User:Kinkreet/Immunology/Structure and Function of Antibodies

An antigen may have more than one epitope, of more than one kind; they are termed linear if the whole epitope are made up of consecutive residues, or discontinuous if made up of residues in close proximity spatially, but not in the 2-dimensional sense. On the polymeric carbohydrates of bacteria, the epitope are often repeated and so many antibodies can bind to the same antigen many times. The epitope must be on the surface of the antigen for it to be recognised; this is one of the reasons why T cells can only bind antigens presented on B cells, because the B cell must break down the antigen to present the epitope originally hidden inside the antigen.

Production

Antibodies are produced in two types - polyclonal and monoclonal. Polyclonal antibodies (antisera) contain a mixture of different types of antibodies that target different epitopes of the same antigen; most polyclonal antibodies are of the IgG subclass. Monoclonal antibodies contain one type of antibody that target one epitope of the antigen.

Polyclonal

Polyclonal antibodies are produced by purifying the serum from inoculated animals such as mice; this is usually in the form of injecting the antigen of interest into the organism to stimulate an immune response by the B cells. The B cells mature into plasma cells which secretes antibodies. Polyclonal antibodies can be produced quickly with requiring little technology, and is relatively inexpensive to produce. It can be used to amplify a signal as the target protein will bind to multiple sites on the antigen instead of just one, giving a larger signal; however, this should not be done if quantification is important, as it becomes inaccurate. It can also be used in immunoprecipitation (IP, and ChIP) to ensure the antigen required is retained, even if the antigen varies due to differences in post-translational modifications, or different conformations due to local environment. Other uses include histochemistry, enzyme linked immunosorbent assays (ELISA),diagnosis of disease , immunoturbidimetric methods, western blots and Biochip technology.

Monoclonal

Monoclonal antibodies contains only one type and subclass of immunoglobulin, specific to only one epitope on the antigen. They are relatively expensive and require technical training to use the machines; the time scale are usually longer, especially for hybridomas.

Previously, monoclonal antibodies are obtained by picking out a single B lymphocyte from either the spleen or lymph nodes of immunized animals, and then culturing it to allow it produce more antibodies, which are then purified. However, due to the 'Hayflick limit', B cells can only divide a number of times before dying out. In 1975, Kohler and Milstein used polyethylglycol (PEG) to fuse immortalized heteromyleoma (tumour cell) with B lymphocytes, allowing the cytosolic and genetic material to mix, creating a cell (hybridoma) with the immortal characteristics of myleoma and the antibody-producing characteristics of B lymphocytes. The mixture is treated to select out the plasma and myeloma cells, so only the hybridomas remain; the selected mixture is then diluted extensively so each aliquot effectively contains only one hybridoma cell. Each aliquot is then cultivated to produce a large population of that hybridoma; this can be frozen in liquid nitrogen for storage, and can make a large amount of antibodies when required. Because the whole cell line is derived from the fusion of one B lymphocyte, all the antibodies produced by the cell line (clone of the original) will be identical to each other, as a single B cell can only make one type of antibody. The advantages of hydridomas are, once set up, a constant, renewable and consistent source of monoclonal antibodies; they are specific and so gives little background noise. It does not suffer from batch-to-batch variation, and so the results are highly reproducible.

Chimeric and Humanized

Antibodies can be used therapeutically against certain immunodiseases; antibodies against tumour necrosis factor (TNF) are used to treat arthritis. However, because the antibodies are from a different organism, the recipient might generate an immune response to it. Chimeric and humanized antibodies are produced to minimize this effect. Chimeric antibodies contain a large portion (usually the constant regions) that are human; and humanized antibodies are where parts of the antibody's amino acid sequence are modified to be more similar to a human antibody.

Structure

Every antibody is made up of two heavy chains and two light chains. A light chain is covalently linked to a heavy chain through a disulphide bond; the two heavy chains are held together by two disulphide bonds at the hinge region. Both the chains are made up of domains of ~110Å, stabilised by a disulphide bond within each domain. The light chain contains two such domains (V_L for Variable Light, and C_L for Constant Light); the heavy chain contains four (γ, α, δ) or five (μ, ε) domains (V_H, C_H1, C_H2, C_H3, C_H4). For antibodies with γ, α, δ, the hinge region is flexible, whereas in μ, ε they are not. The antibody can also be seen as joined fragments - the light chain and V_H and C_H1 domains of the heavy chain makes up the antigen-binding fragment (Fab) and the rest makes up the crystallisable fragment (Fc).

The V_L domain contains 3 hypervariable regions (HV, a.k.a. Complementarity determining region, CDR), which determines the shape of the binding site. In three-dimensions, they are proximal to each other on the tip of the antibody arm. The C_H1 domain also contains a HV region, and together, they form the antigen binding site, which can bind 6-12 residues of the epitope.

There are five classes of antibodies, defined by the heavy chain types (heavy chain μ, γ, α, δ, ε gives IgM, IgG, IgA, IgD and IgE, respectively). Each class of antibody can have variations, dependent on the type of light chain. There are two different types of light chains - κ and λ - and only one type can exist in each antibody.

Different sections of an antibody can be separated using proteolytic cleavage.

Papain

Papain is a cysteine protease first found in papaya; in a cell, it can be secreted or stored in lysosomes. It is synthesised as a 345-residue precursor (including a 18-residue signalling peptide required for membrane association, followed by 115-residue propeptide required for correct folding, and a 211-residue mature peptide). Papain is composed of 2 domains surrounding a cleft; the cleft contains a diad (C25, H159) catalytic site. The whole structure is stabilised by 3 disulfide bonds. It works by deprotonating cys25 by his159's imidazole ring, allowing cys25 to nucleophilically attack the carbonyl carbon on a peptide chain. On an antibody, it cleaves the Fc fragment from the Fab fragment. The cleaved fragments would no longer be able to promote agglutination, precipitation, opsonisation and lysis.

β-mercaptoethanol

β-mercaptoethanol reduce and thus cleaves disulphide bonds. Thus, if an antibody is treated with β-mercaptoethanol, the light chains will dissociate from the heavy chain, and both chains will unfold because the disulphide bonds holding each domain together will be reduced. Depending on the concentration of β-mercaptoethanol, different fragment conformation and associations may be obtained.

Pepsin

Pepsin cleaves below the hinge and the double disulphide bond, giving two separated Fc fragments, and a joined Fab fragment.

Function

The adaptive immune response involves B cells binding to antigens specifically using the antibodies presented on the cell surface. Because the range of antigens the organism may encounter, there are many different antibodies, each on the surface of different B cells. The function of an antibody includes binding to antigens (on pathogens or on infected tissues), bind host tissues, and fixing complement (mostly serine proteases)

Antibodies can bind to complement proteins and causes it to fragment into a large and small fragment. The small fragment is used to attract cells, and the large fragment starts a cascade.

Uses

Antibodies can be used to screen for molecules which cross reacts with it, and also for screening for uncharacterised antigens, such as cell surface markers (CD markers).

Different types of antibodies

IgG

IgG is the major (~75%) type of antibody found in the secondary immune response. IgG can diffuse easily, and thus is evenly distributed in the body between the bloodstream and tissue. There are many subclasses of IgG, categorized by the number and arrangement of the disulphide bonds linking the heavy chains together. IgG1 have 2 disulphide bonds on one side of the hinge, making it flexible; IgG2 and IgG4 have 2 more bonds on the other side of the hinge, making it more inflexible; IgG2 are often used to recognised repetitive and regularly-spaced epitopes, such as the carbohydrates on bacterial surfaces. IgG3 contains many bonds in the hinge region, and is susceptible to proteolytic cleavage, giving it a short half-life.

	IgG1	IgG2	IgG3	IgG4
Proportion of total IgG (%)	45-75	16-48	2-8	1-12
Length of heavy-chain hinge (amino acids)	15	12	62	12
Number of disulfide bonds in the hinge	2	4	11	2
Susceptibility of hinge to proteolytic cleavage	++	+	+++	+
Half-life in serum (days)	21	21	7	21
Capacity to bind C1q and activate compliment	++	+	+++	+
Response to protein antigens	++	+	++	+
Response to carbohydrate antigens	+	++	-	-
Response to allergens	+	-	-	++
Number of allotypes	3	5	19	1

IgM (10%)

IgM is an antibody which can activate complement to the same degree as IgG can. When not bound to a membrane, it exists as a pentamer (and sometimes hexamer) which are held together by disulphide bonds. The pentamer has a high molecular weight (1 MDa) nad thus is restricted to the blood stream. The pentamer contains a J chain, which is added at the ER, this is essential for polymerisation of the IgM. The IgM pentamer is good at binding to bacteria and agglutinating them together. IgM pentamer can have two configuration: in a star or staple configuration. The star configuration is roughly flat and can bind to many antigens; in the staple configuration, the variable arms all fold towards one direction, thus binds to only one or two antigens, allowing for very strong binding, as there are potentially 10 binding sites.

There are no subclasses for IgM.

IgA (15%)

IgA is the major antibody in external secretions, such as seromucous secretion of tears, saliva, and mucus in respiratory, urogenital and intestinal tracts. When antigens enter from the tracts, it is recognised by organised lymphoid follicles in the Lamina propria, and the B cells differentiate into antibody-secreting plasma cells. IgA is produced by the plasma cells and they associate as a dimer. The IgA dimer is recognized by a poly-Ig receptor on basolateral membrane of epithelial cells, and upon binding, internalize the ligand-receptor complex. The receptor undergoes proteolytic cleavage, and is excreted at the apical membrane (into the lumen of the tract) as a mature dimer. Like the IgM, dimeric IgA also contains a J chain which helps in the polymerization. The J chain exists on the secretory component (what remains of the poly-Ig receptor), which holds the two IgA monomers together, as well as to protect against proteolytic degration. The internalization of the antibody and its secretion on the opposite end is termed transcytosis.

IgE

IgE is present at very low concentrations in serum, because it is usually bound to surfaces of basophils and mast cells. When bound, it can last for months, whereas in serum it has a half life of only a few days. When an antigen binds, it crosslinks two IgE together, and this causes the release of allergic mediators such as histamine by degranulation. These allergic mediators cause fluid secretion, vasodilation and smooth muscle contraction.

Recognition

Antibody receptors recognizes the F_C region of antibodies; they are named F_c[α/

γ/ε]RI/II/III.

There are three types of receptors which recognises the F_c region of IgG: F_cγRI (CD64), F_cγRII (CD32) and F_cγRIII (CD16). 2-3 genes encode for each of these types, and are designated A, B and C. F_cγRI binds with high affinity, and is used early on in the immune response to recognise single antibodies; F_cγRII and F_cγRIII bind with lower affinity and is used later on in the immune response to bind aggregates of IgG surrounding multivalent antigens. F_cγRI are usually localized on the cell surface of myeloid cells, especially macrophages, and binds the lower hinge region and the C_H2 domain of IgG1 and IgG3. When the antigen binds to the bound antibody, it activates the cell, which may result in phagocytosis, cytokine secretion or other responses.