Enzyme-linked immunosorbent assay

Competitive ELISA

This type of assay is used when the primary goal is to detect antibodies against pre-known antigens.

The underlying mechanism is based on the principle of direct competition. An antigen is sorbed onto the solid phase (plastic). Two types of antibodies compete to bind to it: the target antibodies (from the patient's serum, unlabeled) and specially added labeled antibodies.

Result evaluation here has an inverse relationship: the degree of inhibition of binding of labeled reagents serves as a measure of the patient's antibody concentration. The more autoantibodies present in the sample, the fewer enzyme labels can attach to the antigen compared to the control sample (where competitors are absent). Chromogenic substrates reacting with the enzyme (usually peroxidase or alkaline phosphatase) are used to visualize the reaction.

Two-site assay (sandwich method)

This format is used to search for an antigen. A mandatory condition for a sandwich assay is that the target antigen molecule must possess at least two different epitopes (binding sites).

Assay steps:

1. Antibodies specific to the first epitope of the target antigen are tightly immobilized on the surface of the plastic well.
2. The test sample containing the antigen is added, and primary binding occurs.
3. "Second antibodies" that recognize the second epitope of the same antigen are layered on top.

To record results in the classical version, anti-isotypic antibodies carrying an enzyme label are used. However, there is also a more modern modification that increases standardization and sensitivity. In this version, the "second antibodies" are pre-labeled with streptavidin, and detection is carried out using biotin, which is already conjugated to an enzyme. The final antigen concentration in the sample is directly proportional to the amount of bound label.

Related methods: flow cytometry

In addition to ELISA, flow cytometry is actively used to analyze immune cells. The method is implemented using a laser flow cytometer: cells line up in a single file and pass through a tiny capillary under a laser beam.

The device automatically records light scattering parameters:

• Forward scatter (FSC) — makes it possible to assess the physical size of the passing cell.
• Side scatter (SSC) — provides information on internal structure, particularly granularity.

Based on these data, a two-dimensional distribution is plotted, allowing specific cell populations to be isolated. Fluorescence staining is used for deeper analysis. Using fluorophore-labeled monoclonal antibodies, marker molecules can be detected on the membrane or inside the cell. Modern devices are equipped with multiple lasers and are capable of distinguishing more than 10 colors simultaneously. This same principle underlies cell sorting based on the expression of desired molecules.

Molecular biology methods

Molecular biology tools are increasingly used in diagnostics to detect immunologically significant factors. The absolute leader in this field is the polymerase chain reaction (PCR), which complements classical immunochemical tests.

Class and exam questions

What else to study on this topic

• Mechanism of the reaction of chromogenic substrates with peroxidase
• Principles of cell fractionation and laser sorting
• Use of monoclonal antibodies in fluorescence analysis
• Polymerase chain reaction (PCR) in immunodiagnostics
• Analysis of two-dimensional distribution plots in flow cytometry

Other topics in the section "Immunity in defense and damage of the organism. Pathology of immunity"

Prepared by WhiteCoat Pro faculty in accordance with the medical school curriculum. For educational purposes only.