosorbent assay (ELISA) is used for the detection and quantification of cytokines secreted by activated cells in culture into tissue culture supernatant. Immobilizing a cytokine-specific capture antibody onto a high protein binding capacity ELISA plate enables capture of target cytokine. The captured cytokine is detected by a biotinylated cytokine-specific antibody which recognizes a distinct epitope. The sandwiched target cytokine is quantified using a colorimetric reaction based on activity of avidin-horseradish peroxidase (bound to the biotinylated detection antibody) on a specific soluble substrate (e.g., ABTS). The colored end-product is read by a spectrophotometer.

Antibody Pairs and Standards Quick Guide

Cytokine ELISA Protocol

The following protocol is a general guideline for using the eBioscience capture/detection antibody pairs.

Materials

• 96-well plate (Corning Costar 9018, Cat. No. 44-2504, or Nunc Maxisorp flat-bottom, Cat. No. 44-2404)
• Capture and detection antibodies for the cytokine of interest (see the Quick Guide Chart)
• Avidin Horse Radish Peroxidase (AV-HRP, Cat. No. 18-4100)

Buffers

• Coating Buffer D-PBS, pH 7.2 (Cat. No. 00-0044)
• Assay Diluent: eBioscience ELISA Diluent (Cat. No. 00-4202)
• Wash Buffer: 1X PBS, 0.05% Tween-20 (or eBioscience ELISA Wash Buffer Powder Cat. No. 00-0400)
• Substrate Solution: Super Aqua Blue (eBioscience Cat. No. 00-4203) or ABTS substrate (Sigma, Cat. No. A-1888)
• Stop Solution for ABTS Substrate: 20% SDS/50% DMF (Pierce Cat. No. 20672)

Instruments

• Pipettes and pipettors
• Refrigerator
• 96-well micro test spectrophotometer (405nm)
• Plate Washer: Wash bottle or automated wash machine

Experiment Duration

• 1 overnight incubation
• 5-5 1/2 hour incubations
• 1 hour washing and analyzing samples

Method

1. Coat plate with 100µl/well of capture antibody in Coating Buffer at concentration of 1-4µg/ml. Seal plate and incubate at 4°C overnight.
2. Aspirate wells and wash 3 times with at least 300µl/well Wash Buffer. Invert the plate and blot on absorbent paper to remove any residual buffer.
3. Block wells with 200µl/well of Assay Diluent. Incubate at room temperature for 1 hour.
4. Aspirate/wash as in step 2.
5. Add 100µl/well of the cytokine standard and your samples to the wells. Seal plate and incubate at room temperature for 2 hours.
6. Aspirate/wash as in step 2. Repeat for a total of 5 washes.
7. Add 100µl/well of biotin-conjugated detection antibody in Assay Diluent at a concentration of 0.5-2µg/ml. Seal plate and incubate at room temperature for 1 hour.
8. Aspirate/wash as in step 2. Repeat for a total of 5 washes.
9. Add 100µl/well of AV-HRP diluted in Assay Diluent at 1/500 dilution and incubate at room temperature for 30 minutes.
10. Aspirate/wash as in step 2. In this wash step, soak wells in wash buffer for 1 to 2 minutes prior to aspiration. Repeat for a total of 7 washes.
11. Follow the instructions for eBioscience Super Aqua Blue single component, enhanced ABTS substrate or use traditional ABTS substrate. For traditional ABTS, thaw ABTS substrate within 20 minutes of use. Add 11µl of 30% H₂O₂ per 11ml of substrate. Add 100µl/well of ABTS substrate solution to each well. Incubate plate at room temperature for 10 to 60 minutes. The color development can be stopped by adding 50µl of Stop Solution.
12. Read plate at 405nm.

Cytokine ELISA Buffers

• 1X PBS:
  • 80.0g NaCl
  • 11.6g Na₂HPO₄
  • 2.0g KH₂PO₄
  • 2.0g KCl
  • DI H₂0 up to 10.0 L
  • pH to 7.0