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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).

elisa

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; Gallus Immunotech), 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.

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