Why variation occurs between ELISA experiments

Antigen Immobilization

Antigen immobilization varies depending on the type of ELISA assay performed.

Direct ELISA – The antigen is directly attached to the microtiter plate and the labelled antibody subsequently added to the plate.

Indirect ELISA – The antigen is directly attached to the plate which is then bound by a primary antibody followed by a labelled secondary antibody.

Sandwich ELISA – Is the most common ELISA used for complex protein samples as only the specific antigen becomes immobilized rather than the entire sample of proteins. Two antibodies are required for sandwich ELISA which bind different epitopes. The first antibody is bound to the plate and is known as the capture antibody. The secondary antibody commonly referred to as the detection antibody, this detects the immobilized antigen which is bound to the capture antibody. Both monoclonal and polyclonal antibodies can be used for sandwich ELISA.

Biotin signal amplification

Biotin is a small vitamin type molecule which can be easily modified so that it can be chemically attached to proteins and antibodies of interest. Avidin and streptavidin though from different sources have identical functions – strong binding specificity for the biotin molecule, this affinity is used in tagging and detection in ELISA.

Signal amplification can be achieved in two ways, using a biotinylated detection antibody which is probed using an avidin/streptavidin protein conjugated to either HRP or AP enzymes. Or using a biotinylated detection antibody probed with a preincubated mixture of avidin and biotinylated enzyme, known as “avidin-biotin complex” (ABC) signal amplification.


The development of stable fluorophores has made fluorescent signal detection an attractive option for ELISA signal detection. Florescent tagging is commonly used for multiplex assays whereby more than one antigen can be detected simultaneously using antibodies which are conjugated to different fluorophores. The antibodies used may be labelled directly, secondary labelled antibodies may also be used.

Enzymatic detection

The most widely used enzymes for ELISA detection are horseradish peroxidase (HRP) and alkaline phosphate (AP). HRP is the most widely used as its small size 40kDa allows it to be coupled easily to antibodies and enables interaction with a variety of substrates. AP is also used but its size 140kDa makes it difficult to conjugate. It is also prone to a number of stability issues.

Substrate Selection

Enzymatic detection requires the catalysis of a substrate to produce a coloured/fluorescent signal which can be quantified either through the use of a spectrophotometer or fluorometer. Colorimetric substrates form a soluble coloured product that accumulates over time relative to the amount of enzyme present in each well. Colorimetric substrates for HRP include TMB, OPD, ABTS and pNPP. Substrates for AP include BCIP/NBT and pNPP.