Home Resources Immunoglobulin Structure

The Immunoglobulin Structure

The Immunogobulin Structure A single immunoglobulin molecule is shown above, with the five main regions FAB, FC, Heavy chain, Light Chain and Complimentary Determining Regions (CDR). The FC region is mainly involved in compliment attachment via later stages of the immune response, which results in clearance of the antigen that the antibody has bound to. The FAB is mainly involved in scaffolding and orientating the CDR to maintain a strong adhesion between antigen and antibody. There are two main arms, which are identical, and promote a valency of 2 allowing agglutination to occur.

Certain immunoglobulins such as Immunoglobulin M form a pentameric structure of immunoglobulin proteins and have a valency of 10, and as a result are much better agglutinators.

The CDR is involved in the binding to the antigen, it does this by presenting amino acids in the correct direction that allow hydrogen bonds and van der Waals attractions to occur between atoms/amino acids on the antigen.

Immunoglobulin Classes

Immunoglobulin Classes Immunoglobulin molecules are comprised of light (L) and heavy (H) chains, each consisting of variable (V) and constant (C) regions.

There are two types of light chains, kappa (k) and lambda (?), only one of which is used in each antibody.

There are five types of heavy chain alpha (a), delta (d), epsilon (e), gamma (?), and mu (µ), only one of which is used in each antibody. The heavy chain denotes the class of antibody that the molecule will end up being, such as IgA, IgM, IgG.

Complimentary Determining Region

Complimentary Determining Region Genetic information required by the immune system is stored in a set of cells known as B-cells. B-cells originate from stem cells in the bone marrow and become terminally differentiated during their development and once matured contain a shortened version of the immunoglobulin genes. At a certain point during their development they remove a large portion of the genetic information that had allowed them to create an extremely diverse range of antibody CDRS and have selected to translate only specific regions of the genes needed for 1 heavy and 1 light chain.

In humans, the genes responsible for the possible chain CDR’s are k, l, a, d, e, g, and m. Kappa (k) resides on lambda (l) resides on chromosome 22 and a, d, e, g, and m reside on chromosome 14. Therefore, a shortening of two out of the three chromosomes mentioned will be evident in a mature B-cell depending on whether the immunglobulin produced has a l or a k light chain.

The B-cell removes a stretch of the chromosome because the DNA contains a large stretch of multiple copies of slightly different Variable (V), Diversity (D) and Joining (J) regions. These differing V, D, J regions (V gene) were created by gene duplication of one V [˜ Lemon battery cell, made with copper and zinc electrodes] , one D, and one J [˜ 1 W·s, watt- second] region followed by further base substitution via natural selection over time. This has created a library of differing V, D, J regions that lie one after another in the stretch of DNA at that particular locus. These can be selected independently and linked to create a unique molecule. CDRs are created via the combination of single copies of each V, D, and J from a library of multiple heterologous V, D, J regions to create a V gene that is now contiguous. The B-cells once matured remove these different copies of V, D, and J from the chromosomes altogether. (The cell still retains the ability to switch class if required; this is achieved via a differing Constant region (C) on the 3’ end following the chosen C selected for that B-cell. In general, the C region is selected via mRNA splicing).

Immunoglobulin diversity is therefore a result of evolutionary determined germline repertoire which is inherited and updated down the history of the genetic line. The somatic association of germline V, D, J segments which can occur at different regions within any of the V, D, J regions allows not only amino acid order to change but also the number of amino acids at that the particular V gene to vary. Once a functional V gene is created, somatic hyper mutation of particular codons will occur to produce differing amino acids in complimentary determining regions which ‘fine tune’ the binding site.

The diagram shows how V, D, J regions can organise to create a large number of antibodies.

This unique somatic rearrangement of DNA is held within the nucleus of a B-cell. This B-cell is only one cell in a large population of B-cells that make up the immune system. These B-cells have been primed early on during foetal development via negative selection to not react with any protein that is ‘self’. The methods through which negative selection occur differ among species and the genes which control this process occur within differing chromosomes within different species. However all species achieve the end result in that any circulating antigen that is recognised as being ‘non-self’ promotes an immune response.

How the Immune System triggers a response

How the Immune System triggers a response Immune systems do this via a molecular cascade, which involves Antigen Presenting Cells (APC), helper T-cells and B-cells. The T-Cell Receptor on the helper T-cell recognises the degraded antigen and the Major Histocompatibility glycoprotein complex (MHC II). This complex of T-cell receptor with degraded antigen fragment and MHC II stimulates the release of the soluble molecule Interleukin –1 (IL-1). This IL-1 molecule binds to the IL-1 receptor on the helper T-cell and is internalised. Whilst IL-1 is non- specific, only helper T-cells with APC bound will react to this molecule. This combined stimulation by IL-1 and the APC causes the expression of a growth factor interleukin 2 (IL-2) and its receptor. IL-2 causes helper T-cells that are expressing IL-2 and the IL-2 receptor and are bound to APC to proliferate.

Now that the IL-1 and IL-2 receptors have IL-1 and IL-2 bound respectively, the next phase of the immune response starts. The helper T-cell is involved in another complex with a B-cell, which has specifically phagocytosed and degraded the antigen. The binding of the helper T-cell to the B-cell MHC II causes the B-cell to synthesize an interleukin 4 (IL-4) Receptor. Helper T-cells then synthesize IL-4. When IL-4 binds to IL-4 receptors, B-cells synthesize Interleukin 5 (IL5) receptors. Helper T-cells also produce IL-5. This complex of helper T-cells + IL-5 + IL-4 + B-cell causes B-cells to split into: 1. a Larger Plasma cell and 2. a Smaller Memory cell

Polyclonal Antibodies

Polyclonal Antibodies The immune response will generally involve the activation of multiple B-cells all which target a particular epitope within an antigen, and therefore a large number of differing antibodies will be produced that will circulate throughout the circulatory system to clear the antigen from the body. It is this population of antibodies, which are purified from whole blood, which is clotted to serum and then purified to release immunoglobulins. These complex mixtures of antibodies, all targeting different epitopes on a protein, are called polyclonal antibodies and are ideal for use in second stage antigen detection in sandwich assays.

Monoclonal Antibodies

Monoclonal Antibodies B-cells can be isolated easily from the spleen and lymph nodes of immunised animals; however, these cells have a limited life span, and can only divide a limited number of times, coined the ‘Hayflick limit’. This prohibits the culture of B-cells directly. For an antibody to be useful in research or industry, it must be readily available in large quantities. Due to the Hayflick limit, this would not be possible using B-cells cultured in vitro as they would eventually stop dividing and the population would die out.

Consequently, in 1975 Köhler and Milstein developed a technology to fuse immortal heteromyleoma cells with lymphocytes, using poly ethylglycol (PEG) to break down cell membranes and allow mixing of the genetic material from both cell types. The resulting cell type is called a hybridoma. This hybridoma takes on the characteristics of both the lymphocyte and heteromyeloma cell, creating an immortal cell with the ability to produce antibody. As the new cell line hybridoma is a product of the fusion of one heteromyeloma cell with one B-cell, the culture only ever has one antibody within the supernatant which, when purified, is called a Monoclonal antibody. This technology allows scientists to extract and purify one antibody from the complex mixture of antibodies present in the in vivo polyclonal response. This cell line, once stabilised via single cell cloning, can be frozen and stored indefinitely under liquid nitrogen, allowing the antibody to be produced in vitro, in large quantities when required.

Monoclonal antibodies can be raised against many targets. Specific antibody characteristics can be identified and selected e.g. sensitivity requirements and cross reactivity levels can be specified and monoclonal antibodies screened to identify any cell lines exhibiting the required characteristics.

Proteins

Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential components of organisms that participate in every process within cells. Many proteins are enzymes that catalyse biochemical reactions that are vital to cell metabolism. Proteins also have structural and / or mechanical functions, such as actin and myosin in muscle. These are the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell integrity. Proteins also play a functional role in cell signalling, immune responses, cell adhesion, and the cell cycle.

Eukaryotic Expression Systems

 

Prokaryotic Protein Expression Systems

Prokaryotic recombinant protein expression systems have several advantages. These include, cost, culture conditions, rapid cell growth, yield and relatively short expression time. Expression can be induced using several methods i.e., IPTG, lactose, cold shock etc.
On the other hand, if the protein is required for functional or enzymatic studies, prokaryotic systems may prove disadvantageous, as many proteins form insoluble aggregates, known as inclusion bodies which after refolding may not retain correct biological functionality. Furthermore, bacteria do not allow for post-translational modifications, sometimes necessary for biological activity.

 

 

Eukaryotic Expression Systems

Eukaryotic genes are not really “at home” in prokaryotic cells, even when they are expressed under the control of the prokaryotic vectors. One reason is that E. coli cells frequently recognise the protein products of cloned eukaryotic genes as foreign and remove them. In addition, prokaryotes do not carry out the same kind of post-translational modification as eukaryotes. For example, a protein that would ordinarily be coupled to sugars in a eukaryotic cell will be expressed as a ‘naked’ protein when cloned and expressed in bacteria. Thus, a protein’s activity or stability, is affected, in addition to its reactivity to its respective antibody. Moreover, the interior of a bacterial cell is not as conducive to correct folding of eukaryotic proteins as that of the interior of a eukaryotic cell. Frequently, the result is improperly folded, biologically inactive products of cloned genes.
Eukaryotic systems, which are currently employed in the expression of recombinant proteins, include:
• Yeast cells
• Mammalian cells
• Baculovirus cells (insect cells)