Types of ELISA: Direct, Indirect, Sandwich, and Competitive
Enzyme-Linked Immunosorbent Assay (ELISA) is a versatile and sensitive immunoassay widely used for the detection and quantification of antigens or antibodies.
There are four primary types of ELISA:
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA
Each format has its unique mechanism, benefits, and best-use scenarios. Understanding the distinctions between these types is critical for selecting the most suitable assay for your research or diagnostic needs.
Direct ELISA
This type of ELISA assay is significantly faster than the rest and has fewer steps. With fewer reagents and steps, there is smaller chance of human error when compared to the other techniques.
On the other hand, because the antigen immobilization is not specific, higher background can be seen. This issue is caused by all proteins including the target one binding to the plate, instead of only the protein of interest. Compared to the rest, this method is less flexible because a different conjugated primary antibody is needed for every protein.
There is no signal amplification with direct ELISA, due to the lack of secondary antibody. This leads to a less sensitive assay, making this technique suitable for analysing the immune response to an antigen.
Indirect ELISA
Indirect ELISA uses an unlabeled primary antibody followed by an enzyme-conjugated secondary antibody that recognizes the primary. This two-step detection offers signal amplification and increased sensitivity.
It's commonly used for measuring antibody concentrations in samples like serum or plasma. Indirect ELISA allows flexibility with different detection systems, but includes more steps and potential for cross-reactivity due to the secondary antibody.
Sandwich ELISA
Sandwich ELISA captures the target antigen between two antibodies: a plate-bound "capture" antibody and a labeled "detection" antibody. This format provides high specificity and sensitivity, especially for complex samples.
It is the preferred method for detecting low-abundance proteins, cytokines, or biomarkers. The main consideration is the need for well-matched antibody pairs that bind different epitopes of the same antigen.
Competitive ELISA
Also known as blocking ELISA, this technique is the most complex of all the previous. This assay is generally used for quantitatively measuring the concentration of an antigen via interference detection in signal output. The higher the sample antigen concentration, the weaker the signal.
It is less sensitive to sample dilution and matrix effects when compared to sandwich ELISA and out of all the variations, competitive ELISA offers the best flexibility and consistency.
This approach is useful for detecting small molecules or antigens with only one epitope, such as hormones or drugs. It requires careful optimization but excels where conventional sandwich formats fall short.
The only drawback to this assay is that it shares the same limitations as a base assay, because all ELISAs can be modified to a competitive variation.
Comparing ELISA Kit Types
| ELISA Type | Mechanism | Advantages | Disadvantages | Best Use Case |
|---|---|---|---|---|
| Direct ELISA | Antigen detected directly by enzyme-labeled primary antibody | Fast and simple, fewer steps | Lower sensitivity, no amplification, higher background | High-purity antigen samples |
| Indirect ELISA | Primary antibody binds antigen; enzyme-labeled secondary antibody binds primary | Signal amplification, versatile detection | More steps, potential cross-reactivity | Detecting antibodies in serum/plasma |
| Sandwich ELISA | Antigen is "sandwiched" between capture and detection antibodies | High specificity and sensitivity, suitable for complex samples | Requires matched antibody pairs, more optimization | Low-abundance targets like cytokines or biomarkers |
| Competitive ELISA | Sample antigen competes with labeled antigen for antibody binding | Ideal for small molecules, independent of antigen size | Less intuitive, lower signal, more complex assay design | Hormones, small peptides, or single-epitope targets |