Enzyme Linked Immunosorbent Assay (ELISA)

Described here is a Direct ELISA, a procedure which will provide you with a titre of your primary IgY antibody:

Reagents and Buffers:

Coating Buffer (0.05 M Carbonate Buffer, pH 9.6)
1.9 g Na₂CO₃*H₂O
2.9 g NaHCO₃

QC to volume of 1 L wtih distilled water and mix thoroughly. Check the pH, adusting if necessary. Store in fridge, indicating a 2-week expiry date.

1 M Potassium Phosphate Buffer, pH 7.3

660 ml 1 M K₂HPO₄
330 ml 1 M KH₂PO₄
Mix thoroughly. Check pH. Adjust if necessary (to lower pH, add more 1M KH₂PO₄; to raise pH, add more 1M K₂HPO₄). Store in fridge.

Phosphate Buffered Saline (PBS)

30 ml 5 M NaCl
10 ml 1 M Potassium Phosphate buffer, pH 7.3

QC to volume of 1 L wtih distilled water and mix thoroughly. Store in fridge.

Phosphate Buffered Saline-Tween, 0.05% (PBS-Tween)

Add 0.5 mL Tween 20 to 1 L PBS.

Blocking Buffer (2% milk in PBS-Tween)

Dissolve 2 g non fat dry milk powder in 100 ml PBS-Tween. Store in fridge for up to a week.

Diluent Buffer

Dissolve 0.3 g non fat dry milk powder in 100 ml PBS-Tween Store in fridge for up to a week.

0.1M Citric Acid

2 g citric acid

QC to volume of 100 mL wtih distilled water and mix thoroughly. Store in fridge.

0.1M Sodium Phosphate Dibasic (Na₂HPO₄)

14.2 g Na₂HPO₄

QC to volume of 100 mL wtih distilled water and mix thoroughly. Store in fridge.

Citrate Phosphate Buffer, pH 4.0

Combine equal amounts of 0.1M Citric Acid and 0.1M Na₂HPO₄.

Mix thoroughly. Check pH. Adjust if necessary (to lower pH, add more citric acid, to raise pH, add more Na₂HPO₄).

Procedure

1. Prepare your experiment by noting on your 96-well ELISA plate, which IgY lots are being tested. Make sure to include a negative control (wells with no antigen, just coating buffer) as well as a positive control (which may be an antibody that you know is positive OR your primary antibody diluted similarly to your antigen).
2. Dilute the antigen to 2.0 ug/ml (peptide, 5 ug/ml) in Coating Buffer. Add 100 ul to each well, cover the plate and incubate overnight at 4° C.
3. Next day, flick out contents of wells and wash once with PBS-Tween. Pat the plate dry on a paper towel.
4. Block the rest of the protein binding sites on your plate by adding 200 ul Blocking Buffer to each well. Cover plate and incubate for 2 hours at room temperature. Wash plates 3 time with PBS-Tween.
5. To each well add 100 ul Diluent Buffer. Dilute test antibodies to 300 ug/ml (in diluent buffer) and add 50 ul to appropriate wells in row A only. Serial dilute the antibody by mixing with micropipette contents of wells in row A, 8 times and removing 50 ul and adding to row B. Repeat down to row G, discarding the last 50 ul/well from row G. Leave row H blank. Incubate plate(s) for 1-2 hours at room temperature.
6. Wash plates 3 times with PBS-Tween. To each well add 100 ul Horseradish Peroxidase (HRP) Donkey anti-IgY (Cat. # DAIgY-HRP), diluted 1 :5000 in Diluent Buffer. Incubate plate(s) for 1 hour at room temperature.
7. Wash plates 3 times with PBS-Tween. Pat the plate dry on a paper towel. Now, develop the plates by adding the enzyme substrate. There are a number of substrates available commercially. We use ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) (Sigma), adding 0.4 mg to Citrate Phosphate buffer. Just before adding 100 ul to each well, add 10 ul 30% hydrogen peroxide (H₂O₂) per 100 ml substrate solution. Incubate approximately 20 minutes or until green colour develops. Measure colour at 410 nm setting in ELISA reader.