What Are Immunoassays? Different Types & Applications

What Are Immunoassays?

Immunoassays are bioanalytical methods used to detect the presence and concentration of a specific biomolecule, ranging from small molecules to macromolecules, in a given biological sample using the antigen and antibody reaction.

Figure: Antigen-antibody interaction.

Antibodies are proteins produced in organisms against foreign substances, such as viruses, toxins, and bacteria. They have antigen-binding sites to bond with the analytes or antigens with specificity. 

During immunoassays, the immunoanalytical reagents, consisting of labeled and unlabelled analytes, are mixed and allowed to incubate. The target analyte in the sample binds to the antibody, forming an immune complex, while others remain in the unbound reagent fraction. The formed immune complex is then separated from the unbound fraction using physical or chemical separation techniques.

The analysis of the separated biomolecules is conducted by measuring the activity of the labels (e.g., radiation, fluorescence, or enzyme) in either the bound or free fraction. To quantify the concentration of the unlabelled analyte in the sample, a standard curve is constructed, plotting the measured signal against the analyte concentration.

In this article, we will cover the formats of immunoassay technology, their working mechanism, and their role in clinical and diagnostic applications. 

The Different Immunoassay Options

Here’re different types of immunoassays used in labs to study different biomolecules:

• Enzyme-linked Immunosorbent Assay (ELISA): A type of enzyme immunoassay (EIA), performed in biochemistry and immunology labs. In this assay, the antigens or capture antibodies are immobilized on the surface of the microplates followed by incubation with target antigen-specific antibodies. 

The antigen-antibody complex is then visualized using a variety of detection methods, such as chemiluminescent, colorimetric, chromogenic, and fluorescent assay. 

The visualization of the complex is possible because of the involvement of enzyme-labeled antibodies. The conjugates extensively used in ELISA assays include horseradish peroxidase (HRP) and alkaline phosphatase (AP). They interact with suitable substrates to initiate a reaction that produces a measurable signal. 

The different types of ELISA assays used in labs include sandwich ELISA, competitive ELISA, direct ELISA, and indirect ELISA. Because of its high sensitivity and high specificity, ELISA is widely used in biomedical applications, such as high-throughput screening of drugs and antibodies. 

Figure: Different types of ELISA assay. 

• Radioimmunoassay (RIA): It’s a high-throughput technique used in labs to quantify drugs, hormones, and viral antigens. It uses radioactive isotopes or radioisotopes to label antibodies that identify target analytes or specific antigens. In this method, an unlabeled antigen competes with a radiolabeled antigen for binding to a specific antibody. The level of free radiolabeled antigen reflects the concentration of unlabeled antigen in the mixture.

Figure: An illustration of the procedure of radioimmunoassay. 

• Chemiluminescence immunoassays (CLIA): It’s a biochemical technique used in labs to detect proteins, peptides, and hormones in a range of samples. In this assay, enzymes convert a substrate into a reaction product that emits light instead of producing a specific color. Luminescence refers to the emission of light when a substance transitions from an excited state to a ground state.
• Fluoroimmunoassays: It’s an immunoassay format that involves the use of fluorescent molecules such as Rhodamine B and fluorescein to detect a range of biomolecules. The fluorescent label antibody binds to the antigen, resulting in measurable fluorescence intensity, which is used to determine the amount of analyte of interest. 
• Counting immunoassays: In this immunoassay, particle beads are covered with antibodies specific to the target antigen. Then, it’s incubated with the sample, which results in the formation of an antigen-antibody complex. The unbound beads are counted using a cell counter, giving the concentration of the antigen of interest. 

How Immunoassays Work

Immunoassays work on the principle of antigen-antibody interaction. Antibodies exhibit high specificity for analytes like hormones, nucleoproteins, and peptides. When pure reagents are used, antigen-antibody complexes form, allowing the successful detection of analytes. A color change indicates the presence of the targeted molecule, with the intensity of the color representing the amount of the analyte in the test solution.

Today, a variety of immunoassays are developed, such as ELISA, chemiluminescent, and fluorescent assay, for different purposes, however, they all have three common components:

• Target analyte: The antigen of interest that you want to detect using the immunoassay. 
• Antibody: These are proteins having high binding affinity for the target antigen. 
• Detection label: A chemical compound that when attached to an antibody or antigen produces a measurable signal after the antigen-antibody reaction. Its examples include fluorescein, HRP, and rhodamine B. 

What Are Immunoassays Used For?

Immunoassay methods play a crucial role in pharmaceutical analysis. They are used for diagnosing diseases, monitoring therapeutic drug levels in the body, and studying how drugs work. These techniques help scientists and doctors better understand and treat illnesses, ensuring safer and more effective medications for everyone.

Drug Monitoring

Immunoassays are incredibly important for disease diagnosis and treatment. They are utilized in various areas such as pregnancy tests, cancer diagnostics, and monitoring the effectiveness of drug therapies. 

These bioanalytical tests help assess the response to medications for diseases like HIV and cancer. Additionally, immunoassays are important for ensuring the safety and compliance of workplace drug testing.

Biopharmaceutical Analysis

Immunoassays provide a powerful and versatile analytical toolset in biopharmaceutical analysis, enabling researchers and manufacturers to assess the quality, potency, and safety of biopharmaceutical drugs. They also have applications in gene and cell therapy and vaccine development. 

The immunoassays utilize the specific binding interactions between antibodies and target molecules to detect and quantify the presence of therapeutic proteins, antibodies, vaccines, and other biologics.

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Immunoassays are bioanalytical techniques performed in life sciences labs to detect and quantify proteins, peptides, and hormones in a given sample using antigen-antibody interaction. Based on the type of label added to the antibody, which is used in workflows, the immunoassay is categorized into different formats, such as enzyme immunoassay, fluorescence immunoassay, and chemiluminescent immunoassay. 

Though all the immunoassay techniques differ in label and the detection methods used in the process, they all have three common components, which include antigen, antibody, and detection label. 

Immunoassays and other high-throughput techniques have a wide range of applications in gene therapies and drug discoveries. However, conducting such research requires access to high-quality reagents and advanced equipment, which can be costly. 

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