Antigens and Epitopes

Antigens

• Antigens are substances recognized by the immune system.
• They can be simple or complex.
• Types of molecules that can be antigens: carbohydrates, lipids, proteins, nucleic acids, and phospholipids.
• B cells recognize any biological antigen.
• T cells recognize peptide antigens presented on MHC.

Epitopes (Antigenic Determinants)

• Epitopes are the smallest part on an antigen that binds with B cell receptors (BCR) and T cell receptors.
• If an antigen contains multiple epitopes, it is called a multivalent antigen.

Functional Types of Antigens/Epitopes

• They are divided into immunogens, haptens, and tolerogens.

Immunogens

• Large antigens with epitopes are capable of binding with immune receptors and inducing an immune response.

Haptens

• Small antigens with epitopes are capable of binding with an immune receptor but cannot induce an immune response on their own.
• Haptens can produce an immune response when conjugated with a large carrier molecule, leading to an immune response against the epitopes of both the hapten and the carrier.

Tolerogens

• Self-antigens (MHC) normally do not stimulate the immune system.

Factors Influencing Immunogenicity

• Size: Proteins > 10 KDs are more immunogenic.
• Complexity: Complex proteins with numerous, diverse epitopes are more likely to induce an immune response compared to simple peptides with one or few epitopes.
• Conformation and Accessibility: Epitopes must be accessible to the immune system.
• Chemical Properties: Proteins are good immunogens, whereas carbohydrates, steroids, and lipids are poor immunogens. Amino acids and haptens are not immunogenic by themselves.

Types of Antigens

T-cell Independent Antigens (TI)

• Activate B cells without T cell help.
• Examples: polysaccharides (Pneumococcal polysaccharide, LPS).

T-cell Dependent Antigens

• Require T cell help for B cell activation.
• Examples: proteins (microbial proteins and non-self or altered-self proteins).

Immunoglobulins (Igs) (Antibodies)

• Glycoproteins that mediate humoral immunity.

Production & Distribution of Antibodies

• In lymph nodes, antigen stimulation of B cells with T helper cytokines leads to B cell proliferation and differentiation into plasma cells.
• Plasma cells secrete antibodies, which enter circulation and travel to the site of infection.
• Mature B cells in bone marrow express membrane-bound antibodies (BCR).
• Antibodies are produced in lymphoid tissue and bone marrow.

Forms of Antibodies

• Membrane Bound Ig
  • Expressed on B cell surfaces (IgM & IgD) as BCR for Ag
  • Binding with Ag initiates B cell response
• Secreted Ig
  • Found in plasma & mucosa & interstitial fluids of tissues

Structure of Antibodies

• Y-shaped molecules comprised of 4 polypeptide chains.
• Two identical heavy chains, each with one variable domain (VH) and 3 or 4 constant domains (CH).
• Two identical light chains, each with one variable domain (VL) and one constant domain (CL).
• Each variable domain (VL or VH) contains three hypervariable regions called complementary determining repeats (CDR).
• Disulfide bonds connect the heavy chain with the light chain and the heavy chains with each other.

Regions of Antibodies Based on Proteolytic Fragments

• Fab (Fragment Antigen Binding): contains the whole light chain + VH + CH1, 2 copies per antibody, involved in antigen recognition and binding.
• Fc (Fragment Crystallizable): tends to crystallize in solution, 1 copy per antibody, consists of the remaining parts of the two heavy chains, constant domain involved in effector and biological functions.
• Hinge Region: Flexible region that lies between Fab and Fc, provides mobility to both Fab arms to accommodate different antigens.

Ig Classes (Isotypes)

Immunoglobulin Classes

• Divided into five different classes based on structural differences in the constant domains of the heavy chain
  • Gamma heavy chains → IgG
  • Alpha heavy chains → IgA
  • Mu heavy chains → IgM
  • Epsilon heavy chains → IgE
  • Delta heavy chains → IgD
• Different classes and subclasses of antibodies perform different effector functions.
• There are two types of light chains: kappa (κ) and lambda (λ). An antibody has either two κ or two λ light chains.
• Heavy chain class (isotype) switching: The switch from one Ig isotype to another. After B lymphocyte activation, a specific B cell clone proliferates and differentiates; some progeny secrete IgM, others produce antibodies of different isotypes.

IgA

- Subtypes: IgA1, 2
- H chain: α1 or α2
- Serum mg/ml: 3.5
- Secreted form: Monomer, dimer, tri
- Functions: Mucosal immunity

IgD

- Subtypes: -
- H chain: δ
- Serum mg/ml: traces
- Secreted form: none
- Functions: B cell receptor

IgE

- Subtypes: -
- H chain: ε
- Serum mg/ml: 0.05
- Secreted form: monomer   
- Functions: Parasite and Allergy

IgG

- Subtypes: IgG1-4
- H chain: γ (1, 2, 3, 4)
- Serum mg/ml: 13.5
- Secreted form: monomer
- Functions: Opsonization, Complement activation, ADCC

IgM

- Subtypes: -
- H chain: μ
- Serum mg/ml: 1.5
- Secreted form: pentamer
- Functions: B cell receptor, Complement activation

Monoclonal Antibodies

• Identical, monospecific antibodies that are produced by one type of immune cell and are all clones of a single parent cell.
• Antibodies from the blood of an immunized host are called polyclonal antibodies.

Production of Monoclonal Antibodies: Hybridoma Technology

- A mouse is immunized with the antigen.
- B cells are isolated from the spleen of the mouse.
- B cells (antibody-producing cells) are fused with myeloma cells (malignant cells) in vitro using a fusion agent such as polyethylene glycol or a virus.
- Cell fusion forms an antibody-producing cell "hybridoma".
- Hybrids (fused cells) are selected for growth in special culture media.
- B cells that fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely. Only hybridomas between B cells and myeloma cells survive.
- Hybridomas secrete a large amount of mAbs.

Applications

• Identification of phenotypic markers: They have been used to define clusters of differentiation (CD markers) on lymphocytes.
• Immunodiagnosis: the diagnosis of many infectious and systemic diseases relies on the detection of specific antigens or antibodies in the circulation or tissues by use of mAbs.
• Tumor diagnosis: tumor-specific monoclonal antibodies are used for detection of tumors by imaging techniques.
• Therapy: a number of mAbs are used therapeutically today, such as Anti-CD3 for immunosuppression and prevention of graft rejection.