The antigen-antibody interaction: Agglutination reaction

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In this article, I briefly describe agglutination reaction, which is a type of 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, a large number of such weak interactions are required. These interactions can only take place if the antigen and antibody molecules are close enough for some of the individual atoms to fit into complementary recesses.

Affinity and avidity

The strength of antigen-antibody interaction depends on a very close fit between the two. Affinity is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody. 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. When comparing affinity and avidity, 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.

The antigen–antibody interaction is highly specific. However, sometimes the antibody elicited by one antigen can cross-react with an unrelated antigen, called cross-reactivity. Cross-reactions arise because the cross-reacting antigen has an epitope, which is structurally similar to one on the immunizing antigen.

Cross-reactivity

Cross-reactivity is often observed among polysaccharide antigens that contain similar oligosaccharide residues. The glycoproteins expressed on red blood cells are the ABO blood group antigens. Subtle differences in the terminal residues of the sugars attached to these surface proteins distinguish the A and B blood group antigens. An individual lacking one or both of these antigens will have serum antibodies to the missing antigens. Thus, individuals belonging to blood groups O and B, have anti-A antibodies in their serum. Similarly, individuals belonging to blood groups O and A, have anti-B antibodies in their serum.

Individuals belonging to blood group AB, are believed not to have anti-A nor anti-B antibodies because they express both antigens on their red cells. The antibodies are induced by exposure to cross-reacting microbial antigens present on common intestinal bacteria. Microbial antigens elicit the blood group antibodies, which will cross-react with similar oligosaccharides present on foreign red blood cells providing the basis for blood typing tests, and accounting for the necessity of compatible blood types during blood transfusions. A number of viral and bacterial antigens elicit antibody that cross-reacts with the host-cell components, which results in a tissue damaging reaction. Some vaccines also exhibit cross-reactivity.

Types of antigen-antibody interaction

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

Agglutination reaction

The interaction of antibody with a particulate antigen results in visible clumping, known as agglutination. Antibodies that agglutinate specific antigens, termed as agglutinin. Agglutination and precipitation, both are highly specific as they depend on the specific antibody and antigen pair. Like precipitation reactions, an excess of antibody also inhibits agglutination reactions, which is known as prozone effect. However, the difference between the two reactions lies in the size of antigens. In precipitation, antigens are soluble molecules, whereas agglutination reactions involve antigens which are large and easily sedimented particles. Agglutination reactions can be used to type blood cells for transfusion, to identify bacterial cultures, and to detect the presence and relative amount of specific antibody in a patient’s serum.

Prozone effect

When antibody concentration is high, the number of antibody binding sites can greatly exceed the number of epitopes. As a result, most antibodies bind antigen only univalently instead of multivalently. The univalent binding does not allow antibodies to crosslink between antigens. This effect is known as prozone effect, which can be observed by performing the assay at a variety of antigen or antibody concentration. At optimum dilution of antibody concentration, higher levels of agglutination can be observed. When using polyclonal antibodies, incomplete antibodies (class IgG) also cause prozone effect. Incomplete antibodies are the antibodies present in high concentration in the antiserum, which bind to the antigen but do not induce agglutination.

Types of agglutination reactions

Agglutination reactions can be classified into three types, such as quantitative agglutination, qualitative agglutination and passive agglutination.

Quantitative agglutination

To know whether a patient has or had bacterial infection, agglutination reaction is considered. This type of agglutination reaction is called quantitative agglutination test as the amount of antibodies to particulate antigen is determined. The presence of serum antibodies in an individual specific for surface antigens on the bacterial cells can be detected by bacterial agglutination reactions. Suppose a patient is suspected of having typhoid fever, then the patient’s serum is mixed with a culture of Salmonella typhi. If there is a visible agglutination marked by clumping of the bacteria, then it can be said that the patient either had or has an S. typhi infection.

Since certain antibodies can persist in a patient’s blood for years after the patent has recovered from the infection, a positive reaction does not confirm that the patient currently has the infection. To determine whether a patient is currently suffering from typhoid fever, the amount or titer of the antibody is determined, both at the onset of illness and two weeks later.

The serum from the suspected patient is serially diluted in an array of tubes to which the bacteria is added. The observation of visible agglutination in the last tube reflects the serum antibody titer of the patient. The agglutination titer is defined as the reciprocal of the greatest serum dilution that elicits a positive agglutination reaction. It is obvious that the higher the titer, the greater is the antibody response to the disease typhoid. If the patient currently suffers from suspected typhoid fever, it shows a significant rise in the agglutination titer to the bacterium Salmonella typhi. Bacterial typing is also done through agglutination reactions.

Qualitative agglutination

The agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody. The antibody is mixed with the specific antigen, and the agglutination of the particulate antigen indicates a positive test. Typing of red blood cells is known as hemagglutination, which is a specific form of agglutination. The ABO blood group antigens are intrinsic red blood cell antigens. The ‘A’ and ‘B’ signs refers to proteins on the surface of red blood cells. The individuals expressing the ‘A’ antigens, have blood type ‘A’ and those expressing ‘B’ antigens, have blood type ‘B’. Individuals expressing both ‘A’ and ‘B’ antigens, have blood type ‘AB’. Those individuals not expressing either ‘A’ or ‘B’ antigen, have blood type ‘O’. Blood type can be determined by using antibodies that bind to the A or B blood group in a sample of blood.

In the process of blood grouping, an individual’s serum is tested against RBCs of known blood groups. The individual’s RBCs are also tested against known serum types. In this way, the individual’s blood group is confirmed from both RBCs and serum. To type for the ABO antigens, red blood cells (RBCs) are mixed on a slide with antisera to the A or B blood-group antigens. If a visible clump is seen on the slide, it denotes the presence of antigens in the cells. If antibodies that bind the A blood group are added and agglutination occurs, then it confirms the blood to be either type A or type AB. To identify between type A or type AB, antibodies that bind the B group are added and if there is no agglutination, the blood is identified as type A.

Passive agglutination

The process of passive agglutination involves soluble antigens. The particles are coated with soluble antigens, and the process of agglutination is carried out by antiserum specific for the adsorbed antigen. Passive hemagglutination is a kind of passive agglutination in which erythrocytes are usually modified by mild treatment with tannic acid or chromium chloride. They are used to adsorb soluble antigen onto their surface, which then agglutinate in the presence of antiserum specific for the adsorbed antigen. Serum containing antibodies is serially diluted into microtiter plate wells. Then the red blood cells coated with antigen are added to individual well. The size of the characteristic spread pattern of agglutinated red blood cells on the bottom of the well is used to assay the agglutination.

Passive agglutination can be performed with tanned erythrocytes or synthetic particles such as latex beads. The synthetic beads have the advantage of consistency, uniformity, and stability. The first step in the process involves the linking of latex particle with the antibody molecules that specifically attach to the antigenic determinants on the surface of the particles. The formation of large lattices is observed through these cross links. These large lattices sediment readily due to the large size of clumps, and are visible to the eye within minutes.

Inhibition of agglutination reaction

The modification of the agglutination reaction is called agglutination inhibition. Before mixing the antibody with latex, if it is incubated with antigen, then agglutination is inhibited. This happens as there are no free antibodies available for agglutination. In agglutination inhibition, the absence of agglutination is a diagnostic of antigen, which provides a high sensitive assay for small quantities of antigen. The home pregnancy test kits, which included latex particles coated with human chorionic gonadotropin (HCG) and antibody to HCG. With the addition of urine of a pregnant woman containing HCG to the kit, agglutination of the latex particles is inhibited with further addition of anti-HCG. The absence of agglutination confirms the pregnancy. If the urine contains no HCG, then visible clumping occurs and agglutination can be observed, which indicates no pregnancy (figure below).

Figure : Agglutination inhibition

To detect the presence of any illegal drugs in an individual’s blood or urine sample, agglutination inhibition assays can be employed. They are also widely used in clinical laboratories to determine whether an individual has been exposed to certain types of viruses that cause agglutination of red blood cells. The immune status of women against Rubella virus can be determined by applying this technique.

Conclusion

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 bonding depends on weak and non-covalent interactions like hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Walls interactions.

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 the avidity. A strong antigen-antibody interaction depends on a very close fit between the antigen and antibody, which requires high degree of specificity. Though the antigen–antibody interaction is highly specific, sometimes the antibody elicited by one antigen can cross-react with an unrelated antigen, known as cross-reactivity.

Agglutination reaction can be categorized into quantitative agglutination, qualitative agglutination, and passive agglutination. Agglutination reaction is performed to determine whether a patient has or had bacterial infection. Quantitative agglutination test determines the amount of antibodies to particulate antigen. Agglutination reaction can be carried out in a qualitative manner, e.g., typing of red blood cells is an example of qualitative agglutination. Passive agglutination involves soluble antigens. The particles are coated with soluble antigens, and the process of agglutination is carried out by antiserum specific for the adsorbed antigen.

Agglutination inhibition is the modification of agglutination reaction. In agglutination inhibition, the absence of agglutination is a diagnostic of antigen, which provides a high sensitive assay for small quantities of antigen.

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