Modern immunodiagnostic methods

Radioimmunoassay (RIA)

The radioimmunoassays are based on a highly sensitive antigen-antibody binding reaction. Radionuclides act as indicator labels here — most commonly isotopes of iodine, carbon, or tritium.

Technically, the research process boils down to separating the formed radioactive immune complex and subsequently measuring the level of beta or gamma radiation using a special counter. The intensity of the recorded radiation is in direct mathematical dependence on the number of bound molecules.

There are two main variants of the reaction setup:

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

The method has colossal sensitivity, capturing minor concentrations of substances, but poses a certain ecological hazard due to the need to work with radiation sources. 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 comprehensive diagnostic approach combining the capabilities of electrophoresis, fraction transfer, and enzyme-linked immunosorbent (or radioimmuno) assay. It is an indispensable tool for confirmatory diagnostics, a classic example of which is the verification of HIV infection.

The study proceeds in three consecutive stages:

1. Electrophoresis. Pathogen antigens are separated in a polyacrylamide gel under the influence of an electric current. Fractions are distributed strictly according to their molecular weight and charge.
2. Blotting (transfer). The separated antigens are replicated from the gel onto a nitrocellulose membrane, preserving the exact topographical arrangement of the invisible bands (blots).
3. Development. At the diagnostic stage, ready-made commercial strips with applied blots are used. The patient's test serum is applied to them. If specific antibodies are present in the blood, they bind to the antigens. After washing away unbound elements, secondary anti-species antibodies (against human immunoglobulins) carrying an enzyme label are added. The introduction of a chromogen causes a color change of the substrate, and clear bands are visualized on the membrane.

Immune electron microscopy

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

When treating viral particles with specific immune serum, large immune aggregates form. Antibodies tightly surround virions, creating a distinctive protein «corona» around them. This layer provides pronounced electron-optical contrasting, making viral particles and their clusters clearly visible.

For additional enhancement of visibility, labeling with electron-dense markers is used. In particular, antibodies are conjugated with ferritin, a special iron-containing protein that effectively blocks the electron beam and helps localize the target antigens.

Flow cytometry

Flow cytometry is an instrumental method for analyzing cell populations, blood cell differentiation, and their identification by surface CD antigens.

Before starting the analysis, the cell suspension is specifically stained with monoclonal antibodies previously labeled with fluorochromes. The cytometer itself implements the principle of hydrodynamic focusing: cells line up in a capillary in a single file and cross the laser irradiation zone strictly one by one.

The laser performs two key tasks:

• Morphology evaluation. The forward scatter detector records the size and internal granularity of the passing cell.
• Marker evaluation. The laser excites the emission of attached fluorochrome labels. The fluorescence intensity reflects the density of CD antigens on the membrane.

The complex optical system of the device uses dichroic mirrors to separate the emitted light by wavelengths (e.g., green spectrum 530–550 nm and red 560–580 nm). The signal directed to photomultipliers is converted into a graphical histogram.

Briefly: answers to topical questions

What else to study on this topic

• Mechanisms of apoptosis and pyroptosis evaluated by flow cytometry
• Spectrum of CD antigens and their role in lymphocyte identification
• Specific protein bands (blots) significant in the diagnosis of HIV infection
• Physical principles of photomultiplier operation in laser cytofluorometry
• Isotopes used in radioimmunoassay and their physiological safety

Other topics in the "Immunodiagnostic reactions" section

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