Most Common Chemiluminescent Immunoassay (CLIA) Formats

CLIA stands for Chemiluminescent Immunoassay. It is a lab test used to detect substances in blood. These can be antigens, antibodies, hormones, or drugs.

The test works on the principle of antigen-antibody binding. CLIA is popular because it is very sensitive, gives results fast, and can measure a wide range of levels.

How CLIA Works:

• Antibodies or antigens are immobilized on a solid surface.
• When a blood sample is added, the target substance binds to them.
• Then, a special chemical called a chemiluminescent molecule is added. These molecules include acridinium esters, luminol, or isoluminol.
• A reaction takes place, and light is produced.
• The amount of light shows how much of the substance is present.

Today, ready-to-use Chemiluminescent Immunoassay (CLIA) kits are available. They are used in diagnostics, research, and biotech labs. These kits save time and make the test easy to use. CLIA has many formats. Let’s look at the most common ones.

CLIA Format Based On How The Immune Complexes Are Separated

1) Solid-Phase Format: 

In this format, antibodies or antigens are immobilized directly on a solid surface, such as microtiter plates, tubes, or wells. After the immune reaction, unbound reagents are washed away, and the chemiluminescent signal is measured.

Pros:

• Stable immobilization
• Low background noise
• High reproducibility

2) Magnetic Bead-Based Format: 

In this method, antibodies or antigens are coated onto magnetic beads. After the immune reaction, magnetic separation is used to isolate the bound from the unbound reagents. This method boosts assay speed and sensitivity.

Pros:

• Faster washing
• Higher surface area for binding
• Excellent compatibility with automated analyzers

CLIA Formats Based On How The Immune Complex Is Detected

1) Direct CLIA:

In the direct format, the antigen is immobilized on a solid surface. In this, a chemiluminescent-labeled antibody binds directly to it. The intensity of the emitted light corresponds to the amount of antigen present.

Advantages

• Simple design with fewer steps
• Faster results compared to other formats

Limitations

• Limited signal amplification, which may reduce sensitivity
• May not be suitable for the detection of very low concentrations of analytes

Direct CLIA is often used for applications where analyte concentrations are relatively high and testing speed is prioritized.

2) Indirect CLIA:

Indirect CLIA involves two antibodies: a primary antibody that binds to the antigen and a secondary antibody conjugated with a chemiluminescent label that binds to the primary antibody. This method helps in signal amplification and thereby increases assay sensitivity.

Advantages

• Higher sensitivity than direct CLIA
• More flexibility, as a single labeled secondary antibody can be used for multiple assays

Limitations

• Longer procedure due to extra incubation steps
• Possibility of cross-reactivity from the secondary antibody

Indirect CLIA is widely used in clinical testing, especially for the detection of antibodies in infectious disease screening.

3) Sandwich CLIA: 

The sandwich format is one of the most popular CLIA designs, especially for the detection of larger molecules like proteins, hormones, and antigens. It is called a “sandwich” because the target analyte is captured between two antibodies.

In this method:

• A capture antibody is immobilized on a solid phase.
• After this, the target antigen from the sample is bound to the captured antibody.
• Now, a second antibody conjugated with a chemiluminescent label is added. This antibody binds to a different epitope (site) on the antigen and forms a sandwich-like structure.
• When the chemiluminescent substrate is added, a light-emitting reaction occurs. The intensity of the light is directly proportional to the concentration of the antigen in the sample.

Advantages

• Extremely high specificity because two antibodies target different sites of the antigen
• High sensitivity. Ideal for detecting very low concentrations
• Ideal for complex biological samples

Limitations

• Requires high-quality antibodies against different epitopes of the antigen
• May be costlier due to dual antibody requirement

Sandwich CLIA is commonly used for hormone assays (e.g., thyroid-stimulating hormone, insulin), cancer biomarkers, and infectious disease antigen testing.

4) Competitive CLIA:

In competitive CLIA, the sample analyte competes with a chemiluminescent-labeled analyte (tracer) for a limited number of binding sites on a specific antibody. The signal generated is inversely proportional to the analyte concentration in the sample. Higher analyte levels result in lower luminescence.

Advantages

• Suitable for small molecules and haptens (e.g., drugs, toxins, steroids) that cannot be detected using sandwich assays
• Can handle a wide dynamic range of analyte concentrations

Limitations

• Lower sensitivity compared to sandwich assays
• Interpretation is slightly more complex because signal intensity is inversely related to analyte concentration
• This format is widely used in therapeutic drug monitoring, toxicology, and hormone analysis

5) Homogeneous CLIA:

Unlike other formats, homogeneous CLIA does not require separation of bound and unbound reagents. Instead, the chemiluminescent signal changes depending on whether the immune complex is formed. This makes the assay simpler and faster.

Advantages

• Rapid results with minimal washing steps
• Simplified automation for high-throughput screening

Limitations

• Reduced sensitivity compared to heterogeneous formats
• May not be ideal for very low analyte concentrations

Homogeneous CLIA is often applied in large-scale clinical screenings where speed and automation are essential.

The Bottom Line

CLIA is widely used in medical diagnostics, pharmaceutical research, food safety, and environmental monitoring. However, the choice of CLIA format depends on the analyte types, required sensitivity, sample complexity, and laboratory workflow. So, make sure you know your needs before using CLIA for your experiments.