ELISA principle
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Understand all the basic principles of ELISA and decide if this is the right technique for your experiments.
Figure 1. The basic setup of an ELISA assay. A capture antibody on a multi-well plate will immobilize the antigen of interest. This antigen will be recognized and bound by a detection antibody conjugated to biotin and streptavidin-HRP.
ELISA stands for enzyme-linked immunosorbent assay, also often referred to as enzyme immunoassay (EIA). An ELISA, like other types of immunoassays, relies on antibodies to detect a target antigen using highly specific antibody-antigen interactions.
In an ELISA assay, the antigen must be immobilized to a solid surface. This is done either directly or via the use of a capture antibody itself immobilized on the surface. The antigen is then complexed to a detection antibody conjugated with a molecule amenable for detection such as an enzyme or a fluorophore.
An ELISA assay is typically performed in a multi-well plate (96- or 384-wells). The multi-well plate provides the solid surface to immobilize the antigen. Immobilization of the analytes facilitates separation of the antigen from the rest of the components in the sample. This characteristic makes ELISA one of the easiest assays to perform on multiple samples simultaneously.
Advantages and disadvantages of ELISA
| Advantages | Disadvantages |
| High sensitivity and specificity: it is common for ELISAs to detect antigens at the picogram level in a very specific manner due to the use of antibodies. | Temporary readouts: detection is based on enzyme/substrate reactions and therefore readout must be obtained in a short time span. |
| High throughput: commercial ELISA kits are normally available in a 96-well plate format. But the assay can be easily adapted to 384-well plates. | Limited antigen information: information limited to amount or presence of the antigen in the sample. |
| Easy to perform: protocols are easy to follow and involve little hands-on time. | |
| Quantitative: it can determine the concentration of antigen in a sample. | |
| Possibility to test various sample types: serum, plasma, cellular and tissue extracts, urine, and saliva among others. |
These are the general ELISA advantages and disadvantages. There are other advantages and disadvantages depending on the type of ELISA used as explained in the next section.
ELISA guide