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Discuss the structure of antibody molecules
The antibody molecule can be defined as a Glycoprotein – meaning the presence of carbohydrate groups attached to a polypeptide chain. The molecule consists of several protein chains. The traditional representation of an antibody molecule is depicted as a Y-shaped structure containing 4 protein subunits. The two longer subunits are called heavy chains, they contain 400 amino acids and have a molecular mass of about 50-75 kDa. The two shorter subunits are called light chains, contain 200 amino acids and hold a molecular mass of 25 kDa.
There are two types of light chains, κ and λ. They are similar in structure, however, coded by different genes. Therefore any individual antibody molecule will consist of two heavy chains and two light chains of either κ or λ, but never both. The heavy chains are linked to each other and also linked to the light chains. These links are provided by disulphide bridges.
Within both heavy and light chains, repeating substructures called domains exist. These domains are approximately 110 amino acids long and can be found in both heavy and light chains. They are flanked by intrachain disulphide bridges. Approximately 4-5 domains exist in the heavy chain and the light chain contains 2. An important property of a domain is its ability to fold correctly even when removed from a protein.
The N-Terminal ends contain what is known as a variable domain. The variable domain is responsible for the detection of a specific antigen. Variable regions are built of distinctive amino acid sequences that are unique to a particular antibody. Variable domains differ between heavy and light chains, therefore, these domains are referred to as VH and VL respectively.
The variable domain can be broken down further into three distinct regions. These regions are between 5-15 amino acids long and are contained In each heavy and light chain. They are called the hyper-variable regions.
The C-Terminal contains constant domains. These regions, unlike variable domains, are common with other antibody molecules. These domains do not perform antigen specific recognition. The heavy chain constant domains are called CH1, CH2, CH3 and CH4; the light chain domain is called CL. The space between the CH1 and CH2 domains of the heavy chain is a region containing several prolines (Amino Acid) making this part of the molecule quite flexible, it is known as the hinge region.
IgG is the basic monomer structure of all immunoglobulin’s. Its 4 polypeptide chains can separate by reducing disulphide bonds. This occurs in the presence of a strong acid such as 2-Mercaptoethanol. After bonds have been broken, the molecule possesses strong non-covalent attractions.
The whole antibody structure can be proven via the breaking of IgG by various chemicals. Incubation of Ig with enzyme papain produces two fragments, one of 45 kDa (Fab - fragment antigen binding) and one of 50 kDa (Fc- fragment crystallisable). Pepsin is another enzyme that generates a single fragment of 100 kDa F(Ab)2. A final enzyme, 2-Mercaptoethanol, is capable of breaking intrachain disulphide bonds generating two fragments, the two heavy chains at 50 kDa and the two light chains at 25 kDa each.
Besides the most abundant IgG class, there exists IgM, IgA, IgE and IgD.
Page 68 DIAGRAM
The antibody molecule binds to a specific part of the antigen, called an epitope. This structure ranges from 8-22 amino acids long. Epitopes can be linear or conformational. Linear sequences are continuous while conformational involves folding of the protein.
Describe how antibodies are produced for the detection of antigen in immunoassays
Antibody is produced by plasma cells that differentiate from antigen-specific B cells. B lymphocytes that have never encountered antigen before have IgM and IgD on their surface. B lymphocytes can bind to antigen via IgM or IgD but cannot secrete antibody straight away.
To secrete large amounts of antibody, B-Cells must undergo differential processes. These processes increase the quality and range of antibodies.
- Affinity maturation of antibody - Increases affinity of antibodies towards antigen.
- Antibody class switch. B-Cells may change the antibody on their surface from IgM and IgD to another class. Importantly, an individual plasma cell will only secrete one type of antibody.
PAGE 143 DIAGRAM
The CD4 T cell (Helper T Cell) is an important part of Antibody production. They do not directly create antibodies, however, they are necessary for most B-Cells to produce antibody.
Stages in antibody production:
- Antigen presents itself to antigen-specific CD4 T cells to proliferate and differentiate into Helper T Cells.
- Stimulation of B cells by antigen and interaction with helper T Cells.
- Proliferation of B cells and their differentiation into plasma or memory cells.
This occurs primarily in the spleen, although lymph nodes play a major role in production also. Antigen in the spleen comes from blood, whereas antigen in the lymph nodes arrives via lymphatic vessels.
PAGE 132 DIAGRAM
Polyclonal antibodies arise from multiple B-Cell clones that have been activated by the immune response of an immunized animal. B-Cells produce somewhere between 1x108 and 1x1010 IgG antibodies with different binding sites. IgG is the most abundant antibody in serum.
The name Polyclonal derives from the many antibodies that bind to multiple epitopes on any one antigen. Because of this, cross reactivity can occur. This is a process whereby some antibodies in mixture will bind to epitopes found in other antigens.
The Goat, Sheep and Rabbit have been used in the production of polyclonal antibodies. The animal is injected with a specific antigen that elicits a primary immune response. This is followed by a secondary and tertiary immunization that produces higher titers of antibody against the particular immunising antigen.
Serum is then collected from the animal, containing the antibodies of interest. This serum can be refined through affinity purification in order to enrich the antibodies. This process ultimately lends itself to the production of high titer, high affinity polyclonal antibodies against the antigen of interest.
For example IgG can be purified through protein G Affinity Chromatography. This technique essentially recovers the target analyte from an immobilised ligand. Protein G is a wall component of Staphylococcus Aureus that specifically binds to the Fc portion of IgG. This binding is brought about by electrostatic interaction, Van der Waals forces or Hydrogen bonding and hydrophobic interactions.
Non-specific proteins are washed and then the bound target is eluted using an elution wash, the elution wash involves an elution buffer and a change in the ionic strength of the column to force elution.
When the antigen of interest is sometimes too small to be detected, it can bind to a bigger molecule e.g Albumin, which is also immunogenic. When this occurs, the smaller molecule is defined as a Hapten.
This Conjugated Hapten is essentially a low molecular weight antigen. Without binding to a larger molecule, the immune response would be too small. The conjugated Hapten is useful in this instance. Anything less than 3kDa means the antigen is probably not immunogenic, in which case conjugation is required.
- Commonly used carriers:
-BSA (Bovine Serum Albumin)
-HSA (Human Serum Albumin)
-KLH (Keyhole limpet haemocyanin)
Rabbit thyroglobulin and KLH are better carriers for smaller molecules, giving rise to antibodies with high affinity. BSA also gives satisfactory results and is commonly used.
The injection of antigen into a Rabbit over a 12 week period is a method used to produce antibodies. If an inject of antigen alone does not work, adjuvant is added along with antigen. This Helps activation of B-Cells and increases the concentration of antibody. This antiserum will contain antibodies generated by the animal and may interfere (background binding). Affinity purification can solve this. Monoclonal antibodies don’t have this problem.
Monoclonal Antibodies are produced when the animal is injected with antigen, the Spleen is then removed, the B-Cell source, and fused with myeloma cells resulting in hybridomas. Cell fusion with tumour cells grown in culture is performed in the presence of 50% polyethylene glycol. Myeloma cells lose ability to make antibodies, however, immortal hybridoma cells can grow indefinitely and make antibody. Hybridomas are selected via HAT medium (hypoxanthine, aminoprotein and thymidine) this is because only B-Cells survive in this environment, Myeloma cells will die. Monoclonal antibodies recognise one epitope only.