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ELISA Tests: Beyond Just Antibody Detection

ELISA's versatility, sensibility, and specificity make it an efficient method to detect and quantify biomolecules in different settings

Morgana Moretti, PhD

Morgana Moretti, PhD, is an active scientist and freelance medical writer with more than 12 years of research and writing experience. She holds a doctoral degree in biochemistry, has published...

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ELISA, which stands for enzyme-linked immunosorbent assay, has become a fundamental tool in basic sciences, clinical diagnostics, and food safety testing. This article overviews ELISA's essential principles and various applications to give you a comprehensive understanding of this versatile and cost-effective method to detect and quantify biomolecules.

Principles of ELISA

ELISA uses antibodies that recognize and bind to specific proteins or molecules of interest called antigens. In an ELISA, the antigen is immobilized on a microplate and then complexed with an antibody linked to a reporter enzyme. To detect the presence of a target molecule in an ELISA, scientists measure the activity of the reporter enzyme by incubating it with the appropriate substrate, which produces a colorimetric or fluorescent signal that indicates the antigen's presence. The amount of signal is proportional to the amount of antigen present in the sample.

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Formats: direct, indirect, sandwich, and competitive ELISA

There are four primary types of ELISA: direct, indirect, sandwich, and competitive. Overall, the choice of ELISA type will depend on the available budget, training of lab staff, and the specific needs of the experiment, including the target molecule and required sensitivity.

ELISA TypeDetection MethodSensitivityAdvantagesDisadvantages
Direct ELISADirectly detects the antigenLow to moderateSimple and quick, with no secondary antibody cross-reactivityLow sensitivity compared to other types of ELISA
Indirect ELISADetects primary antibody-antigen complexHighIncreased sensitivity compared to direct ELISA; lower antigen concentration requiredRisk of cross-reactivity between the secondary detection antibodies
Sandwich ELISAAntigens are sandwiched between two layers of antibodies (capture and detect on antibodies)HighDetects low concentrations of antigens; the highest sensitivity among all the ELISA typesTime-consuming and expensive
Competitive ELISACompetition between labeled and unlabeled antigensHighLess sample purification is needed; can measure a large range of antigens in a sample; can detect small antigensLow specificity and cannot be used in dilute samples

ELISA applications: from basic sciences to environmental analysis

ELISA has many uses. Some applications of this technique include: 

Diagnostic testing and investigation of disease biomarkers

In the diagnostic space, ELISA can detect and quantify analytes in biological samples like serum, plasma, and urine, with applications ranging from viral and bacterial detection to pregnancy testing.

ELISA can also be used to investigate disease biomarkers. For example, in an article published in Nature Aging in February 2023, researchers used sandwich ELISA to measure circulating growth differentiation factor 11 (GDF11) levels in the blood of patients with depression.2 The authors found that people with depression have a decrease in GDF11 compared to healthy controls and raised the possibility that serum GDF11 levels can be a potential biomarker for depression in humans.

Additionally, ELISA is a method used to measure the concentration of disease biomarkers over time, which can provide insights into disease progression and the effectiveness of treatment.

For example, ELISA-based assays use specific antibodies that recognize and bind to HIV proteins, allowing for the measurement of HIV concentration in the patient’s blood. By using ELISA to measure viral load over time in HIV-positive patients, healthcare providers can monitor disease progression and treatment effectiveness.

ELISA can also detect and estimate the levels of tumor markers, such as the prostate-specific antigen, which has revolutionized the diagnosis, treatment, and monitoring of patients with prostate cancer. In addition, ELISA can identify autoantibodies indicative of conditions such as lupus and rheumatoid arthritis.

Food safety

ELISA is intensively applied in food safety as it can detect bacteria, parasites, pesticides, and other food contaminants.3

For example, ELISA is a popular method to detect allergenic substances, including egg whites, peanuts, and milk. Commercial kits can detect allergen proteins in various sample types, including clean-in-place final rinse water, food ingredients, and processed food products.

By detecting allergens and other food contaminants, manufacturers can take appropriate action to prevent cross-contamination and ensure that their products remain safe. 

Environmental monitoring

ELISA has proven to be a valuable tool to quantify surfactants, endocrine disruptors, estrogens, and persistent organic pollutants such as dioxins and polychlorinated biphenyls in environmental and industrial wastes.4 This can help identify potential pollution sources and develop strategies to protect public health and the environment.

Compared to instrumental analytical methods such as high-performance liquid chromatography and liquid chromatography-mass spectrometry, ELISA offers considerable advantages because of ease of handling, fast measurement, high sample turnover, and acceptable costs.

Emerging ELISA techniques: potentials and pitfalls

ELISA has advanced significantly in recent years to meet consumers' demands for higher productivity, greater sensitivity, and faster results. One such advancement is the development of multiplex ELISA, which allows multiple analytes to be simultaneously detected and quantified in a single sample.5

There are four primary types of ELISA: direct, indirect, sandwich, and competitive.

In a multiplex ELISA, multiple capture antibodies, each specific to a different target analyte, are immobilized onto a solid support such as a microplate. After the sample is added, detection antibodies labeled with fluorescent or luminescent tags are introduced to the mixture. If a specific analyte is present, the detection antibody will bind to it, allowing for its identification.

The availability of commercial multiplex immunoassays for research applications is expanding rapidly and offers the benefit of reduced time and smaller volumes needed to conduct an analysis. However, the transition to clinical use requires rigorous validation and standardization to ensure reliable and accurate results.

Nanoparticle-based ELISA is another recent modification of traditional ELISA.6 In this approach, nanoparticles are coated with antibodies or antigens to capture target molecules from a sample. The captured molecules are then detected using a secondary antibody conjugated to an enzyme or fluorescent molecule. 

Nanoparticle-based ELISA offers several advantages over traditional ELISA, including increased sensitivity and specificity due to the enhanced binding capacity of nanoparticles, as well as the ability to detect multiple analytes simultaneously. This technique has shown promising results but is still in the early stages of development and requires further optimization and validation before it becomes widely used in clinical and environmental analysis.

Recent significant innovations, coupled with the extensive global expertise in ELISA, promise to expand this technique's capabilities, sensitivity, and automation processing, making it an even more attractive choice for analyte detection in labs worldwide.


1.    “Enzyme Linked Immunosorbent Assay”.

2.    “Systemic GDF11 attenuates depression-like phenotype in aged mice via stimulation of neuronal autophagy”.

3.    “Application of nano-ELISA in food analysis: Recent advances and challenges”.

4.    “The use of enzyme-linked immunosorbent assays (ELISA) for the determination of pollutants in environmental and industrial wastes”.

5.    “ELISA in the multiplex era: Potentials and pitfalls”.

6.    “Recent improvements in enzyme-linked immunosorbent assays based on nanomaterials”.