The antigen-antibody interaction- Immunofluorescence

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In this article, I briefly describe the method of immunofluorescence, which is an antigen-antibody interaction.

Antigen-antibody interaction

The Interaction between antigen and antibody is a bimolecular association, which does not lead to an irreversible chemical alteration in either the antibody or the antigen. The antigen-antibody association involves many non-covalent interactions between the antigenic determinant (epitope) of the antigen and the variable-region (VH/VL) domain of the antibody molecule. The antigen-antibody binding depends on weak and non-covalent interactions like hydrogen bonds, hydrophobic interactions, electrostatic forces, and Van der Walls interactions. Thus, to make a sturdy antigen-antibody interaction, many such weak interactions are required. These interactions can only occur if the antigen and antibody molecules are close enough for some individual atoms to fit into complementary recesses.

Affinity and avidity

A very close fit between antigen and antibody increases the strength of the bond. The sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody determines the affinity. The affinity of an antibody for a specific epitope is the combined strength of the non-covalent interactions between a single antigen-binding site on an antibody and the epitope.

The strength of multiple interactions between a multivalent antibody and antigen is called avidity. When complex antigens containing multiple repeating antigenic determinants are mixed up with antibodies containing multiple binding sites, the interaction of an antibody with an antigen at one site will increase the probability of a reaction between those two molecules at a second site. Avidity is more than the sum of the individual affinities. Affinity defines the strength of interaction between antibody and antigen at single antigenic sites, whereas avidity defines the overall stability or strength of the antibody-antigen complex. The strength of the antibody-antigen complex is controlled by three major factors, i.e., antibody-epitope affinity, the valence of both the antigen and antibody and the structural arrangement of the interacting parts.

Specificity and cross-reactivity

The specificity of an antigen-antibody reaction is the ability of an individual antibody combining site to react with only one antigenic determinant. It also defines the ability of a population of antibody molecules to react with only one antigen. An antibody can interact with its antigen, thus making the antigen-antibody reactions highly specific. A strong antigen-antibody interaction depends on a very close fit between the antigen and antibody, which requires a high degree of specificity.

Sometimes, the antibody elicited by one antigen can cross-react with an unrelated antigen, called cross-reactivity. The cross-reacting antigen has an epitope, which is structurally similar to one, on the immunizing antigen.

Types of antigen-antibody interaction

There are mainly six types of antigen-antibody interaction and can be categorized as

Immunofluorescence

It is an antigen-antibody interaction where the antibodies are tagged (labeled) with a fluorescent dye. Then, the antigen-antibody complex is visualized using an ultra-violet (fluorescent) microscope. Fluorescent molecules absorb and emit light of different wavelengths. When excited by light of appropriate wavelength, Immune complexes containing fluorescently labeled antibodies can be detected by colored light emission. This method commonly uses fluorescent compounds such as fluorescein and rhodamine. Phycoerythrin, an intensely colored and highly fluorescent pigment obtained from the algae, is also routinely used. Immunofluorescence can be categorized into direct and indirect immunofluorescence.

Direct immunofluorescence

This technique is used to detect antigens in clinical specimens using specific fluorochrome-labeled antibodies. In direct immunofluorescence, the specific antibody is directly conjugated with a fluorescent dye. The method begins with the fixation of cells with membrane antigens (mAg) to a slide (figure 1a). Then, the cells are stained with anti-mAg antibodies that are labeled with fluorochromes. The slide is kept for a period of incubation, after which the slide is washed to remove any unbound excess labeled antibody. Then, the slide is viewed under a fluorescent microscope. Under a fluorescent microscope, it is observed that the field is dark, and areas with bound antibodies fluoresce green. This technique can be used to detect viral, parasitic, and tumor antigens from patient specimens or monolayers of cells.

Figure 1: Direct and indirect methods of immunofluorescence

Indirect immunofluorescence

This method is used to detect antibodies in a patient’s serum. It starts with the reaction of unlabeled primary antibodies with cells having membrane antigens. After an incubation period, the slide is washed to remove any unbound antibodies. Then, the cells are stained with fluorochrome-labeled secondary antibodies (fluorescein-labeled goat anti-mouse antibodies). This antibody binds to the Fc portion of the first antibody and persists despite washing. When observed under an electron microscope, the presence of secondary antibodies is detected (Figure 1b).

Difference between indirect immunofluorescence and direct immunofluorescence

Indirect immunofluorescence staining has some advantages over direct staining. The supply of primary antibodies is often a limiting factor and loss of primary antibody occurs during conjugation reaction. Indirect methods avoid the loss of Primary antibody, which is not conjugated with fluorochrome.

Indirect methods also increase the sensitivity of staining because multiple molecules of the fluorochrome reagent bind to each primary antibody molecule and increase the amount of light emitted at the location of each primary antibody molecule.

Application of immunofluorescence

The CD4+ and CD8+ T-cell populations can be identified by applying the technique of immunofluorescence. Antigen-antibody complexes in autoimmune diseases can be detected by immunofluorescence. It is also suitable for detecting complement components in tissues. The major application of it is localizing antigens in tissue sections or sub-cellular compartments.

Fluorescent antibody techniques are important qualitative tools, but they do not provide any quantitative data. The flow cytometer uses a laser beam and a light detector to count single intact cells in suspension. Whenever a cell passes the laser beam, light is deflected from the detector, and this interruption of the laser signal is recorded. The cells having a fluorescently labeled antibody bound to their cell surface antigens get spurred by the laser and emit light that is detected by a second detector system located at a right angle to the laser beam.

The flow cytometer has multiple applications in clinical and research areas. The cell populations that have been labeled with two or three different fluorescent antibodies can be identified by flow cytometry.

Conclusion

The antigen-antibody binding depends on weak and non-covalent interactions like hydrogen bonds, hydrophobic interactions, electrostatic forces, and Van der Walls interactions. A very close fit between antigen and antibody increases the strength of the bond. The sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody determines the affinity. The specificity of an antigen-antibody reaction is the ability of an individual antibody combining site to react with only one antigenic determinant.

Immunofluorescence is an antigen-antibody interaction where the antibodies are tagged (labeled) with a fluorescent dye. Then, it is followed by the visualization of the antigen-antibody complex under a fluorescent microscope. Direct immunofluorescence is used to detect antigens in clinical specimens using specific fluorochrome-labeled antibodies. In this process, the specific antibody is directly conjugated with a fluorescent dye. Indirect immunofluorescence starts with the reaction of unlabeled primary antibodies with cells having membrane antigens. It has more advantages than direct immunofluorescence.

Antigen-antibody complexes in autoimmune diseases can be detected by immunofluorescence. It is also suitable for detecting complement components in tissues.

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