Elisa Protocols

IL-12 p40/p70 ELISA

1. Dilute capture anti-IL-12p40/p70 purified (10 ug/ml) in 0.1 M NaHCO3 (pH 8.2) and add 50 ul to appropriate # of weels of Coster EIA medium binding plates. Incubate at 4C for overnight or in the incubator for 2 hrs.

2. Wash with nanopure ater 1X. Pat Dry.

3. Add 150 ul of Blocking Buffer (PBS with 3.0% BSA, 0.05% Tween 20) Incubate 60 min at 37C.

4. Remove. Pat Dry.

5. Add 50 ul of Diltuion Buffer (DB) to the 1st 2 rows. Then add 50 ul of sups (or 25 ul of serum + 25 ul of DB) to appropriate wells and 50 ul of diluted STD to well 406 of the first row. Add 50 ul of IL-12 STD at 12,500 pg/ml to well 4-6 and do 1:2 serial dilution in triplicate for teh rows 1 and 2. Wells 1-3 of the 1st row are the blanks for the ELISA. Incubate at 37C for 60-120 min.

6. Wash with nanopure water 2X. Pat Dry.

7. Add 50 ul of biotin-anti-IL-12 (2ug/ml) (Detection Ab) dilute in DB and incubate 37C for 60 min.

8. Wash with nanopure water 2X pat Dry.

9. Add 50 ul of streptavidin-HRP (1:1000). Place at 37C for 30 min.

10. Wash with nanopure water 4-5X. This wash is the most important. Pat Dry.

11. Add 50 ul of TMB Substrate and allow the color to develop at Rm Temp (generally 5-30 min)

12. Add 50 ul of Stop (1N H2SO4) Read at 450.

BLOCKING BUFFER

    PBS     + 3.0% bsa 0.05% tWEEN20

    DILUTION BUFFER (DB)

        PBS + 0.05% Tween 20 + 0.5% BSA

    SUBSTRATE

        Sigma one-step TMB (T-8540)

TNFα ELISA

1. Dilute capture anti-TNFα purified (8 ul/ml) in 0.1 M NaHCO3 (pH 9.5) and add 50 ul to appropriate # of weels of Coster EIA medium binding plates. Incubate at 4C for overnight or in the incubator for 2 hrs.

2. Wash with nanopure ater 1X. Pat Dry.

3. Add 150 ul of Blocking Buffer (PBS with 1.0% lipid-free BSA, 0.05% Tween 20) Incubate 60 min at 37C.

4. Remove. Pat Dry.

5. Add 50 ul of Diltuion Buffer (DB) to the 1st 2 rows. Then add 50 ul of sups (or 25 ul of serum + 25 ul of DB) to appropriate wells and 50 ul of diluted STD to well 406 of the first row. Add 50 ul of IL-12 STD at 12,500 pg/ml to well 4-6 and do 1:2 serial dilution in triplicate for teh rows 1 and 2. Wells 1-3 of the 1st row are the blanks for the ELISA. Incubate at 37C for 60-120 min.

6. Wash with nanopure water 2X. Pat Dry.

7. Add 50 ul of biotin-anti-IL-12 (2ug/ml) (Detection Ab) dilute in DB and incubate 37C for 60 min.

8. Wash with nanopure water 2X pat Dry.

9. Add 50 ul of streptavidin-HRP (1:1000). Place at 37C for 30 min.

10. Wash with nanopure water 4-5X. This wash is the most important. Pat Dry.

11. Add 50 ul of TMB Substrate and allow the color to develop at Rm Temp (generally 5-30 min)

12. Add 50 ul of Stop (1N H2SO4) Read at 450 nm.

BLOCKING BUFFER

    PBS + 1.0% Lipid-free BSA  0.05% tWEEN20

    DILUTION BUFFER (DB)

        PBS + 1 + 0.05% Tween 20 + 0.5% BSA

    SUBSTRATE

        Sigma one-step TMB (T-8540)

Sample EILSA Protocol for IL-12 p40/p70

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