Direct ELISA

Enzyme-linked immunosorbent assay

Direct ELISA detects immobilized antigen using a single conjugated primary antibody. Direct detection can be performed with antigen that is immobilized onto the assay plate. The advantage of direct detection is that only one antibody is used, fewer steps are needed and nonspecific signal from secondary antibody cross-reactivity is eliminated.

Reagents

  • Bicarbonate/Carbonate Coating Buffer (100 mM)
    • 3.03 g Na2CO3
    • 6.0 g NaHCO3
    • 1000 ml distilled water
    • pH 9.6
  • Phosphate- Buffered Saline
    • 1.16 g Na2HPO4
    • 0.1 g KCl
    • 0.1 g K3PO4
    • 4.0 g NaCl (500 ml distilled water) pH 7.4
  • Blocking Buffer
    • 1% BSA/serum/non-fat dry milk/casein/gelatin in PBS
  • Wash Buffer
    • 0.05% (v/v) Polysorbate 20 in PBS

A. Plate coating

  1. Dilute the antigen to a final concentration of 20 µg/ml using the coating buffer. (NB. A concentration of 20 µg/ml will saturate available sites on the well.)
  2. Coat a 96 well plate with 100 ?l/well of the antigen. Serially dilute down the plate.
  3. Cover plate and incubate at 4°C overnight.
  4. Discard coating solution.
  5. Wash plate 3 times using wash buffer.

B. Plate blocking

  1. Block any remaining protein-binding sites in the coated wells using 200 µl/well of blocking buffer.
  2. Cover and incubate at room temperature for 1-2 hours.
  3. Wash plate 3 times using wash buffer.

C. Antibody incubation

  1. Add 100 µl/well of antibody diluted in blocking buffer. (NB. Antibody dilutions vary considerably and should be determined by the end user. As a guide, use 0.1 µg/ml final concentration for purified antibodies.)
  2. Cover and incubate at room temperature for 1-2 hours.
  3. Wash plate 3 times using wash buffer.

D. Detection

  1. Add 100 µl/well of the substrate solution.
  2. Incubate for development (if necessary).
  3. Add 100 µl/well of stop solution.
  4. Read the absorbance (optical density) using a plate reader

E. Analysis

  1. Plot a standard curve from the data obtained from the plate reader. Show concentration on the x axis (log scale) vs. absorbance on the Y axis (linear). Use the plotted standard curve graph to interpolate the concentration of the sample.