The Sandwich ELISA measures the amount of antigen between two layers of antibodies (i.e. capture and
detection antibody). The antigen to be measured must contain at least two antigenic sites capable of
binding to antibody, since at least two antibodies act in the sandwich.
Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in
Sandwich ELISA systems. Monoclonal antibodies recognise a single epitope that allows fine detection and
quantification of small differences in antigen. A polyclonal is often used as the capture antibody to pull down
as much of the antigen as possible.
Coating with Capture antibody
Step 1.
Coat the wells of a PVC microtiter plate with the capture antibody at a concentration of 1-10 μg/ml in
carbonate/bicarbonate buffer (pH7.4).
Step 2.
Cover the plate with an adhesive plastic and incubate overnight at 4°C.
Step 3.
Remove the coating solution and wash the plate twice 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.
Blocking and Adding Samples
Step 4.
Block the remaining protein-binding sites in the coated wells by adding 200 μl blocking buffer, 5% non fat
dry milk/PBS, per well.
Step 5.
Cover the plate with an adhesive plastic and incubate for at least 1-2 h at room temperature or, if more
convenient, overnight at 4°C.
Step 6.
Add 100 μl of appropriately diluted samples to each well. For accurate quantitative results, always compare
signal of unknown samples against those of a standard curve. Standards (duplicates or triplicates) and
blank must be run with each plate to ensure accuracy. Incubate for 90 min at 37°C.
Step 7.
Remove the samples and wash the plate twice by filling the wells with 200 μl PBS.
Incubation with Detection antibody and then Secondary antibody
Step 8.
Add 100 μl of diluted detection antibody to each well.
Step 9.
Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.
Step 10.
Wash the plate four times with PBS.
Step 11.
Add 100 μl of secondary antibody conjugated, diluted at the optimal concentration (according to the
manufacturer) in blocking buffer immediately before use.
Step 12.
Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature.
Step 13.
Wash the plate four times with PBS.