Immunoglobulins: Its Structure and Classes (1932 Words)

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Antibodies are specific glycoproteins produced by В-cells and constitute main components of humoral immunity.

Antigen or immunogen present in the foreign body (microorganism or parasite) are mainly protein molecules which are responsible for the production of specific antibody or immunoglobin in the host.

Some antigen molecules have low molecular weight so called haptens which do not bind to antibodies but stimulate antibody formation.

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Specific regions of antigen molecules having 5-8 amino acids are called epitope and are required for antibody production. About 10,000 different antibodies have been found in a human body formed by plasma cells. These antibodies are categorized into five main kinds namely IgG, IgM, IgA, IgD and IgE.

Structure of Immunoglobulins:

Generally, immunoglobulins contain one or more basic units (monomers) having four polypeptide chains united by disulphide bonds (S – S). Immunoglobulin G or IgG is the most common and thoroughly studied antibody molecule. It has Y – shaped structure with following domains:

1. Heavy chains:

Entire immunoglobulin is a tetramer consisting of two identical high molecular weight or heavy chains and two low molecular weight or light chains. Heavy chain is made of about 500 amino acids, (mol. weight of 50,000 to 70,000 Daltons). There are 5 types of heavy chains designated α (alpha), δ (delta), Ɛ (epsilon) γ (gamma) and µ (mu) in IgA, IgD, IgE, IgG and IgM respectively.

Both chains of antibody molecule have variable (V) and a constant (C) portion. Generally, half of each light chain consisted of a variable region (V_L) whereas only one-quarter of each heavy chain was variable (V_H) among different patients. Remaining three-quarters of the heavy chain (C_H) was constant for all IgGs.

The constant portion of heavy chain is subdivided into three sections of almost equal length. It is believed that each of the three sections of С part of IgG heavy chain arose during evolution by the duplication of an ancestral gene which encoded for an Ig unit of approximately 110 amino acids. The variable regions (V_H or V_L) are also thought to have arisen by evolution from same ancestral Ig units.

2. Light chains:

They include two low molecular weight chains designated K (Kappa) and λ. (lambda) present in five different antibodies. About 200 – 250 amino acids are present in each chain with mol. weight of 23,000 Daltons). Both chains show following domains:

(a) Antigen-binding domain:

They consist of F_v and Fab segments. Among them, Fab segment is heterodimer being made of entire light chain + V_H and C_H1 parts of heavy chains. F_v segment is also heterodimer containing variable domains of heavy chains (V_H) and light chains (V_L).

(b) Effector domains:

They are also called Fc segment which helps in the elimination of antigen. F_c region consists of homodimer with С_H2 and C_H3 domains of both heavy chains.

Structurally, terminal regions of both light and heavy chains contain three small hyper variable regions forming antigen binding sites. These are also called CDR or complementarity-determining regions. These hyper variable portions of the chains contain deletions and insertions of amino acids as well as substitutions of one amino acid for another.

About 5-10 amino acids in such hyper variable region form the antigen binding site. Therefore, variable domains of an antibody determine the molecule’s combining site specificity.

Proximal remaining parts of the chains are not variable and are known as framework regions. These constant domains facilitate anchorage of antibody in the plasma membrane, activation of the complement system by which bacterial cells are punctured and destroyed and stimulation of lymphocyte pro iteration.

Classes of Immunoglobulins:

1. IgG:

It is the major class of serum immunoglobulins. It takes part in various antibacterial, antiviral and antitoxic activities. It passes through placenta and gives passive immunity to the new born child for about six months.

2. IgM:

It is roughly less than 10% of normal serum immunoglobulins. It is made up of five-four-chain subunits. Its molecular weight is 900,000 Daltons. IgM develops primary immunity against most antigens. IgM antibodies are effective against toxins from diptheria, tetanus, botulism, anthrax, snake venoms.

3. IgA:

IgA antibody is about 13 to 15% of the total Igs in human serum. It is a predominant immunoglobulin in body serum and secretions e.g. saliva, tears, nasal fluids, sweat, colostrum, milk and lung secretions, gastrointestinal and genitourinary secretions. Here it provides defence against microorganisms.

Amniotic fluid also has IgA and it provides passive immunity to the foetus. Serum IgA contains several types of antibodies including isoagglutinins, antibodies of anti-diptheria, anti-Brucella, anti-insulin and anti­poliomyelitis.

Secretory IgA is structurally different from serum IgA. Serum IgA does not pass from serum to exocrine glands or to other mucosal sites. Serum IgA is produced by plasma cells in the bone marrow and Secretory IgA is produced by local plasma cells distributed in Secretory tissues.

4. IgD:

It is found in traces in human serum, i.e. about 1% of total serum Igs. It was first discovered in the serum of patients of myeloma tumors or malignant lymphomas. Its function is not yet established. Its structure is like that of IgG having two L chains and two H chains joined by S-S bonds. Its molecular weight is about 180,000 Daltons.

5. IgE:

IgE in normal serum is lowest amongst all other Igs. Its molecular weight is about 200,000 Daltons. It has also two L chains and two H chains joined by one S-S bonds. Allergens stimulate the plasma cells for the synthesis of IgE antibodies.

Igs are also further sub dived into subclasses. Such as IgG1, IgG2, IgG3 and IgG4 and each of them have a distinct H chain. IgM has IgM1 and IgM2. IgA is also classified into IgA1 and IgA2.

Immunoglobulins are highly antigenic or immunogenic. Antibodies can be produced against different classes or subclasses of Igs, after injecting them into another heterologus species. The different Igs differ in antigenic determinants mainly on А-chains. The differences are mainly due to amino acid sequences.

Immunocytochemistry:

It is based on the detection of antigens by antibodies. Antigens are large circular molecules such as proteins, polysaccharides and nucleic acids. When these are injected into another animal, they activate lymphocytes to produce antibodies.

Hapten (a small molecule) such as 5-hydroxy tryptamine, when coupled to a larger molecular species can also produce antibodies. Basically each antibody is composed of four peptides: two identical light chains and two identical heavy chains. The specificity for the antigenic determinants depends on the variability of the amino acid sequence at the binding site.

Most antibodies belong to the immunoglobulin G (IgG) class present in the y-globulin fraction of the serum. IgG has a molecular weight of 145,000 to 156,000 Daltons and is Y-shape. It has two binding sites at the variable parts (Fab) and a constant region (Fe).

Structure of antibody molecule:

It consists of two light and two heavy chains joined together by disulphide (-S-S-) bridges. Each light or heavy chain consits of two parts: a variable region, which differs for each antibody specificity, and a constant region, which is same in many antibody molecules.

The composition of two variable regions (VL and VH) will determine the properties of the antibody combining site. The constant part of the heavy chain (CH) determines other properties related to the elimination of the antigen. The heavy chain has three globular protein domains (CH₁, CH₂, CH₃) of related amino acid composition which presumably arose in evolution through duplications of a common DNA sequence.

At the DNA level each domain (including the hinge region) is encoded in a different Exon, and the introns are then removed by RNA splicing. The antibody molecule provides one of the best examples of how different functional domains of proteins can be brought together by the splicing mechanism.

In Immunocytochemistry the antibody that interacts with the tissue antigen is known as the primary antibody. This first antigen-antibody complex can be revealed by suitable markers for observation in the light or electron microscope.

1. Direct method-Albert Coons:

In early fifties conjugated IgG with a fluorescent dye (fluorescein), which permitted direct detection of the complex in a tissue section under fluorescence microscope,

1. In one case IgG was labelled with a radioactive marker such as tritiated amino acid added during synthesis of antibody, and then revealed by radio autography.

2. In other case an enzyme, such as peroxidase, was linked to the IgG and then reacted with diaminobensidine in the presence of H₂O₂ (DAB reaction), giving an opaque deposit that can be seen microscopically. In addition of peroxidase, alkaline phosphatase and β-galactosidase are also used.

3. In another case antibodies coupled with ferritin (iron-containing protein) is used. It is opaque to electrons.

2. Indirect methods:

Here the antigen-antibody interaction is further amplified by the introduction of a second labelled antibody. For example, if the primary antibody is IgG made in a rabbit, an anti-rabbit IgG made in a goat or a sheep is introduced. The reaction can be observed by fluorescence, autoradiography or by electron microscope.

Complex methods:

Besides the use of peroxidase or antibodies labelled with fluorescent dye (tritium or peroxidase), more complex methods are used.

1. Biotin-avidin-peroxidase method:

The constant region (Fc) of the IgG is bound to biotin and then reacted with an avidin-peroxidase conjugate. This enzyme is often coupled to anti IgG is peroxidase, which is reacted with 3′, 3′-diamino benzidine (DAB) in the presence of H₂O₂. This gives an electron- dense deposit that can be detected with light or electron microscopes.

2. PAP method:

This is peroxidase-antiperoxidase (PAP) method of Sternberger. After steps 1 and 2 (Fig. 12) in which unlabelled antibodies are used. In step 3, peroxidase rabbit antiperoxidase complex is introduced. It also binds to the sheep antirabbit IgG. The peroxidase – antiperoxidase complex is made by interacting the enzyme with the corresponding IgG made in the rabbit. The peroxidase is finally detected by DAB method.

3. Protein А-Gold technique:

It is based on the use of colloidal gold particles, which are very opaque to electrons of EM. These particles are surrounded by protein A, extracted from Staphylococcus aureus. It has the property of interacting with IgG immunoglobulins. This interaction is with Fe region of IgG, and thus it does not affect the antibody site which can react with antigen.

The protein А-gold complex can be used for any antigen provided it has interacted with the specific antibody. The gold particles are easily detectable under electron microscope (EM) and because of its small size, the cellular structure containing antigen can be clearly identified. It is also possible to make a quantitative estimation of antigenic sites.

4. Gold anti-IgG method:

In this method colloidal gold may be used as a marker of an IgG for a tissue antigen, either direct or by using a secondary body. Gold particles of 5 to 40 nm are used for tagging the IgG.

The gold particles deposited at the antigen site can be enlarged by reacting them with silver for increasing the contrast. By using gold particles of different sizes associated to different IgG, it is possible to tag two or more antigens in the same tissue. (Pickel and Teitelman, 1984).

Monoclonal antibodies:

These are produced by using cell fission between lymphocytes and cells of myeloma (i.e., a tumor of the bone marrow). These monoclonal antibodies derive from a clone (i.e., a colony of single lymphocyte) are very pure and recognize a single immunogenic determinant in the protein. These have many important medical applications. These can be used in tissues for immunochemical methods (Milstein, 1980).