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.
An ELISA assay is typically performed in a multi-well plate (96- or 384-wells), which provides the solid surface to immobilize the antigen. Immobilization of the analytes facilitates the 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.
For more information, view our webinar on ELISA principles.
ELISA advantages and disadvantages
Advantages
- 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.
- 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.
- 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.
Disadvantages
- Temporary readouts: detection is based on enzyme/substrate reactions and therefore readout must be obtained in a short time span.
- Limited antigen information: information limited to the amount or presence of the antigen in the sample.
Typesof ELISA
There are four main types of ELISA: direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA. Each has unique advantages, disadvantages and suitability.
Figure 2.The different types of ELISA (direct, indirect, sandwich, and competitive)
Direct ELISA
In a direct ELISA, the antigen is immobilized to the surface of the multi-well plate and detected with an antibody specific for the antigen The antibody is directly conjugated to HRP or other detection molecules.
Indirect ELISA
Indirect ELISA is a technique that uses a two-step process for detection, whereby a primary antibody specific for the antigen binds to the target, and a labeled secondary antibody against the host species of the primary antibody binds to the primary antibody for detection. As for direct ELISA assays, the antigen is immobilized to the surface of the multi-well plate.
The method can also be used to detect specific antibodies in a serum sample by substituting the serum for the primary antibody.
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Sandwich ELISA
Sandwich ELISA (or sandwich immunoassay) is the most commonly used format. This format requires two antibodies specific for different epitopes of the antigen. These two antibodies are normally referred to as matched antibody pairs. One of the antibodies is coated on the surface of the multi-well plate and used as a capture antibody to facilitate the immobilization of the antigen. The other antibody is conjugated and facilitates the detection of the antigen.
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Competitive ELISA
Also known as inhibition ELISA or competitive immunoassay, competitive ELISA assays measure the concentration of an antigen by detection of signal interference. Each of the previous formats can be adapted to the competitive format.
The sample antigen competes with a reference antigen for binding to a specific amount of labeled antibody. The reference antigen is pre-coated on a multi-well plate and sample is pre-incubated with labeled antibody and added to the wells. Depending on the amount of antigen in the sample, more or less free antibodies will be available to bind the reference antigen. This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the signal.
Some competitive ELISA kits use labeled antigen instead of a labeled antibody. The labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody. The lower the amount of antigen in the sample, the stronger the signal due to more labeled antigen in the well.
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Which type of ELISA should I use?
| Advantages | Disadvantages | |
| Direct ELISA | Short protocol: saves time and reagents. No cross-reactivity from secondary antibody. | Potential high background: all proteins in the sample bind to the surface. No signal amplification. Low flexibility: the primary antibody must be conjugated. |
| Indirect ELISA | Signal amplification: several secondary antibodies will bind to the primary antibody. High flexibility: the same secondary antibody may be used for several primary antibodies. | Long protocol if compared to direct ELISA. Potential cross-reactivity from secondary antibody. |
| Sandwich ELISA | High specificity: involves two antibodies detecting different epitopes on the same antigen. Suitable for complex samples. High flexibility and sensitivity: both direct and indirect methods can be used. | Demanding design: finding two antibodies against the same target that recognize different epitopes and work well together can be challenging at times. |
| Competitive ELISA | Depends on base ELISA selected. Suitable for small antigens. | Depends on base ELISA selected |
ELISA guide
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