Basic Immunology - Antibodies

Front 1

The structure of a typical antibody

Back 1

Antibodies are the products of activated B-cells (plasma cells) and are produced in response to infection during the adaptive immune response.

They are also known as immunoglobulins.

They are y-shaped, made up of four polypeptide chains.

> Heavy (H) chains - the two heavy chains are made up of four structural domains, three have a constant (C) structure between molecules and one is highly variable (V) between molecules.

> Light (L) chains - made up of one constant domain and one variable domain. There are gamma and kappa light chains, that have the same function.

Light chains are linked to heavy chains by disulphide bonds.

Antibodies are big molecules, molecular weight of 150,000.

Front 2

What are the functions of a typical antibody?

Back 2

• To recognise and bind antigens derived from the infective pathogens. A highly specific sequence in the fragment antigen binding (Fab) variable region is complementary to a single antigenic determinant, or epitope (the part of an antigenic protein that is recognised by antibody), of an immunogenic protein that binds in the cleft between Vh and Vl.

• To interact with other parts of the immune system to destroy the pathogen once the antibody is bound to it. This is the function of the constant region (Fc).

The 5 main types of heavy chain (isotypes) are specialised to activate different effector functions. The five functional classes of antibody are IgM, IgD, IgG, IgA and IgE

Front 3

Antibody repertoire definition

Back 3

The collection of different antibodies within an individual. It consists of at least 10^11 different molecules.

Antibodies can recognise all classes of molecules including proteins, polysaccharides and lipids.

Front 4

How are so many different antibodies generated?

Back 4

1. Multiple different copies of each gene segment that make up the variable region of an antibody - heavy chains are coded for by variable, diversity and joining segments and light chains are coded for by variable and joining segments. There are multiple copies of each gene segment that make up the variable region of each chain. Different combinations arise by random gene rearrangement.

2. Different combinations of heavy and light chains to form antigen binding site - total number of combinations is 2.61 x 10^6

3. Somatic hypermutation - high rate of point mutation in the variable region genes when B-cells respond to antigen and start producing antibodies at a high rate

Front 5

Antibodies differ in the structure of the constant (Fc) region - antibody isotypes

Back 5

Immunoglobulin isotypes are distinguished by the structure of their heavy chain constant regions.

The differences are in the:

number of constant domains,

the number and distribution of disulphide groups

length of hinge region

number and distribution of carbohydrate groups.

Front 6

Antibodies differ in the structure of the constant (Fc) region - antibody isotypes

Back 6

Immunoglobulin isotypes are distinguished by the structure of their heavy chain constant regions.

The differences are in the:

number of constant domains,

the number and distribution of disulphide groups

length of hinge region

number and distribution of carbohydrate groups.

Front 7

Isotype switching definition

Back 7

During an immune response, the same variable region can be expressed in IgG, IgD, IgA, IgM or IgE isotypes, i.e. With different constant regions

Front 8

IgM

Back 8

Low affinity of binding, but in secreted form it forms pentamers with a total of 10 antigen binding sites.

Because of its large size, it is usually confined to the blood and is the first antibody to be synthesised during an immune response.

The heavy chain isotype then switches to another depending on the cytokine environment

Front 9

IgM

Back 9

Low affinity of binding, but in secreted form it forms pentamers with a total of 10 antigen binding sites.

Because of its large size, it is usually confined to the blood and is the first antibody to be synthesised during an immune response.

The heavy chain isotype then switches to another depending on the cytokine environment

Front 10

IgG and IgE

Back 10

Monomers in the secreted form

IgG is the main isotype found in blood

IgE is present in low levels in the blood

Due to their smaller size, they can diffuse out of the blood and into tissues

Front 11

IgA

Back 11

Usually found as a dimer in the secreted form and a specialised transport system at epithelial surfaces means that IgA is the main isotype found in secretions such as saliva, tears and mucus in the airway.

Monomers are joined by the J-chain

Front 12

IgA

Back 12

Usually found as a dimer in the secreted form and a specialised transport system at epithelial surfaces means that IgA is the main isotype found in secretions such as saliva, tears and mucus in the airway.

Monomers are joined by the J-chain

Front 13

IgD

Back 13

Little IgD is produced at any time

Front 14

Transcytosis of IgA across epithelia into external secretions

Back 14

Mediated by Poly-Ig receptor, a specialised transport protein.

Most IgA is synthesised by plasma cells lying just beneath the gut, airways, tears and salivary and mammary glands.

The dimer is bound by J-chain and diffuses and binds to the Poly-Ig receptor on the basal aspect of epithelial cells. This complex undergoes endocytosis into a vesicle and transport across the epithelial cell to the apical surface. At this surface, the receptor is cleaved to leave the extracellular IgA-binding component (secretory component) bound to the dimer.

This form is known as secretory IgA and is found in all external secretions and is important in mucosal defence. The residual piece of the Poly-Ig receptor is non-functional and is degraded.

Front 15

Antibodies are found in bodily fluids where they have three main functions:

Back 15

• inhibition of bacterial adhesion to host cells - bacteria colonise cell surfaces via specific cell surface molecules (adhesins) which allow them to bind to ligands on the host cell. Specific antibodies bind to adhesins and prevent colonisation and uptake of bacteria. On their own, antibodies can not prevent bacteria replicating outside of cells - IgA important here

• neutralisation of bacterial exotoxins - bacteria release exotoxins (endotoxin when they die). A toxin must bind a specific molecule that acts as a cellular receptor which allows it to be internalised and poison the cell. Specific antibodies that bind to the receptor-binding site on the toxin and prevent uptake are called neutralising antibodies and prevent toxic effect. IgG can diffuse rapidly to neutralise

• inhibition of viral infection - viral infection follows binding of virus to receptors on the cell surface, endocytosis, fusion of viral and cell membranes either on the cell surface or inside of the cell which triggers entry of viral DNA. Antibodies inhibit infection

Front 16

Stimulation of opsonisation - isotypes

Back 16

Antibodies can opsonise bacteria enabling phagocytic cells to ingest and destroy the bacteria. Macrophages have receptors for the constant (Fc) region of antibodies on their surface and bind Fc and internalise it, together with the pathogen.

IgG is the main isotype for opsonisation and aggregation of IgG on the bacterial surface allows binding to Fc receptors.

Free IgG does not bind to Fc receptors, allowing the phagocyte to distinguish between the majority of free antibody and antibody that is bound to pathogen

Front 17

Stimulation of complement C1 activation

Back 17

There are no receptors for IgM. However, the complement system is activated by complexes of antigen with IgM and IgG antibodies and is able both to opsonise and directly destroy bacteria.

IgM is a powerful activator of the complement cascade. It activates complement C1 and enhance opsonisation with C3b

Front 18

Main components and actions of complement

Back 18

• classical pathway - activated by antigen binding to antibody. IgG or IgM in the blood bind to pathogens.

• lectin pathway - activated when lectins bind to carbohydrates on bacterial cell walls

• alternate pathway - spontaneously activated complement binds to surface of pathogen, where it forms C3 convertase (C3bBb) that is stabilised by properdin (factor P).

• recruitment of inflammatory cells - C3a and C5a which recruit neutrophils to help clear bacteria

Front 19

Virus infected cells in the absence of MHC-1 molecules

Back 19

In the absence of MHC-1 molecules, virus infected cells can also signal the presence of intracellular infection by expressing viral proteins on their cell surface that can be recognised by IgG.

Cells bound by IgG can be killed by NK cells which are large lymphoid cells with prominent granules that contain perforin and granzymes that kill the target cell.

The destruction of antibody coated target cells is called antibody-dependent cell-mediated cytotoxicity and is triggered when antibody bound to the cell surface interacts eith FcyIgG receptors on NK cell

Front 20

Immunological memory response

Back 20

• after a B-cell has been activated, it takes 4-5 days for clonal expansion before proliferation is complete and B-cells have become antibody-producing plasma cells. These cells have a life span of 4 weeks, and once antigen is removed, the antigen-specific cells die. However, some do persist after antigen is removed to ensure rapid and effective response on second exposure for lasting immunity.

• observed with antibody response with first immunisation and a secondary booster immunisation with same antigen. Second response occurs after shorter lag phase, higher plateau and antibodies of higher affinity.

Front 21

Immunological memory response

Back 21

• after a B-cell has been activated, it takes 4-5 days for clonal expansion before proliferation is complete and B-cells have become antibody-producing plasma cells. These cells have a life span of 4 weeks, and once antigen is removed, the antigen-specific cells die. However, some do persist after antigen is removed to ensure rapid and effective response on second exposure for lasting immunity.

• observed with antibody response with first immunisation and a secondary booster immunisation with same antigen. Second response occurs after shorter lag phase, higher plateau and antibodies of higher affinity.

Front 22

Active immunisation

Back 22

Killed or heat attenuated live organisms to induce a state of specific immunity

Attenuation makes the organism innocuous but still immunogenic. Lifelong protection can be achieved, especially if boosted by a repeat immunisation

Front 23

Immunological memory response

Back 23

• after a B-cell has been activated, it takes 4-5 days for clonal expansion before proliferation is complete and B-cells have become antibody-producing plasma cells. These cells have a life span of 4 weeks, and once antigen is removed, the antigen-specific cells die. However, some do persist after antigen is removed to ensure rapid and effective response on second exposure for lasting immunity.

• observed with antibody response with first immunisation and a secondary booster immunisation with same antigen. Second response occurs after shorter lag phase, higher plateau and antibodies of higher affinity.

Front 24

Active immunisation

Back 24

Killed or heat attenuated live organisms to induce a state of specific immunity

Attenuation makes the organism innocuous but still immunogenic. Lifelong protection can be achieved, especially if boosted by a repeat immunisation

Front 25

Passive immunity

Back 25

Transfer of serum containing specific antibodies to an organism