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.

eBioscience Ready-SET-Go! ELISA reagent sets (with or without high-affinity binding microwell plates) contain the necessary reagents, buffers and diluents for performing quantitative enzyme linked immunosorbent assays (ELISA). These ELISA reagent sets are specifically engineered for accurate and precise measurement of cytokine protein levels from samples including serum, plasma, and supernatants from cell cultures.

Materials Provided in the Reagent Set

• Capture Antibody: Pre-titrated, purified antibody
• Detection Antibody: Pre-titrated, biotin-conjugated antibody
• Standard: Recombinant cytokine for generating standard curve and calibrating samples
• Coating Buffer: 10X concentrated
• Assay Diluent: 5X concentrated
• Detection enzyme: pre-titrated Avidin-HRP
• Substrate Solution: Tetramethylbenzidine (TMB) Substrate Solution
• Certificate of Analysis: Lot-specific instructions for dilution of antibodies and standards
• 96 Well Plate: NUNC Maxisorp flat-bottom or Corning Costar 9018 (included with product catalog numbers ending in suffixes -22, -44, -76, -86)

Other Materials Needed

• 96-well plate (not included in sets with catalog numbers ending in suffixes -77 or -88): Corning Costar 9018 (Cat. No. 44-2504) or NUNC Maxisorp flat-bottom (Cat. No. 44-2404)
  IMPORTANT NOTE: The use of ELISA plates which are not high affinity protein binding plates will result in suboptimal performance, e.g., low signal or inconsistent data. Do not use tissue culture plates or low protein absorption plates. Use only the Corning Costar 9018 or NUNC Maxisorp 96 well plates provided or suggested.
• Buffers
  • Wash Buffer
  • Stop Solution
• Pipettes and pipettors
• Refrigerator
• 96-well ELISA plate reader (microplate spectrophotometer)
• ELISA plate washer

Stability

eBioscience ELISA sets are guaranteed to perform as specified at least 12 months from date of receipt if stored and handled as instructed according to the accompanying datasheet and Certificate of Analysis.

Storage Instructions for Cytokine Standards

The frozen cytokine standard is already aliquoted at 20 µl per vial. Upon receipt, frozen cytokine standard should be immediately stored at -80°C; stable for at least 12 months. After thawing, quick-spin vial prior to opening. Do not re-aliquot into smaller fractions. These are single use vials. Use one time and discard. For dilution of the standard, please see instructions on the Certificate of Analysis and follow these as written.

Storage Instructions for Other Set Reagents

Store at 4°C.

IMPORTANT NOTE: Be certain that no sodium azide is present in the solutions used in this assay, as this inhibits HRP enzyme activity.

Time Requirements

• 1 overnight incubation
• 4½-hour incubations
• 1 hour washing and analyzing samples

Antibody Pairs Used in eBioscience Ready-SET-Go! Sets

Experimental Procedure

1. Coat Corning Costar 9018 or NUNC Maxisorp 96 well ELISA plate with 100 µl/well of capture antibody in Coating Buffer* (dilute as noted on Certificate of Analysis, which is included with the reagent set). Seal the plate and incubate overnight at 4°C.
   *Important: Warm the 10X Coating Buffer to room temperature prior to dilution.
2. Aspirate wells and wash 5 times with >250 µl/well Wash Buffer (diluted to 1X). Allowing time for soaking (~ 1 minute) during each wash step increases the effectiveness of the washes. Blot plate on absorbent paper to remove any residual buffer.
3. Dilute 1 part 5X concentrated Assay Diluent with 4 parts DI water.* Block wells with 200 µl/well of 1X Assay Diluent. Incubate at room temperature for 1 hour. Aspirate/wash as in step 2.
   *Important: Do not include sodium azide in any buffers, as this will inactivate the HRP.
4. Aspirate/wash as in step 2.
5. Using Assay Diluent, dilute standards as noted on the Certificate of Analysis (C of A). Add 100 µl/well of standard to the appropriate wells. Perform 2-fold serial dilutions of the top standards to make the standard curve. Add 100 µl/well of your samples to the appropriate wells. Cover or seal the plate and incubate at room temperature for 2 hours (or overnight at 4°C for maximal sensitivity).
6. Aspirate/wash as in step 2. Repeat for a total of 5 washes.
7. Add 100 µl/well of detection antibody diluted in 1X Assay Diluent (dilute as noted on C of A). Seal the 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 100 µl/well of Avidin-HRP* diluted in 1X Assay Diluent (dilute as noted on C of A). Seal the plate and incubate at room temperature for 30 minutes.
   *Important: Do not include sodium azide in any buffers, as this will inactivate the HRP.
10. Aspirate and 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. Add 100 µl/well of Substrate Solution to each well. Incubate plate at room temperature for 15 minutes.
12. Add 50 µl of Stop Solution to each well.
13. Read plate at 450 nm. If wavelength subtraction is available, subtract the values of 570 nm from those of 450 nm and analyze data.

References

Ready-SET-Go! Cytokine ELISA Set Buffers

ELISA Wash Buffer

• 1X PBS
• 0.05% Tween-20

Stop Solution

• 1M H₃PO₄ or 2M H₂SO₄

1X PBS

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

Standard Calibration

The recombinant cytokine standard of the Ready-SET-Go! is calibrated against NIBSC standards.

ELISA Troubleshooting Guide