ELISA Formats

Enzyme-Linked Immunosorbent Assay (ELISA) is an immunoenzyme technology developed rapidly after immunofluorescence and radioimmunoassay and has been widely used in various research fields such as biology and medical science. ELISA can be used to measure both antigens and antibodies. According to the different experimental principles and operations of ELISA, it can be roughly divided into four types: direct method, indirect method, sandwich method, and competition method.

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Introduction to ELISA

ELISA utilizes the principle of specific reaction of antigen and antibody. After the solid phase of the antigen or antibody and the enzyme labeling of the antigen or antibody are added to the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product, and the amount of the product is directly related to the amount of the substance to be tested in the sample, thus performing qualitative or quantitative analysis.

During the experiment, it is necessary to coat the antigen or capture antibody on the solid phase carrier, convert the chemical signal into an electrical signal by detecting the molecule (enzyme or fluorescent group), and quantify the target protein with the result of OD value or luminescence value.

Types of ELISAs

ELISA can be used for the determination of antigens and antibodies. Necessary reagents in this assay include solid-phase antigen or antibody, enzyme-labeled antigen or antibody, and a substrate for the enzyme. According to the source of reagents, the properties of specimens, and the conditions of detection, ELISA is divided into various categories.

Fig. 1 Types of ELISA. (Engvall, 2010)

Direct ELISA

In direct ELISA, the antigen is diluted in a certain ratio and coated on a solid phase carrier, and then detected with an antigen-specific antibody that is directly coupled to HRP or other detection molecules. Direct ELISA is a straightforward and time-saving method but may have lower sensitivity compared to other types.

Advantages

Disadvantages

Fewer experimental steps and faster detection speed.
It does not need to use secondary antibodies, which avoid cross-reactivity and makes the results less prone to errors.
Suitable for the detection of small molecule antigens.

Each target protein requires the preparation of a primary antibody that can bind specifically to it, resulting in low experimental flexibility.
The antigen of direct ELISA is not specifically fixed, and target proteins and other impurity proteins in the sample will bind to the ELISA plate, resulting in a higher experimental background.
No signal amplification, reducing the sensitivity of the assay.

Indirect ELISA

The indirect ELISA offers enhanced sensitivity by using a two-step detection process. After antigen immobilization, a primary antibody specific to the target antigen is added. Subsequently, a secondary antibody, conjugated to a detection molecule, is introduced. This secondary antibody recognizes and binds to the primary antibody, amplifying the signal. Indirect ELISA allows for signal amplification, making it a preferred choice when sensitivity is crucial.

Advantages	Disadvantages
Indirect ELISA involves enzyme-labeled secondary antibodies, providing greater sensitivity and requiring fewer labeled antibodies. Indirect ELISA offers greater flexibility since different primary antibodies can be used with a single labeled secondary antibody.	Indirect ELISA has the possibility of cross-reactivity (enzyme-labeled secondary antibody binds directly to the antigen), which may increase the background. The indirect ELISA experiment involves an additional step of secondary antibody incubation, resulting in a longer experimental period compared to direct ELISA.

Sandwich ELISA

In a sandwich ELISA, the capture antibody is immobilized in the wells of the ELISA plate, then the sample is added, followed by the detection antibody. If the detection antibody is an enzyme-labeled antibody, it can be referred to as direct sandwich ELISA; if the detection antibody is not labeled, it is also necessary to use an enzyme-labeled secondary antibody to bind to the detection antibody, which is called indirect sandwich ELISA.

Advantages	Disadvantages
Sandwich ELISA has high sensitivity, which is 2-5 times more sensitive than direct or indirect ELISA. Sandwich ELISA involves two antibodies detecting different epitopes on the same antigen, offering high specificity. This assay is suitable for complex samples.	Sandwich ELISA has high requirements for paired antibodies. If there is no standardized kit or paired antibody that has been tested, it is necessary to customize and optimize the paired antibody. Understand our ELISA Matched Pair Development Services

Competitive ELISA

Competitive ELISA is used to measure the concentration of small molecules, such as hormones or drugs. In this method, the antigen of interest, which is present in the sample, competes with a labeled antigen for binding to a limited amount of specific antibodies immobilized on the microplate. The amount of labeled antigen bound to the antibody is inversely proportional to the concentration of the antigen present in the sample. Competitive ELISA is commonly used for quantitative analysis of low molecular weight compounds.

Advantages	Disadvantages
Suitable for small antigens. This method is applicable to impure samples and is fast and efficient.	The sensitivity and specificity of this method are poor.

In recent years, advancements in ELISA technology have led to the development of novel variations and improvements in sensitivity, multiplexing capability, and automation. Multiplex ELISA platforms allow simultaneous detection of multiple analytes, enhancing efficiency and reducing sample volume requirements. Furthermore, the integration of microfluidics, nanomaterials, and advanced detection systems has propelled ELISA toward miniaturization and point-of-care applications. As the field of ELISA continues to evolve, it holds great promise for revolutionizing diagnostics and personalized medicine.

Reference

Engvall E. The ELISA, enzyme-linked immunosorbent assay. Clinical Chemistry. 2010, 56(2): 319-320.

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