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 pipetted onto the plate at less than 2μg/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 100μl 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 curve.
8. Incubate for 2h at room temperature.
Note: 2hours is usually enough to obtain a strong signal. Stronger staining will often be 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 pipet.
11. After sufficient color development adds 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
· BluePhos: 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.
1% BSA, serum, non-fat dry milk, casein, gelatin in PBS.
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 binding.