ELISA Protocol Modifications

An ELISA protocol can be modified in a variety of ways such as the type of antigen immobilization used, the detection method chemiluminescence, fluorescence or chemifluorescence. Different modifications and applications to ELISA protocols are outlined below.

Modification of the ELISA protocol with different technologies allows researchers increase the range of detection methods to complete their study. Below, we highlight some popular techniques that researchers use.


Chemifluroescence systems require HRP, unlike chemiluminescence. The final concentration of the enzyme HRP conjugate used should be within the range of 25-50 ng/ml. Chemifluorescence is measured using a fluorometer with appropriate filters.


Chemiluminescence is considered to be one of the most sensitive ELISA detection techniques, with the ability to detect sub-picogram amounts of antigen. The working concentration of enzyme conjugate can vary between 10-100 ng/ml depending on the specifics and sensitivity of the assay.

Competitive ELISA

A competitive ELISA is based on the competition between the antigen in a standard/sample and an enzyme conjugated form of the same antigen. The two antigens compete for a limited of antibody bound to the pre-coated plate in the the same well. There is an inverse relationship between the optical density (OD) and the amount of analyte. As the concentration of antigen in the sample increases, the amount of labelled antigen captured by the coating antibody decreases.

Direct Antigen Immobilization

When performing direct antigen immobilization to the plate there is no need for a capture antibody. The protocol should be performed as described with the use of a detection antibody, enzyme conjugate and substrate.

Enzyme labeled secondary antibody

When using a non-biotinylated detection antibody it is necessary to use a slightly higher concentration of secondary antibody enzyme than would be used for a steptavidin-enzyme conjugate. Reduced amplification of the enzyme is noted when using non-biotinylated detection antibodies when compared to using a biotinylated detection antibody with streptavidin-HRP.


No enzyme conjugate or substrate is necessary when determining analyte concentration via fluorescence. The fluorophore is attached directly to the secondary antibody and in some instances to the detection antibody or avidin. The signal is measured using a plate reader equipped to measure fluroescence or by a fluorometer.

Alkaline Phosphatase

If alkaline phosphatase (AP) is used instead of Horseradish peroxidase (HRP) an AP-specific substrate must be used. The most widely used AP substrate is p-nitrophenol (PNPP). PNPP has a detection limit of around 10 ng per well. Enzyme conjugates are normally used at a working concnetration of ~100/200 ng/ml.