Direct ELISA Protocol

Reagent of Direct ELISA Protocol

Bicarbonate/carbonate coating buffer (100 mM)

Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:

3.03 g Na₂CO₃,

6.0 g NaHCO₃

1000 ml distilled water

pH 9.6,

PBS

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

Commonly used blocking agents are 1% BSA, serum, non-fat dry milk, casein, gelatin in PBS.

Wash solution

Usually PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

Antibody dilution buffer

Primary and secondary antibody should be diluted in 1x blocking solution to reduce non-specific binding.

Method of Direct ELISA Protocol

Coating antigen to microplate

• Coat microtiter plate wells with 100 µl of the appropriate coating antigen, at a concentration of between 1-10 µg/ml in coating buffer.
• Cover the plate with an adhesive plastic and incubate for 2 h at room temperature, or 4°C overnight. The coating incubation time may require some optimization.
• Remove the coating solution and wash the plate 3 times by filling the wells with 200 µl PBS. 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.

Test samples containing pure antigen are usually pipeted onto the plate at less than 2ug/ml. Pure solutions are not essential, but as a guideline, over 3% of the protein in the test sample should be the target protein. (antigen). Antigen protein concentration should not be over 20ug/ml as this will saturate most of the available sites on the microtitre plate.

Blocking

Add 150 µl of blocking solution to each well. Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or overnight at 4°C. Wash 4 times in wash buffer. The process blocks the remaining protein-binding sites in the coated wells.

Incubation with the antibody

• Add 100 µl of the antibody, diluted at the optimal concentration (according to the manufacturer's instructions) in blocking buffer immediately before use.
• Cover the plate with an adhesive plastic and incubate for 2 h at room temperature. This incubation time may require optimization. Although 2 hours is usually enough to obtain a strong signal, if a weak signal is obtained, stronger staining will often observed when incubated overnight at 4°C.
• Wash the plate four times with PBS.

Detection

• Add 100 µl of biotin-conjugated detection antibody (appropriately diluted in wash buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.
• Add 100 µl of enzyme-conjugated streptavidin (appropriately diluted in wash buffer) to each well. Incubate for 60 minutes at 37°C. Wash 3 times in wash buffer.
• Add 100 µl of the appropriate substrate solution to each well. Incubate at room temperature (and in the dark if required) for 30 minutes or until desired color change is attained.
• Read absorbance values immediately at the appropriate wavelength. Gently tap plate to ensure thorough mixing. Measure absorbance within 30 minutes.

Analysis of data

Prepare a standard curve from the data produced from the serial dilutions with concentration on the x axis (log scale) vs absorbance on the Y axis (linear). Read the concentration of the sample from this standard curve by the OD of sample.