Modern Methods of Immunodiagnostics

Radioimmunoassay (RIA)

Radioimmunoassay is based on the highly sensitive binding reaction between an antigen and an antibody. The indicator label is a radionuclide—most commonly isotopes of iodine, carbon, or tritium.

Technically, the procedure involves separating the formed radioactive immune complex and measuring the beta or gamma emission using a specialized counter. The radiation intensity is directly proportional to the number of bound molecules.

There are two main variations of the assay:

• Solid-phase RIA. One of the reacting components (antigen or antibody) is pre-adsorbed onto a solid support, such as the wells of a polystyrene microplate.
• Competitive RIA. Used for precise quantitative measurement. The target antigen from the patient's sample and a standard labeled antigen compete for a limited pool of antibodies.

While the method possesses exceptional sensitivity and detects trace concentrations of substances, it presents radiation safety hazards requiring careful handling. It is used in laboratories to determine levels of hormones, enzymes, drugs, immunoglobulins, and microbial antigens.

Immunoblotting (Western Blot)

Immunoblotting, also known as Western Blot, is a complex diagnostic approach combining electrophoresis, membrane transfer, and immunoassay or radioimmunoassay. It is an indispensable tool for confirmatory testing, a classic example being the verification of HIV infection.

The procedure consists of three sequential steps:

1. Electrophoresis. Pathogen antigens are separated in a polyacrylamide gel using an electric current. Fractions are distributed strictly according to their molecular weight and charge.
2. Blotting (Transfer). The separated antigens are transferred from the gel onto a nitrocellulose membrane, preserving the exact topographical arrangement of the bands.
3. Probing (Detection). Commercial strips containing the blotted antigens are incubated with the patient's serum. If specific antibodies are present, they bind to the corresponding antigens. After washing away unbound elements, enzyme-linked secondary anti-species antibodies (directed against human immunoglobulins) are added. Addition of a chromogenic substrate causes a color change, visualizing distinct bands on the membrane.

Immune Electron Microscopy

This method combines the resolving power of electron microscopy with the specificity of serological reactions. Its primary purpose is the detailed visual study of microorganisms, predominantly viruses.

When viral particles are treated with specific immune serum, large immune aggregates form. Antibodies densely coat the virions, creating a characteristic protein 'halo' around them. This layer provides strong electron-optical contrast, making viral particles and their clusters clearly visible.

• To further enhance visibility, electron-dense labels are employed. Specifically, antibodies can be conjugated with ferritin, an iron-rich protein that strongly scatters electron beams and helps localize target antigens.

Flow Cytometry

Flow cytometry is an analytical technique used to evaluate cell populations, differentiate blood cells, and identify them based on surface CD antigens.

Before analysis, a cell suspension is stained with fluorochrome-conjugated monoclonal antibodies. The cytometer utilizes hydrodynamic focusing to align cells in a single-file line within a microcapillary, passing them through a laser intersection point one by one.

The laser performs two key functions:

• Morphological assessment. Forward scatter detects the size and internal granularity of the passing cell.
• Marker assessment. The laser excites the attached fluorochrome labels. Fluorescence intensity reflects the density of surface CD antigens.

The optical system uses dichroic mirrors to split the emitted light by wavelength (e.g., green spectrum 530–550 nm and red spectrum 560–580 nm). The signals captured by photomultiplier tubes are converted into graphical histograms.