Antigen

Concept of Antigen• Antigens are substances that induce a specific

immune response and subsequently react with the products of a specific immune response.

• An antigen is a molecule that stimulates an immune response.

• The word originated from the notion that they can stimulate antibody generation. We now know that the immune system does not only consist of antibodies.

• The modern definition encompasses all substances that can be recognized by the adaptive immune system.

• Tolerogen - An antigen that invokes a specific immune non-responsiveness due to its molecular form. If its molecular form is changed, a tolerogen can become an immunogen.

• Allergen - An allergen is a substance that causes the allergic reaction. The (detrimental) reaction may result after exposure via ingestion, inhalation, injection or contact with skin.

Antigens can be classified in order of their origins

• Exogenous antigens Exogenous antigens are antigens that have

entered the body from the outside, for example by inhalation, ingestion, or injection. By endocytosis or phagocytosis, these antigens are taken into the antigen-presenting cells (APCs) and processed into fragments.

• Endogenous antigens

Endogenous antigens are antigens that have been generated within the cell, as a result of normal cell metabolism, or because of viral or intracellular bacterial infection.

• Autoantigens An autoantigen is usually a normal

protein or complex of proteins (and sometimes DNA or RNA) that is recognized by the immune system of patients suffering from a specific autoimmune disease.

These antigens should under normal conditions not be the target of the immune system, but due to mainly genetic and environmental factors the normal immunological tolerance for such an antigen has been lost in these patients.

• Tumor antigens Tumor antigens are those antigens that are

presented by the MHC I molecules on the surface of tumor cells. These antigens can sometimes be presented only by tumor cells and never by the normal ones. In this case, they are called tumor-specific antigens (TSAs) and typically result from a tumor specific mutation.

Characteristics of Antigen

★ Immunogenicity The capacity to stimulate the production

of antibodies or cell-mediated immune responses.

★ Antigenicity The ability to bind antibody.

♣ Complete antigen♣ Incomplete antigen, also known as hapten

Incomplete antigens have antigenic determinants, but cannot induce immune responses because they lack one or more of the important attributes needed for this function (one example of an incomplete antigen is a hapten, which is an artificial monovalent epitope)

Properties of antigen• Foreignness• Specificity

• High molecular weight: Min 750 Da

• Structural Stability• Degradability• Route of administration• Host genetics• Dose of Antigens

Molecule of Hemoglobin

Antigenic epitopes

Epitope,or,Antigenic determinants, arethe portions of antigen molecules that physically interact with paratopes (combining sites) of immune response molecules and therefore actually "determine" antigen specificity

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Antigenic epitopes

Types of Epitopes

1. Linear epitopes ♣ continuous and found in

polysaccharides as well as in both native (nondenatured) and denatured proteins, especially fibrillar proteins.

♣ specificity depends upon primary sequence.

♣ typical size is 5-6 subunits in length.

2. Conformational epitopes

♣ Discontinuous (involve multiple subunits, often located far apart in the primary sequence of the antigen molecule) and are thus found only in native (globular) proteins.

♣ Specificity depends upon conformation, or three-dimensional shape, which is a combination of tertiary and quaternary structure ... supported by primary and secondary structure, of course.

♣ Typical size is hard to pinpoint, but sequences of up to 16 amino acids in certain protein antigens have been shown to interact with their complementary paratope.

Two different epitopes

• B cell epitope, a portion of antigen molecule that is recognized by B cell receptors.

• T cell epitope, the region of antigen molecules that are recognized by T cell receptors.

T cell epitope and B cell epitope

Classification of Antigens• Thymus-dependent antigen(TD-Ag) • Thymus-independent antigen(TI-Ag)

Superantigen

• Molecules that are potent T lymphocyte mitogens and simultaneously bind to class II MHC molecules. They are often associated with staphylococcal products and are involved in enterotoxemias and toxic shock syndrome in humans.

• Superantigens (SAgs) are secreted proteins (exotoxins) that exhibit highly potent lymphocyte-transforming (mitogenic) activity directed towards T lymphocytes.

• Compared to a normal antigen-induced T-cell response where 0.001-0.0001% of the body’s T-cells are activated, SAgs are capable of activating up to 20% of the body’s T-cells. This causes a massive immune response that is not specific to any particular epitope on the SAg.

Conventional Ag

Mitogen

• An agent that induces mitosis. Here means to activate T cells and/or B cells without help from APCs.

• Lectin, for example, concanavalin A (ConA).

• LPS(lipopolysaccharide)• Staphylococcal protein A(SPA)

Adjuvant

• Adjuvant: The Latin "adjuvans" means to help, particularly to reach a goal.

• An adjuvant is a substance that helps and enhances the pharmacological effect of a drug or increases the ability of an antigen to stimulate the immune system.

Classification of Adjuvant

• Freund’s adjuvant ♥ Complete Freund’s adjuvant(CFA) ♥ Incomplete Freund’s adjuvant(IFA)• Liposome• Inorganic compound• Cytokine• Biodegradable nanoparticles

Mechanisms of adjuvants• Prolonged persistence of immunogen

molecules at the site of injection.

• Enhancement of co-stimulatory signals.

• Induction of granuloma formation.

• Stimulation of lymphocyte proliferation in a non-specific manner.

Other antigens1 Heterophilic antigen A kind of common antigen, existing in

human, animals, and microbes. e. g. Fossman antigen.2 Xenogenic antigen This antigen comes from different genus

and generic. e. g. pathogenic antigen.

3 Allogenic antigen The specific antigen exists in different

individuals. Blood type antigens

4 Autoantigen A pathological term. BUT, sperm antigen

5 Idiotypic antigen

An antibody molecule is some sort of foreign molecule when generated in animal body. Such that immune system recognizes it as Antigen, which is known as Idiotypic antigen.

Antibody•Antibody is also termed as Immunoglobulin•Antibody is globulin (gamma) fraction of serum proteins•Antibodies are generated by the immune system of the body against a specific foreign antigen•Antibodies main are component of Humoral Immunity (HI)•Rodney Porter first described the detail structure of antibody molecule and awarded the Nobel Prize in 1972.

Structure of Immunoglobulin

Immunoglobulin Classes

• Ig class Heavy chain Light chain• IgM mukappaorlambda

• IgG (gamma)kappaorlambda

• IgA (alpha)kappaorlambda

• IgE (epsilon)kappaorlambda

• IgD (delta)kappaorlambda

The major Immunoglobulin ClassesProperties IgG IgM IgA IgE IgD

Mol wt (kDa) 160 900 360 200 160-180

% Carbohydrate 3 12 7 12 12

Electrophoretic mobility

γ β β - γ β - γ γ

Heavy chain γ μ α ε δ

Heavy chain domains

4 5 4 5 4

Subclasses 4 None 2 None None

Half life (days) 21 5 6 2 3

Antigen binding Valency

2 5(10) 2,4 2 2

Immunoglobulin Structure• Each light chain contains about 214 amino acids in two domains.• The heavy chains of IgG contain about 445 amino acids in 4

domains.• The variation in amino acid sequence is largely restricted to three

smaller regions within the entire variable region, these regions are known as hypervariable regions.

• Between the hypervariable regions, the amino acid sequences are relatively constant and termed as framework regions.

• The hinge region consists of about 12 amino acids located between CH1 and CH2 domains. The hinge region contains many hydrophilic and proline residues. The hydrophilic residues make the peptide chains unfold and thus make this region readily accessible to proteolytic enzymes. The proline residues make the chain flexible. This is the region, where papain and pepsin act. This region also contains all the interchain disulfide bonds.

IgG• Molecular wt: 160 kDa• Secrets as secondary antibody• Major secretory antibody in animals• Found in highest concentration in blood• Subclasses: IgG1, IgG2, IgG3 and IgG4

IgM

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C2

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C

C4

C3

C2

C C

C4

C3

C2

CC

C4

C3C2

C

C

C4

C3

C2

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•IgM is a pentamer.

•It is primary immunoglobulin.

•Available as a second highest concentration in mammalian serum

•All the monomers are bind together by a cysteine-rich polypeptide called the J-chain (15 kDa) binds two of the units to complete the circle.

•Heavy chain of IgM molecule contains an additional CH4 domain

IgA

• IgA is major secretory antibody of non-ruminants• IgA produced in body surfaces may either pass through

epithelial cells into external secretions or diffuse into blood stream. Thus, most of the IgA made in the intestinal wall is carried into the intestinal fluid.

• The IgA is transported through intestinal epithelial cells bound to a receptor of 71 kDa protein known as the polymeric Ig receptor (pIgR) or secretory component. Secretory components binds covalently to IgA dimers to form a complex molecule called secretory IgA (SIgA) and protects the IgA from digestion by intertinal proteases.

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SS

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SS

SS

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IgE and IgD• IgE is a monomer with an extra CH4 domain in their heavy

chains.• IgE is largely responsible in immunity against parasitic worms.• They are also responsible in Type-I hypersensitivity reaction.• IgE molecules generally found bound on the surface of mast

cells and basophils.

• IgD is primarily a B cell receptor.• IgD molecule does not have CH2 domain. The CH1 and CH3

domain is separated by a long exposed hinge region.• It does not have any disulfide bonds, because of that IgD is

highly susceptible to proteolytic enzymes.

Immunoglobulin variation• Isotypes: The structural variations that result in the

production of the different Ig classes occur in all animals of a species. These variants are called classes or isotypes.

• Allotypes: When variation occurs because of the minor sequence variations between the proteins of different individuals. These variations, which are inherited, are called allotypes.

• Idiotypes: The structural variations in proteins is unique to the antigen-binding proteins such as Igs and the TCRs. These are formed by the variable regions on light and heavy chains. These variations are individually called idiotopes and collectively known as idiotypes.

Affinity and avidity of antibodies

Affinity

The tightness with which the antigen binding site attaches to an antigen determinant (epitope)

Avidity

The tightness of binding when several antigen binding sites attach to several antigenic determinants

Physical forces holding antibodies and antigen together

• Plasma cells make over a billion different types of antibodies

• Each cell, however, only contains 100,000 genes that code for these polypeptides

• To code for this many antibodies, somatic recombination takes place– Gene segments are shuffled and combined in different

ways by each B cell as it becomes immunocompetent– Information of the newly assembled genes is expressed

as B cell receptors and as antibodies

Mechanisms of Antibody Diversity

• Random mixing of gene segments makes unique antibody genes that: – Code for H and L chains – Account for part of the variability in antibodies

• V gene segments, called hypervariable regions, mutate and increase antibody variation

• Plasma cells can switch H chains, making two or more classes with the same V region

Antibody Diversity

• Antibodies themselves do not destroy antigen; they inactivate and tag it for destruction

• All antibodies form an antigen-antibody (immune) complex• Defensive mechanisms used by antibodies are

neutralization, agglutination, precipitation, and complement fixation

Antibody Targets

• Complement fixation is the main mechanism used against cellular antigens

• Antibodies bound to cells change shape and expose complement binding sites

• This triggers complement fixation and cell lysis• Complement activation:

– Enhances the inflammatory response– Uses a positive feedback cycle to promote phagocytosis– Enlists more and more defensive elements

Complement Fixation and Activation

• Neutralization – antibodies bind to and block specific sites on viruses or exotoxins, thus preventing these antigens from binding to receptors on tissue cells.

• Agglutination – antibodies bind the same determinant on more than one antigen, makes antigen-antibody complexes that are cross-linked into large lattices.

• Cell-bound antigens are cross-linked, causing clumping (agglutination).

• Precipitation – soluble molecules are cross-linked into large insoluble complexes

Other Mechanisms of Antibody Action

Mechanisms of Antibody Action