Canine ELISAs

IL-10

Capture Antibody:

1. Dilute capture anti-IL-10 purified (2 ug/ml) in PBS and add 100 ul to appropriate # of wells of Coster EIA medium binding plates. Seal plate and Incubate at room temp overnight.

2. Wash with wash buffer 3 times. Pat Dry. (complete removal of liquid at each step is important)

3. Add 300 ul of Blocking Buffer (PBS with 1% BSA, 5% sucrose and 0.05% NaN₃) Incubate for minimum 60 min at room temp.

4. Repeat wash as in step 2.

Samples:

5. Add 100 ul of Dilution Buffer (DB) to the 1st 2 rows. Then add 100 ul of sups to appropriate wells and 100 ul of diluted STD to wells 4-6 of the first row. Add 100 ul of IL-10 STD at  pg/ml to well 4-6 and do 1:2 serial dilution in triplicate for rows 1 and 2. Start STD at 8 ng/ml. Cover with adhesive strip and  Incubate at room temp for 2 hours.

6. Repeat the aspiration/wash as in step 2.

Detection Antibody

7. Add 100 ul of biotin-anti-IL-10 (100ng/ml diluted in diluent), cover with adhesive and incubate room temp for 2 hours.

8. Repeat the aspiration/wash as in step 2

Streptavidin-HRP

9. add 100 ul steptavidin HRP, cover plate and incubate at 20 min at room temp

10. Repeat the aspiration/wash as in step 2

Substrate

11. Add 100 ul of substrate solution (TMB) to each well, incubate for 20-30 minutes at room temp (avoid placing in direct light)

12. Add 50 ul of stop solution to each well. Gently tap the plate to ensure thorough mixing.

13. Determine the optical density within 30 minutes, at 450 nm.

Wash Buffer  0.05% Tween 20 in PBS, pH 7.4

Blocking Buffer    PBS     + 1.0% BSA, 5% sucrose (for 50 ml, use 0.5 g BSA & 2.5 g sucrose) (same as BB for IFN-α)

Diluent        1% BSA in PBS, pH 7.4

Substrate  (Tetramethylbenzidine)

Stop solution  1M H₂SO₄

INF-gamma

Capture Antibody

1. Dilute capture INF-gamma purified (1 ug/ml) in PBS and add 100 ul to appropriate # of wells of Coster EIA medium binding plates. Seal plate and Incubate at room temp overnight.

2. Wash with wash buffer 3 times. Pat Dry. (complete removal of liquid at each step is important)

Blocking

3. Add 300 ul of Blocking Buffer (Reagent Diluent) (1% BSA in PBS, pH 7.2-7.4, 02 um filtered) (for 50 ml use 0.5 g BSA) Incubate for minimum 60 min at room temp.

4. Repeat wash as in step 2.

Samples

5. Add 100 ul of Reagent Diluent (RD) to the 1st 2 rows. Then add 100 ul of sups to appropriate wells and 100 ul of diluted STD to wells 4-6 of the first row. Add 100 ul of IFN-gamma STD at  2000pg/ml to well 4-6 and do 1:2 serial dilution in triplicate for rows 1 and 2. Cover with adhesive strip and  Incubate at room temp for 2 hours.

6. Repeat the aspiration/wash as in step 2.

Detection Antibody

7. Add 100 ul of biotin-anti-INF-gamma (100ng/ml diluted in Reagent Diluent), cover with adhesive and incubate room temp for 2 hours.

8. Repeat the aspiration/wash as in step 2

Streptavidin-HRP

9. Add 100 ul of Streptavidin-HRP solution to each well, cover the plate and incubate for 20-30 minutes at room temp (avoid placing in direct light)

10. repeat the aspiration/wash as in step 2.

Substrate

11. Add 100 ul substrate solution (TMB) and  incubate 20-30 min

12.  Add 50 ul of stop solution to each well. Gently tap the plate to ensure thorough mixing.

13. Determine the optical density immidiately, at 450 nm.

Wash Buffer 0.05% Tween 20 in PBS, pH 72.-74

Reagent Diluent (Blocking Buffer)   1% BSA in PBS, pH 7.2-74., 0.2 um filtered

Substrate  (Tetramethylbenzidine)

Stop solution  1M H2SO4

IL-4

Capture Antibody

1. Dilute capture anti-IL-4 purified (1 ug/ml) in PBS and add 100 ul to appropriate # of wells of Coster EIA medium binding plates. Seal plate and Incubate at room temp overnight.

2. Wash with wash buffer 3 times. Pat Dry. (complete removal of liquid at each step is important)

Blocking

3. Add 300 ul of Blocking Buffer (Reagent Diluent) (1% BSA in PBS, pH 7.2-7.4, 02 um filtered) (for 50 ml use 0.5 g BSA) Incubate for minimum 60 min at room temp.

4. Repeat wash as in step 2.

Samples

5. Add 100 ul of Reagent Diluent (RD) to the 1st 2 rows. Then add 100 ul of sups to appropriate wells and 100 ul of diluted STD to wells 4-6 of the first row. Add 100 ul of IFN-gamma STD at  50ng/ml (or other appropriate concentration) to well 4-6 and do 1:2 serial dilution in triplicate for rows 1 and 2. Cover with adhesive strip and  Incubate at room temp for 2 hours.

6. Repeat the aspiration/wash as in step 2.

Detection Antibody

7. Add 100 ul of biotin-IL-4 (100ng/ml diluted in Reagent Diluent), cover with adhesive and incubate room temp for 2 hours.

8. Repeat the aspiration/wash as in step 2

Streptavidin-HRP

9. Add 100 ul of Streptavidin-HRP solution to each well, cover the plate and incubate for 20-30 minutes at room temp (avoid placing in direct light)

10. repeat the aspiration/wash as in step 2.

Substrate

11. Add 100 ul substrate solution (TMB) and  incubate 20-30 min

12.  Add 50 ul of stop solution to each well. Gently tap the plate to ensure thorough mixing.

13. Determine the optical density immidiately, at 450 nm.

Wash Buffer 0.05% Tween 20 in PBS, pH 72.-74

Reagent Diluent (Blocking Buffer)   1% BSA in PBS, pH 7.2-74., 0.2 um filtered

Substrate  (Tetramethylbenzidine)

Stop solution  1M H2SO4