ELISA Protocol

  1. Dilute the antigen in PBS and coat the wells of a PVC microtiter plate with 50 µl of the antigen dilution per well.
  2. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature or 1 hour at 37 degrees Celsius. (Tip: place 96 well plate in a plastic zip bag with a moist towel to help keep the plate from drying out).
  3. Remove the antigen coating solution and wash the plate three times by filling the wells with 300 µl PBS (or PBST). The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. (Tip: remember, washing is the key to good ELISA readings).
  4. Block the remaining protein-binding sites in the coated wells by adding 300 µl blocking buffer, 5% non-fat dry milk/PBS, per well.
  5. Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or, if more convenient, overnight at 4°C.
  6. Wash the plate three times with PBS.
  7.  Make 10-fold dilutions (1:100, 1:1,000, 1:10,000, 1:100,000 and 1:1,000,000) of serum and pre-immune serum (the latter as a negative control) in blocking buffer. Add 50 µl of each dilution to an antigen-coated well in duplicate. If target dilution window is narrow, 2-fold dilutions can be made instead.
  8. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.
  9. Wash the plate four times with PBS.
  10. Add 50 µl of secondary antispecies antibody conjugated to alkaline phosphatase, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use.
  11. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.
  12. Wash the plate four times with PBS.
  13. Dissolve p-Nitrophenyl phosphate at a concentration of 1 mg/ml in substrate buffer (1M diethanolamine, 0.5 mM MgCl2, pH 9.8). Add 50 µl of the substrate solution per well with a multichannel pipette.
  14. Measure the absorbance at 405 nm, using a microtiter plate spectrophotometer. Perform an end-point measurement after 1 h. Calculate the titer of the sera. The titer can be defined as the dilution of serum giving an optical density (OD) of 0.2 above the background of the ELISA after a 1-h reaction.

More Resources:

ELISA Illustrated Assay