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eBioscience: Cytokine neutralizing protocols

Neutralizing Bioassay Protocols

 

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 will result in inhibition of binding of cytokine to its receptor on the responding cells, either established indicator cell lines or cells harvested from tissues, and thence inhibition of 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-A: Antibody Neutralization of Cytokine-induced Proliferation of Indicator Cell Lines

Protocol B-B: Antibody Neutralization of TNFa-Induced Killing of L929 Cell Line
Protocol C-C: Antibody Neutralization of IFNg-Protection from Viral Infection of L929 and A549 Cell Lines

 

Anti-Cytokine Neutralization Assay Quick Guide Chart:

 

MOUSE

Clone

Specificity

Indicator

Cytokine Conc.

(ng/ml)

Ab Top conc.

(ug/ml)

ED50

(ng/ml)

Incubation Time

(hours)

JES6-1A12

mIL-2

CTLL-2

1

1

250

24

11B11

mIL-4

CTLL-2

1

1

125

24

TRFK5

mIL-5

MC/9

1

1

60

48

MP5-20F3

mIL-6

B9

0.01

1

30

48

JES5-2A5

mIL-10

MC/9

1

1

4

48

XMG1.2

mIFN-g

L929

1

1

15

40

R4-6A2

mIFN-g

L929

1

1

15

40

MP6-XT3

mTNF-a

L929

1

10

600

24

MP1-22E9

mGM-CSF

MC/9

1

10

1250

48

 

 

HUMAN

Clone

Specificity

Indicator

Cytokine Conc.

(ng/ml)

Ab Top conc.

(ug/ml)

ED50

(ng/ml)

Incubation Time (hours)

JES4-25D2

hIL-4

TF-1

1

1

30

48

JES1-5A10

hIL-5

TF-1

1

1

15

48

MQ2-13A5

hIL-6

B9

0.05

0.05

0.8

48

JES3-9D7

hIL-10

MC/9

5

1

60

48

C8.6

hIL-12

Human PBMC

1

1

60

24

NIB42

hIFN-g

A549

1

100

5000

40

Mab1

hTNF-a

L929

1

1

60

24

 

*Protocol A-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 ul/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 ul/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 degrees 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 2-3.5x10(5) cells/ml (see Quick Guide Chart).

 

6) Add 100 ul of cell suspension to each well.

 

7) Incubate cells for 24-48 hours (see Quick Guide Chart) at 37 degrees C, 5% CO2 in a humidified incubator.

 

8) Add 10 ul/well of 5 mg/ml MTT solution to the plate and incubate for 4 hours.

 

9) Add 50 ul/well of MTT Lysing Solution to the plate and incubate overnight.