Immunology and Virology - seconda parte

Structure and Characteristics of Immunoglobulins

Non-coding sequences adjacent to the coding segments of Ig loci play an important role in the recombination process and the expression of the various genetic loci.

The CDRs are Hypervariable Regions or Complementary Determining Regions: segments of about 10 aa, three of the VH region and three of the VL region, which combine in a three-dimensional structure that constitutes the binding site. CDR3 has a great variability.

CDR aa form multiple contacts with the antigen: the most extensive contact is with CDR3; they are also involved in the bonds of conserved regions.

Basic characteristics of classes of Immunoglobulins

There are 5 different classes of Immunoglobulins, with different structures and different roles in the response of the immune system:

• IgA
• IgG
• IgE
• IgG
• IgM

Immunoglobulins can be monomeric,

dimeric or pentameric ones.

IgG Structure

All IgG are monomers: the subclasses differ in the number of disulphide bonds and length of the hinge region.

The heavy chains and light one of the antibody are link together by disulphide bonds!

Properties

• IgG is the major Ig in serum - 75% of serum Ig is IgG
• IgG is the major Ig in extra vascular spaces
• Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer is mediated by a receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally well; IgG2 does not cross well.
• Fixes complement - Not all subclasses fix equally well; IgG4 does not fix complement
• Binding to cells - Macrophages, monocytes, neutrophils and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc receptors.

IgM Structure

IgM normally exists as a pentamer but it can also exist as a monomer. In the pentameric form all heavy chains are

identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra domain on the mu chain (CH4) and it has another protein covalently bound via a S-S bond called the J chain (a protein chain). This chain functions in polymerization of the molecule into a pentamer.

Properties:

• IgM is the third most common serum Ig.
• IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen.
• As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of microorganisms.
• IgM binds to some cells via Fc receptors.

B cell surface IgM exists as a monomer and lacks J chain but it has an extra 20 amino acids at the C-terminus to anchor it into the membrane. Cell surface IgM functions as a receptor for antigen on B cells. Surface IgM is noncovalently associated with two additional proteins in the membrane of the B

1. cell called Ig-alpha and Ig-beta that act as signal transducing molecules since the cytoplasmic tail of the Ig molecule itself is too short to transduce a signal.
2. IgA Structure
• Serum IgA is a monomer but IgA found in secretions is a dimer. A J chain is associated with it.
• When IgA is found in secretions (sIgA) is also has another protein associated with it called the secretory piece or T piece. Unlike the remainder of the IgA which is made in the plasma cell, the secretory piece is made in epithelial cells and is added to the IgA as it passes into the secretions. The secretory piece helps IgA to be transported across mucosa and also protects it from degradation in the secretions.
• The IgA in the epithelia there are some receptor that can link the antibody, then it is transcytosed and got the T piece.
3. Properties
• IgA is the 2nd most common serum Ig.
• IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since it is found in secretions secretory IgA is important in

local (mucosal) immunity.

• Normally IgA does not fix complement, unless aggregated.
• IgA can binding to some cells (like neutrophils) - PMN's and some lymphocytes.

IgD

Structure

IgD exists only as a monomer.

Properties

• IgD is found in low levels in serum; its role in serum uncertain.
• IgD is primarily found on B cell surfaces where it functions as a receptor for antigen. IgD on the surface of B cells has extra amino acids at C-terminal end for anchoring to the membrane. It also associates with the Ig-alpha and Ig-beta chains.
• IgD does not bind complement.

IgE

Structure

IgE exists as a monomer and has an extra domain in the constant region.

Properties

• IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen.
• Involved in allergic reactions - As a consequence of its binding to basophils an mast cells, IgE is involved in allergic reactions. Binding of the allergen to the

IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms.

IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite.

IgE does not fix complement.

Monoclonal Antibodies

They are largely used in research and in therapy.

Monoclonal antibodies (mAb or moAb) are antibodies that are made by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies can have monovalent affinity, in that they bind to the same epitope (the part of an antigen that is recognized by the antibody). In contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several different plasma cell (antibody-secreting immune cell) lineages. Bispecific monoclonal antibodies can also

Hybridoma technology is a method for producing large numbers of identical antibodies (also called monoclonal antibodies). This process starts by injecting a mouse (or other mammal) with an antigen that provokes an immune response. A type of white blood cell, the B cell, produces antibodies that bind to the injected antigen. These antibody producing B-cells are then harvested from the mouse and, in turn, fused with immortal B cell cancer cells, a myeloma, to produce a hybrid cell line called a hybridoma, which has both the antibody-producing ability of the B-cell and the longevity and reproducibility of the myeloma. The hybridomas can be grown in culture, each culture starting with one viable hybridoma cell, producing cultures each of which consists of genetically identical hybridomas which produce one antibody per culture (monoclonal) rather than mixtures of different antibodies (polyclonal).

The myeloma cell line that is used in this process is selected for its ability to grow in tissue culture and for an absence of antibody synthesis. In contrast to polyclonal antibodies, which are mixtures of many different antibody molecules, the monoclonal antibodies produced by each hybridoma line are all chemically identical.

The production of monoclonal antibodies was invented by César Milstein and Georges J. F. Köhler in 1975. They shared the Nobel Prize of 1984 for Medicine and Physiology with Niels Kaj Jerne, who made other contributions to immunology.

Polyclonal Antibodies

Polyclonal antibodies are produced from different clones of B cells, each of them having specific binding site for different epitopes on the antigen.

Same antigens have different epitopes on them.

Polyclonal antibodies are different antibodies that each has specific epitopes to bind on.

Monoclonal Antibody Application

By mean of specific HAT medium in vitro selection, are taken cells recognized by

Specific antigen, then separated in order to obtain isolated clones: we obtain just one monoclonal antibody, which derives from one B cell.

The use of monoclonal antibodies is numerous and includes the prevention, diagnosis, and treatment of disease. For example, monoclonal antibodies can distinguish subsets of B cells and T cells, which is helpful in identifying different types of leukaemias. In addition, specific monoclonal antibodies have been used to define cell surface markers on white blood cells and other cell types. This led to the cluster of differentiation series of markers. These are often referred to as CD markers and define several hundred different cell surface components of cells, each specified by binding of a particular monoclonal antibody. Such antibodies are extremely useful for fluorescence-activated cell sorting, the specific isolation of particular types of cells.

- Identification of Cell Markers

- Immune Diagnosis of Infections

- Tumor Diagnosis

- Functional Analysis of Soluble or

Those antibodies are already used against different kinds of tumors:

Some of them are chimeric, while others are humanized.

HAMA = Human Anti-Mouse Antibody

Mouse antibodies cannot be used in therapy as our immune system recognizes them as antigens, and that tries to fight them. That's why another antibody is obtained, in which the variable part is of the mouse, and the constant chain is human.

Those antibodies have been improved in technology, in order to reduce the parts of the mouse and increase the human ones: in the chimeric antibody, the variable domains are of the mouse, while the constant domains are human ones; a completely human antibody has been made in the laboratory thanks to the substitution of all the variable domains with human parts, in order to obtain antibodies exclusively from human B cells.

Ig expression during B Lymphocyte maturation

Immunoglobulins are produced by B lymphocytes, which mature in the bone marrow and migrate to secondary organs.