Neutralizing Bioassay Protocols

Introduction

Antibodies that block binding of cytokines to their specific receptors and neutralize their effects are critical in studies of cytokine function. The following three protocols describe in vitro bioassays using neutralizing anti-mouse and anti-human cytokine antibodies. In general, the cytokine bioassay protocols are modified to pre-incubate the cytokine of interest with the specific neutralizing antibody prior to addition to the responding cells. This prevents cytokine binding to its receptor on the responding cells, thence inhibiting the cytokine effect. The chart below summarizes the general optimized experimental conditions for the neutralization assays using indicator cell lines. When working with other cell types, cytokine concentrations, neutralizing antibody concentrations and incubation times may need to be determined by the investigator.

Protocol A: Antibody Neutralization of Cytokine-induced Proliferation of Indicator Cell Lines

What You Need

Materials

• Indicator cell line (see Quick Guide Chart for a given cytokine)
• Culture Medium (RPMI supplemented with 10% FBS)
• Assay Medium (RPMI supplemented with 10% FBS)
• 96-well flat-bottom culture plate (Costar Cat. No. 3595)
• MTT solution (Sigma Cat. No. M5655) 5mg/ml stock in PBS kept at room temperature (protect from light)
• MTT Lysing Solution 20% SDS/50% DMF

Instruments

• Pipettes and pipettors
• Humidified incubator
• 96-well micro test spectrophotometer

Experiment Duration

• 24-48 hour incubation (see Quick Guide Chart)
• 1 hour assay preparation

Method

1. Add 50 µl/well of Assay Medium to each well of the 96-well Assay plate.
2. Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the Assay plate from row 3 to 12. Leave rows 1 and 2 blank (control rows).
3. Add 50 µl/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the assay plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 blank (cells alone).
4. Incubate the plate at 37°C, 5% CO₂ in a humidified incubator for 2 hours.
5. Wash indicator cells 3 times with RPMI1640 and resuspend in Assay Medium at a density of 2-3.5x10⁵ cells/ml (see Quick Guide Chart).
6. Add 100 µl of cell suspension to each well.
7. Incubate cells for 24-48 hours (see Quick Guide Chart) at 37°C, 5% CO₂ in a humidified incubator.
8. Add 10 µl/well of 5 mg/ml MTT solution to the plate and incubate for 4 hours.
9. Add 50 µl/well of MTT Lysing Solution to the plate and incubate overnight.
10. Read plate at 570-650 nm.
11. Graph standard curve and analyze data.

Protocol B: Antibody Neutralization of TNFa-Induced Killing of L929 Cell Line

What You Need

Materials

• L929 mouse fibroblast line (ATCC Cat. No. CCL-1)
• Culture Medium (RPMI supplemented with 10% FBS)
• Assay Medium (RPMI supplemented with 2% FBS)
• 96-well flat-bottom culture plate (Costar Cat. No. 3595)
• Actinomycin D, 500 µg/ml stock aliquot kept at minus 80°C (protect from light)
• MTT solution (Sigma Cat. No. M5655) 5 mg/ml stock in PBS kept at room temperature (protect from light)
• MTT Lysing solution, 20% SDS/50% DMF

Instruments

• Pipettes and pipettors
• Humidified incubator
• 96-well micro test spectrophotometer

Experiment Duration

• 48-hour incubation (see Quick Guide Chart)
• 1 hour assay preparation

Method

1. Prepare L929 cell suspension at a density of 3.5x10⁵/ml in assay medium. Add 100 µl/well to the 96-well Assay Plate and incubate overnight at 37°C, 5% CO₂ in a humidified incubator.
2. Add 50 µl/well of Assay Medium to each well of another dilution plate.
3. Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the plate from row 3 to 12. Leave rows 1 and 2 blank (control rows).
4. Add 50 µl/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the dilution plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 blank (cells alone).
5. Incubate the dilution plate at 37°C, 5% CO₂ in a humidified incubator for 2 hours.
6. Transfer 50 µl/well of the Ab/Ag mixed solution to the corresponding well of the Assay Plate containing cells.
7. Prepare a 4 µg/ml working solution of the Actinomycin D by diluting the 500 µg/ml stock 125 times in the Assay Medium. Keep Actinomycin D solution protected from light. Add 50 µl of this working solution of Actinomycin D to each well.
8. Incubate plate for 24 hrs at 37°C, 5% CO₂ in a humidified incubator.
9. Add 10 µl/well of 5 mg/ml MTT solution to each well and incubate for 4 hours.
10. Add 50 µl of MTT Lysing Solution to each well and incubate overnight.
11. Read plate at 570-650 nm.
12. Graph standard curve and analyze data.

Protocol C: Antibody Neutralization of IFNg-Protection from Viral Infection of L929 and A549 Cell Lines

What You Need

Materials

• L929 mouse fibroblast line (ATCC Cat. No. CCL-1) or A549 human lung carcinoma (ATCC Cat. No. CCL-185)
• Culture Medium (RPMI supplemented with 10% FBS)
• Assay Medium (RPMI supplemented with 2% FBS)
• 96-well flat-bottom culture plate (Costar Cat. No. 3595)
• EMC virus, 10⁷ pfu/ml aliquots stock kept at minus 80°C
• MTT solution (Sigma Cat. No. M5655) 5 mg/ml stock in PBS kept at room temperature (protect from light)
• MTT Lysing solution, 20% SDS/50% DMF

Instruments

• Pipettes and pipettors
• Humidified incubator
• 96-well micro test spectrophotometer

Experiment Duration

• 24 hour incubation (see Quick Guide Chart)
• 1 hour assay preparation

Method

1. Prepare L929 or A549 cell suspension at a density of 3.5x10⁵/ml in assay medium. Add 100 µl/well to the 96-well Assay Plate and incubate overnight at 37°C, 5% CO₂ in a humidified incubator.
2. Add 50 µl/well of Assay Medium to each well of anther dilution plate.
3. Dilute Functional Grade (FG) anti-cytokine antibody by 2-fold serial dilution in the plate from row 3 to 12. Leave rows 1 and 2 blank (control rows).
4. Add 50 µl/well of samples and recombinant cytokine (see Quick Guide Chart for concentration) to the dilution plate from row 2 to row 12 (row 2 will be indicator cells and cytokine). Leave row 1 blank (cells alone).
5. Incubate the dilution plate at 37°C, 5% CO₂ in a humidified incubator for 2 hours.
6. Transfer 50 µl/well of the Ab/Ag mixed solution to the corresponding well of the Assay Plate containing cells.
7. Incubate the Assay Plate for 6 hours.
8. Aspirate supernatant from the wells carefully and add 100 µl/well of EMC virus solution at a density of 1-4x10⁴ pfu/ml (see Quick Guide Chart for human IFN-g: 1/250 dilution of stock of 10⁷ pfu/ml, for mouse IFN-g, 1/1000 dilution of stock of 10⁷ pfu/ml).
9. Incubate plate for 40 hours at 37°C, 5% CO₂ in a humidified incubator.
10. Add 10 µl of 5 mg/ml MTT solution to each well and incubate for 4 hours.
11. Add 50 µl of MTT Lysing Solution to each well and incubate overnight.
12. Read plate at 570-650 nm
13. Graph standard curve and analyze data.