Antigen and antibody

Dr P Pramod BHMS,MD(Hom)
Antigen : The immune response is characterized by the production of proteins and specifically reactive lymphocytes when an animal encounters foreign macromolecule or cell. The inducing substances are called antigens-antibody generators or immunogens.

Definition
The term antigenicity implies both the ability to induce an immune response (immunogenicity) and the ability to react with products of that response.
Examples: blood group antigens A,B,D; bacterial capsular antigen; bacterial cell wall etc

Epitope
Sites either on or within the antigen with which antibodies and T-cell receptors react are called epitopes. The epitopes are antigenic determinants. They are small areas in antigen having four or five amino acids that has a specific chemical structure, electrical charge and stearic configuration capable of sensitizing immunocyte and of reacting with its complimentary site on specific antibody or T-cell receptor. The epitopes are generally very small. They may have a linear or conformational arrangement. Linear means continuous in amino acid sequence. Conformational denotes spatial arrangement is close, but discontinuous in amino acid sequence. Each antigen may have multiple epitopes, each coding for different antibody. The number of such epitopes determines the valence of the antigen. New antigens can be produced by altering just the epitope. This is possible by conjugating molecules to antigen or by enzymatically removing portion of antigen.

Types of antigen

• Autologous antigen:Antigens that are native to body do not induce immune reaction in the same body. Example: skin graft from thigh to chest of same person.
• Syngeneic antigen:Antigen in genetically identical twins.
• Allogeneic antigen: Antigens in genetically different members of a species. Example: graft from mother to son.
• Xenogeneic antigen: Antigen found in different species. Example monkey kidney transplant to human being.
• Heterophile antigen:Antigens that have one or more epitopes in common. Since antibodies identify epitopes only, antibodies against one antigen will cross-react with other antigen having identical epitope.

Examples:
Syphilis screening test: Cardiolipin antigen is a lipid with which spirochaetes share epitope.So this antigen is used to test antisyphilitic antibodies – Khan test, VDRL Test
Infectious mononucleosis screening tests Ebstein Barr virus that causes infectious mononucleosis induces antibody in the host that cross react with similar epitopes present on sheep RBC membrane.
Weil Felix reaction: serum from patients with Rickettsial infection agglutinate certain strains of Proteus vulgaris antigen. This heterophile reaction is used in the diagnosis of Rocky mountain spotted fever.
Organ specific antigen: antigens seen only in certain organs of the body. Eg thyroglobulin ( thyroid), myelin basic protein ( brain).
Sequestrated antigen: they are organ specific antigens that are not normally exposed to the immune system.

But once they are available in the circulation antibodies develop against them. Eg. Puncture wound to left eye causes release of certain ocular antigens that result in sympathetic uveitis in the right eye.
Thymus dependent antigen: they require T cells to induce antibody production by B cells. They have a complex structure. Their immunologic action is active over a wide range of doses. They can induce production of different classes of antibodies. They induce good immunologic memory. These antigens require processing by macrophages. Examples: erythocytes. serum proteins etc.

Thymus independent antigen: those that can directly stimulate B cell proliferation.They are generally simple in structure. They are active only in high doses. These antigens induce production of certain antibodies (IgG3,IgM).They do not require prior processing by macrophages. They are poor in inducing immunologic memory. Examples: flagellin, pnueumococcal capsular polysaccharide.

Factors determining antigenicity
1. Immunogenicity of antigen
2. Foreignness
3. Chemical nature
4. Size of antigens
5. Biodegradability
6. Structural stability
7. Route of administration

chemical nature of antigen

• Proteins are the strongest immunogens.
• Lipoproteins are complex proteins seen in cell membranes. They are also strongly immunogenic.
• Capsular polysaccharides seen in bacterial capsules.
• Lipopolyasccharides seen in the cell wall of gram negative bacteria.
• Glycoproteins blood group antigens A and B.
• Nucleoprotein especially significant in systemic lupus erythematosus.

Size of antigen
The larger the size, the greater the number of epitopes and hence the antigenicity.
Larger molecules are easily phagocytosed by antigen presenting cells, thereby inducing antibody production.

Haptens:
Antigens with low molecular weight ( 5-10 k Da) cannot induce antibody response. But they can react with preformed antibodies. Such partial antigens are called haptens. Eg. Antibiotics, analgesics etc.
If haptens are coupled to an immunogenic carrier, the immunogenicity is amplified.
Eg penicilline if degraded in the body produce penicilloyl moiety that joins with serum albumin ( immunogenic carrier) inducing allergic response.

Adjuvants:
They are immunogenic carriers and used in vaccines.
Examples: aluminium hydroxide,aluminium potassium tartrate, aluminium phosphate,calcium phosphate.

Biodegradability
To become an antigen it is not sufficient to be a macromolecule,but it should also be capable of being metabolised by body.Polystyrene is a large molecule but it cannot be metabolized.so it is non-antigenic.

Stability
The stability of molecule is also essential to induce immunogenicity.Gelatin is good protein, but it is quite unstable.So it is non-antigenic.In fact the adjuvants are stable in serum for long periods and their action lasts longer.

Antibody
Immunoglobins are defined as proteins of animal origin endowed with known antibody activity and for certain other proteins related to them by chemical
structure including myeloma protein, macroglobulin, cryoglobulin etc. Antibodies are glycoproteins present in gamma globulin fraction of the serum.
The structure of antibody was studied by Rodney Porter,Gerald Edelman and co.They were awarded Nobel prize for this discovery.Rabbit antibody to egg albumin was treated with proteolytic enzyme pappain that yielded two fragments-Fc and Fab.
Antibodies are produced by modified Bcells called plasma cells.

STRUCTURE
The basic structural unit of an antibody consists of four polypeptide chains covalently linked by disulphide bonds.
The monomeric immunoglobulin is composed of two identical heavy polypeptide chains (H chain) and two identical light polypeptide chains
( L chain)
Chain H chain L chain
Mol. Weight 50-75 k Da 25 k Da
Amino acid 400 200
Types 5 ( alpha,gamma, delta, mu, epsilon) 2 (kappa, Lambda)

Each H chain and L chain has two parts, a variable portion and a constant portion. The variable portion is the amino terminal while the constant portion is the carboxyl terminal. The variable portions of heavy and light chains are close together. The lambda chain has three hypervariable areas: aminoacid sequence-30-35,50-55,95-100 in the initial 107 aminoacids which constitute the variable portion. Similarly the hypervariable areas of heavy chain is located in same three corresponding areas of heavy chain. Within the variable portion lies a hypervariable region called complementarity determining regions ( CDR ). The amino acid sequence in CDR is highly variable and hence multiple antibodies according to specificity can be produced.

Both L and H chains have peptide loops enclosed by disulphide bonds. These loops are called domains. Each L chain has two domains – one variable and one constant. H chains of alpha, delta and gamma have one variable and three constant domains. H chains of Mu and epsilon have one variable and four constant domains. The given immunoglobulin has a pair of either kappa or lambda light chains.

HINGE
The junction of the domains which are at an angle to each other is highly flexible and susceptible to fragmentation by enzymes. This junction is called hinge.

FUNCTIONAL DIVISION
Antibodies are functionally divided into two fragments – Fab and Fc fragments. Fab fragment consists of entire L chain and amino terminal of H chain. This is the antigen binding site called Paratope
Fc fragment is the carboxy terminal of heavy chain. It activates complement cascade via classical or alternate pathway. It decides the biological activity of the antibody. This portion also contains the carbohydrate moiety of the glycoprotein.
J Chain : these are the connecting proteins in a polymeric immunoglobulin. Eg J chain joins the two Ig A molecules in a secretory Ig A

Ig G

• Heavy chain: gamma
• Ig G monomer consisting of one pair of L chain. 75 % of total serum immunoglobulin is IgG. It is subdivided into IgG1, IgG2, IgG3, IgG4, based on the gamma chains 1,2,3,4, respectively. Most common Ig G is IgG1. It has a half life of 21 days. Molecular weight – 150000. Serum concentration of 7-19 gm/lit. It has four domains.
• Functions : 
• the only antibody that crosses the placenta
• complement fixation, precipitation, neutralization of toxins
• intracellular killing by NK cells require IgG.
• Is a major opsonising immunoglobin

IgA

• Heavy chain alpha.
• Has a a half life of 6 days. There are two types of IgA – IgA1 ( alpha1) and IgA2 (Alpha 2). It has four domains. Serum concentration is 1-5 gms/lit.It is usually a dimer. 
• Serum IgA_ It constitutes mainly of IgA1. it can be inactivated by gonococci, pneumocci, meningiococci, Haemophilus infleuenzae.
• Secretary IgA : this is usually a dimer. It is the liner of the epithelial tract. It is seen in secretions like the saliva, tears, colostrums, bronchial, genitourinary, intestinal. The dominant class is IgA2.
• Functions:-
• The epithelial cells have receptors with affinity for secretory IgA.
• Secretory IgA protects mucosal surfaces by reacting with adhesion molecules on the potential pathogens and interfering with their adherence and colonization on the epithelium. Neutrophils have receptors for IgA.

IgM

• Heavy chain Mu. Has a half life of 5 days. Serum concentration is 0.5 – 2 gms/litre.
• Molecular weight is 90000. it is also called millionaire molecule. It has 5 domains . there are 2 classes, IgM1, and IgM2. it is a pentamer with 5 monomeric units linked by J chain and by disulphide bonds in the Fc fragment.
• Functions: 
• It is the first antibody that an immunologically competent B lymphocyte can produce.
• It is the predominant antibody in the primary immune response. The predominant antibody produced in the fetus from 20th week onwards.
• This is 1000 times more effective than IgG in immune hemolysis.
• It is 500- 100 times more effective than IgG in opsonisation.
• It is 100 times more effective than IgG in bactericidal action.
• It is 20 times more effective than IgG in bacterial agglutination.
• Some IgM secreted by glands.
• Monomeric IgM is an antibody receptor on the surface of B lymphocyte.

IgD

• Heavy chain Delta. 
• Half life 2-8 days. Serum concentration is 100 microgram /litre. It exists as a monomer. Molecular weight is 160000. it has four domains. 
• Function:
• Antigen receptor in B cell membrane.

IgE

• Heavy chain epsilon
• Serum concentration is 5 microgram /litre. It has a molecular weight of 170000. it is a monomer with 5 domains. It is produced by B cells and plasma cells in the spleen, lymphoid tissue, tonsils adenoids, respiratory tract and gastrointestinal tract. The production begins early in the fetus. It is the only heat labile antibody (56degree Celsius)
• Functions:
• It is seen associated with atopic diseases like, asthma, anaphylaxis. On combining with allergen, they induce histamine release from mast cells and basophils. They have affinity for attaching to membranes of basophils and mast cells. 
• They have an important role in immunity against helminthic diseases

APPLICATIONS
Multiple myeloma:
This defined as abnormal proliferation of neoplastic plasma cells resulting in the production of monoclonal antibodies associated with multifocal lytic lesions through out the skeletal system. The serum electrophoresis which normally shows a raised peak of albumin, here there is an additional peak of gammaglobulins. In 60% cases Ig G is raised while in 16% Ig A and in 14% Ig M is raised .Both heavy and light chains are increased here. The increased light chains are called Bence Jones proteins. This protein is excreted in the urine .It is soluble at room temperature ,precipitates at 60 degree C and redissolves at 80 degree C.
Waldenstrom’s macroglobulinaemia: In this condition there is increased production of Ig M class antibodies.
Cryoglobulinaemia: Here the antibodies are precipitated on cooling and redissolves on warming.
Agammaglobulinaemia:  Deficiency in the production of antibodies.

Hybridoma: It is a modern technique of large scale production of specific antibodies. The spleen cells from a mouse previously sensitized with chosen antigens is grown with mouse myeloma cell in a culture medium. The culture media like polyethylene glycol are destabilizing. So the myeloma cells fuse with the splenic cells and this is then transferred to a tissue culture medium where only hybrid myeloma cells survive. These hybridoma cells continue to produce specific antibodies.