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LifeTein ELISA Protocol

ELISA Protocol

Coating the appropriate antigen to microplate

1. Dilute the antigen to a final concentration of 20 μg/ml in PBS. Fill the microwells of a

Nunc Maxi-Sorp Immuno Plate with 50 μL of the diluted antigen.

Note: Test samples containing pure antigen are usually pipeted onto the plate at less

than 2 μg/ml. Antigen protein concentration should not be over 20 μg/ml as this will

saturate most of the available sites on the microtitre plate.

2. Incubate at 4 °C overnight or 2 h at room temperature.

3. Wash the unbound antigen off the plate by flicking the contents of the plate into the

sink, fill the wells with DI water, flick again, repeat 2X with PBS-Triton.


4. Block the remaining protein-binding sites in the coated wells by adding 200 μl

blocking buffer, 1% BSA/PBS or other blocking reagents.

5. Incubate for 30-60 minutes at Room Temperature (RT) or 4 °C overnight.

6. Wash plate as above.

Incubation with the antibody

7. Add 100 μl of the antibody, diluted at the optimal concentration in blocking buffer

immediately before use.

Note: Be sure to include positive and negative controls, and, if necessary, a standard


8. Incubate for 2 h at room temperature.

Note: 2 hours is usually enough to obtain a strong signal. Stronger staining will often

observed when incubated overnight at 4 °C.

9. Wash the plate 4 times with PBS.


10. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel


11. After sufficient color development add 100 μl of stop solution to the wells.

12. Read the absorbance (optical density) of each well with an ELISA plate reader.

Common Substrates and the appropriate plate reader setting

ABTS: 405-410 nm

TMB: non-stopped 620-650 nm, stopped 450 nm

OPD: non-stopped 450 nm, stopped 490 nm

pNPP: 405-410 nm

BluePhosTM: 595-650 nm

Buffers and reagents

Bicarbonate/carbonate coating buffer (100 mM)

3.03 g Na2CO3,

6.0 g NaHCO3

1000 ml distilled water

pH 9.6,


1.16 g Na2HPO4,

0.1 g KCl,

0.1 g K3PO4,

4.0 g NaCl (500 ml distilled water) pH 7.4.

Blocking solution

1% BSA, serum, non-fat dry milk, casein, gelatin in PBS.

Wash solution

PBS or Tris-buffered saline (pH 7.4) with 0.05% (v/v) Tween20 (TBST) or Triton.

Antibody dilution buffer

Primary and secondary antibody should be diluted in 1x blocking solution to reduce nonspecific