ELISA Types: Direct, Indirect, Sandwich, Competitive, Multiplex ELISA

Enzyme-Linked Immunosorbent Assay (ELISA) is a versatile and widely used biochemical technique for detecting and quantifying specific molecules such as proteins, peptides, antibodies, and small molecules. Different types of ELISA—Direct, Indirect, Sandwich, Competitive, and Multiplex—offer distinct methodologies tailored to various research, diagnostic, and clinical applications.

In our blog, we discuss each type of ELISA as well as their unique advantages, considerations, and applications.

ELISA can be categorized into several types based on the method of detection and purpose.

Direct ELISA

In direct ELISA, the antigen is directly immobilized onto the surface of the microtiter plate. A labeled enzyme-linked antibody specific to the antigen is then added, which binds directly to the antigen. This type of ELISA is relatively simple and quick but may have lower sensitivity compared to other methods.

Advantages:

• Simple and straightforward procedure.
• Requires fewer steps compared to other ELISA types.
• Typically results in lower background noise.

Disadvantages:

• Generally less sensitive than indirect or sandwich ELISA.
• Limited to detecting antigens that can bind directly to antibodies.

Applications: Direct ELISA can be used for screening antigens in biological samples, quantifying antigens that can directly bind to antibodies without interference, and rapid diagnostic tests due to its simplicity and quick turnaround time.

Indirect ELISA

For indirect ELISA, the antigen is pre-coated similarly to direct ELISA. However, in this method, an unlabeled primary antibody specific to the antigen is added first. Then, a labeled secondary antibody that binds to the primary antibody is introduced. This amplifies the signal, increasing sensitivity. Indirect ELISA is versatile and widely used for detecting antibodies in serum.

Advantages:

• Amplifies signal due to multiple secondary antibodies binding to each primary antibody.
• Higher sensitivity compared to direct ELISA.
• Allows for detection of a wide range of antigens.

Disadvantages:

• More complex than direct ELISA, involving an additional incubation step with secondary antibody.
• Increased risk of nonspecific binding due to multiple antibody interactions.

Applications: Indirect ELISA can detect and quantify antibodies in serum or other biological fluids, screen large numbers of samples for antibody responses (e.g., in serological surveys), and determine antibody titers in vaccine development and immune response studies.

Sandwich ELISA

Sandwich ELISA involves immobilizing a capture antibody specific to the antigen of interest onto the microtiter plate. The antigen-containing sample is added, and if the antigen is present, it binds to the capture antibody. Then, a detection antibody that is specific to a different epitope of the antigen is added, forming a "sandwich" with the antigen. A labeled enzyme-linked secondary antibody is then added, which binds to the detection antibody, enabling detection. This method is highly sensitive and specific, making it a suitable and popular choice for detecting antigens in complex samples.

Advantages:

• Highly specific and sensitive, as it involves two antibodies binding to the antigen.
• Less prone to interference from non-specific binding.
• Suitable for detecting antigens in complex biological samples.

Disadvantages:

• Requires two specific antibodies that recognize different epitopes on the antigen.
• More technically demanding and may be costly due to the need for paired antibodies.

Applications: Examples of applications for sandwich ELISA include quantifying specific antigens in complex biological samples (e.g., serum, tissue lysates), detecting biomarkers in disease diagnostics (e.g., detecting tumor markers, cytokines), and monitoring protein expression levels in research and clinical settings.

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Competitive ELISA



In competitive ELISA, also called inhibition ELISA, the reference antigen is pre-coated on the microtiter plate. The sample containing an unknown amount of antigen is incubated with a known amount of labeled antibody (conjugated to an enzyme), and then added to the wells. The reference antigen and the sample antigen compete for binding to the limited quantity of labeled antibodies. The amount of labeled antibody bound to the plate is inversely proportional to the concentration of antigen in the sample. In other words, the more antigen in the sample, the less reference antigen will be detected and the weaker the output signal.

For some competitive ELISA kits, antibodies, instead of antigens, are pre-coated onto the plate. Afterwards, a known amount of labeled antigen and an unknown amount of sample antigen compete for binding to the antibodies coated on the plate.

Although inhibition ELISA can be more complex due to its competitive nature, inhibition ELISA can be flexibly adapted to use principles from direct, indirect, and sandwich ELISA. The competitive ELISA is useful for quantifying small molecules or detecting inhibitors.

Advantages:

• Useful for detecting small molecules or inhibitors.
• Can quantify antigen concentration based on the competitive binding.
• Provides a direct measurement of analyte concentration relative to a standard curve.
• Can be based on direct, indirect, or sandwich ELISA

Disadvantages:

• Typically less sensitive than sandwich or indirect ELISA.
• Requires careful optimization of antigen and antibody concentrations.
• More susceptible to variations in assay conditions affecting results.

Applications: Some applications of competitive ELISA include detecting and quantifying small molecules or inhibitors (e.g., drugs, hormones), assessing antibody neutralization in virology and vaccine research, and screening for contaminants or residues in food, environmental samples, or pharmaceutical products.

Multiplex ELISA



Multiplex ELISA allows for the simultaneous detection and quantification of multiple analytes (antigens or antibodies) within a single sample. It is particularly useful in high-throughput screening and diagnostics where multiple biomarkers need to be analyzed simultaneously.

Advantages:

• Enables simultaneous detection of multiple analytes within a single sample.
• Reduces sample volume and labor compared to running multiple singleplex assays.
• Suitable for high-throughput screening and diagnostics.

Disadvantages:

• Requires specialized equipment and reagents.
• More complex data analysis and interpretation compared to singleplex assays.
• Higher initial setup costs and may require more optimization.

Applications: Multiplex ELISA is used for simultaneous detection and quantification of multiple analytes (e.g., cytokines, biomarkers) in a single sample, high-throughput screening in drug discovery and development, and biomarker profiling for disease diagnosis, prognosis, and monitoring. If you’re looking to test your samples with multiplex ELISA assays, check out Boster’s Multiplex Assay Service to design your research experiment.

Below, we’ve summarized each type of ELISA in a table, along with their respective advantages, disadvantages, and typical applications.

ELISA Type	Description	Advantages	Disadvantages	Typical Applications
Direct ELISA	Antigen directly immobilized on plate; labeled antibody binds directly to antigen.	Simple procedure, fewer steps, lower background noise.	Less sensitive, limited to antigens binding directly to antibodies.	Screening for antigen presence, rapid diagnostic tests.
Indirect ELISA	Antigen immobilized; primary antibody binds antigen, secondary antibody (labeled) binds primary.	Amplifies signal, higher sensitivity, detects a wide range of antigens.	More complex, increased risk of nonspecific binding.	Antibody detection, quantification in serum, antibody titration.
Sandwich ELISA	Capture antibody pre-coated; antigen binds, detection antibody (labeled) binds to antigen "sandwich".	Highly specific and sensitive, less nonspecific binding.	Requires two specific antibodies, technically demanding and costly.	Biomarker detection, quantification of antigens in complex samples.
Competitive ELISA	Antigen immobilized; labeled antigen competes with sample antigen for limited antibody binding sites.	Quantifies small molecules, direct measurement of analyte concentration, flexible.	Less sensitive, requires careful optimization of conditions.	Detection of inhibitors, quantification of small molecules.
Multiplex ELISA	Simultaneous detection of multiple analytes using labeled antibodies specific to each analyte.	Simultaneous detection, reduced sample volume and labor.	Requires specialized equipment and reagents, complex data analysis and interpretation.	Biomarker profiling, high-throughput screening in drug discovery, simultaneous detection of multiple analytes.

The range of ELISA techniques—Direct, Indirect, Sandwich, Competitive, and Multiplex—provides researchers and clinicians with powerful tools for precise detection and quantification of biomolecules across various fields. Each ELISA type offers unique advantages, such as simplicity, sensitivity, specificity, or multiplexing capabilities, accompanied by specific challenges like assay complexity or optimization requirements.

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