Antigen/Antibody Reactions 9/3

Question 1

Role of Antibodies (Ab)

Answer 1

• circulating Ab’s are soluble glycoproteins that recognize and bind antigens (Ag)
• they also function as membrane-bound surface Ag receptors on B cells and play a key role in B cell differentiation
• Consist of a unit of 4 poplypeptide chains: 2 heavy, 2 light
• Five classes of Abs in mammals:
• IgG, IgM, IgA, IgE, IgD

Question 2

Antibody Structure

Answer 2

Heavy chains = classes

• variable regions (Fab)
• Constant regions (Fc)

LIght Chains = types

• Variable regions (Fab)
• Constant regions (Fc)
• antigen binding happens at variable regions on both ends.
• biological activity/function of antibody happens at Fragment constant regions

Question 3

Hypervariable Regions

Answer 3

= Complimentary Determining Regions (CDR) = Idiotopes

• these are areas within the variable regions where poplypeptides show exceptional variablity.
• They are intimately involved in Ag binding by creating an interaction site that is complementary in shape, charge, and hydrophobicity to the epitope it binds.
• There are 6 CDR’s per Ab and TCR

Question 4

Isotypes

Answer 4

Classes = isotype (each has own constant region gene)

• differ in size, charge, aa sequence, carbohydrate content (have structural and functional differences)
• determined by C region of the H chain, defines class and subclass.
• 9 isotypes in humans: each mediates a distinct set of effector functions
• ALL IMMUNOGLOBULIN ISOTYPES ARE BIFUNCTIONAL (with the exception of IgD)
• Abs recognize and bind Ag and then promote killing and/or removal of the immune complex formed through the activation of effector mechanisms.
• Effector functions include binding of the Ab to:

1. Receptors expressed on host tissues
2. The first component of the complement system (C1q) to initiate the classical pathway.

Question 5

Allotype

Answer 5

= Allelic differences - H chains

• subtle differences in isotypes
• We all have IgG (an isotype)- some of us have IgG with suble differences than others. Called Gm markers

Question 6

Idiotype

Answer 6

• Antigenic differences on the Variable regions
• We all may see an antigen, but we respond with slightly different V region determinants
• Idiotypic network

Question 7

IgM

Answer 7

• first antibody produced in a primary response: 2 weeks
• first antibody produced by neonates: this is because it is the first C region on the gene
• basic Y in pentamer form: Has J piece- part of mucosal immunity
• mostly found in serum(5-10%)
• efficienty at binding Ags with multiple repeating epitopes
• efficient at binding complement (conformational change)
• expressed on B cells as a monomer

Question 8

IgG

Answer 8

• predominant in secondary immune response
• 4 subclasses: IgG1, IgG2, IgG3, IgG4 (differences on H chain)
• all four cross placenta
• IgG3 = activates C’
• IgG2 = restricted to carbohydrate Ags
• IgG1/IgG3: bind with affinity to Fc receptors on phagocytic cells –> opsonization

CD markers (Fc receptors for IgG)

• CD16: NK cells, Monocytes/macrophages and granulocytes

Question 9

IgA

Answer 9

• prominent in secretory: main in mucosal immunity (colostrum, saliva, tears, mucus) - bronchial, GU, digestive tracts
• constituent of of secondary immune response
• IgA plasma cells along mucous membrane surfaces are important for entry point immunity and newborn immunity after birth through colostrum

Question 10

IgE

Answer 10

• main player in asthma/allergies: its main function originally was for worms
• binds to blood basophils and tissue mast cells by Fc receptor with very high affinity: this increases half life of mast cells: results in allergic reactions
• Powerful pharmacologic reactions
• Asthma, hay fever, peanut allergies
• Helminth infections

Question 11

IgD

Answer 11

• only Ig isotype that is not bifunctional
• Has no known function in serum

It is present as an Ag-specific receptor on mature B cells: and has same Ag specificity as IgM (same idiotope/)

Question 12

Immunoglobulin Superfamily

Answer 12

IgM, Igalpha/Igbeta heterodimer, T-cell Receptor, MHC molecules

Question 13

Antigens vs. Immunogens

Answer 13

• Antigens are foreign molecules that bind to an antibody or TCR or B cell receptor whether or not they induce an immune response.
• Immunogens are antigens that cause an immune response. All immunogens are antigens but not all antigens are immunogens.
• Pathogen is an organism that causes disease.

Question 14

Epitope

Answer 14

Epitope is the specific part of the Ag(antigen) that contacts the Ag-binding sites of an Ab or TCR. AKA: antigenic determinate.

(Epitope binds the Idiotope by binding the Complementary Determining Region)

Question 15

Haptens

Answer 15

• Haptens are small molecular weight molecules that can bind to an antibody but must be attached to a large carrier macromolecule to stimulate an immune response specific for the small molecule – use this process in vaccination. Also seen in a lot of allergies, i.e. penicillin allergy.
• Haptens are antigens, not immunogens, that must be attached to carrier macromolecule to stimulate specific response.

Question 16

Types of Antigens (Ags)

Answer 16

• Endogenous: The body’s own cellular components or intracellular pathogens. These are further classified into:
• Autoantigens: Self-antigen, which under certain circumstances Induces autoantibody formation. Ex: Autoimmune diseases
• Alloantigens: tissue specific antigen, which is present in one individual of a species but not in others. Ex: ABO, HLA etc.
• Intracellular Pathogens: viruses, intracellular bacteria and parasites. Ex: Chlamydia, Rickettsia, Plasmodia, etc.

Exogenous: These antigens enter the body or system and freely circulate in the body fluids and are trapped by the APCs. The uptake of these exogenous antigens by APCs are mainly mediated by the phagocytosis.

• Allergens
• Iatrogenic - doctor induced
• Microbial

Question 17

Factors Influencing Immunogenicity

Answer 17

> this means more immunogenic

• Molecular mass: small molecules are does not provoke immune system. (why hapdens need to be coupled)
• Foreignness: The immune system normally discriminates between self and non-self such that only foreign molecules are immunogenic.
• Chemical Composition: more complex the substance is chemically = more immunogenic it will be.

Physical form: particulate antigens> soluble antigens and denatured antigens> native form.

Degradability: Antigens that are easily phagocytosed are generally more immunogenic.

Genetic Factors: Some substances are immunogenic in one individual but not in others (i.e. responders and non-responders).

Age: very young and the very old have a diminished ability to mount an immune response

Question 18

Methods of Administration: Dose, Route, Adjuvants

Answer 18

Dose - The dose of administration of an immunogen can influence its immunogenicity. There is a dose of antigen above or below which the immune response will not be optimal.

Route - Generally the subcutaneous route is better than the intravenous or intragastric routes. The route of antigen administration can also alter the nature of the response. (because phagocytes are residing in subcutaneous)

Adjuvants - Substances that can enhance the immune response to an immunogen are called adjuvants. The use of adjuvants, however, is often hampered by undesirable side effects such as fever and inflammation.

Question 19

Chemical Nature of Immunogens/Antigens

Answer 19

*** Proteins are vast majority, make good immunogens (includes pure proteins, glyco/lipoproteins)

*Polysaccharides: Pure Polysaccharides and lipopolysaccharides are good immunogens: have multiple repeating epitopes that bind well to IgM

– Nucleic Acids: generally poorly immunogenic- but will become immunogenic when single stranded or complexed with proteins

• Lipids: NOT usually immunogenic: unless they are haptens

Question 20

Epitopes Recognized by B cells

Answer 20

• typer of interaction is highly dependent upon the 3D conformation of the globular antigen
• Epitopes tend to be accessible and on the exposed surface of the immunogen
• Epitopes may be associated with soluble immunogens or particulate immunogens.
• Composition: epitopes usually contain hydrophilic amino acids
• Size: The size of a B cell epitope is determined by the size of the antigen binding site on the Ab. Conformationally determined epitopes tend to require a larger idiotype. Smaller ligands tend to fit within a deep concave pocket or crevice. Larger globular antigens tend to interact with Ab across a large planar face.

**- B cells and Abs see soluble particulate and extracellular particles **

Question 21

Epitopes Recognized by T Cells

Answer 21

Composition: T cells recognize are the primary sequence of amino acids in proteins.

• do not recognize polysaccharide or nucleic acid antigens.
• epitopes need not be located on the exposed surface of the antigen since recognition by T cells requires that the antigen be proteolytically degraded into smaller peptides.

Size: In general epitopes are small and are limited to approximately 8-15 amino acids.

Number: 8-15 residues can constitute a separate epitope , in practice, the number of antigenic determinants per antigen is much less. The antigenic determinants are limited to those portions of the antigen that can bind to MHC molecules. This is why there can by differences in the responses of different individuals.

Free peptides are not recognized by T cells. T cells only see peptides that have been presented to them in context of HLA

Question 22

Mitogens

Answer 22

• Substances that cause cells, particularly lymphocytes to undergo cell division.

Endotoxin protein (LPS) is a polyclonal activator of humanB cells. Does not need T cell help.

Question 23

Superantigens

Answer 23

• activate a large fraction of T cells non-specifically (up to 25%)

Question 24

Ab/Ag Binding

Answer 24

• bound via Formation of non-covalent bonds
• Hydrogen Bonds
• Electrostatic Bonds
• Van der Waals Forces
• Hydrophobic Forces

Each bond is weak compared to covalent bonding but together generate a high-affinity interaction.

Interaction is reversible.

Question 25

Affinity vs Avidity

Answer 25

The strength of the interaction between an univalent Ag (epitope) & univalent Ab (idiotope) is loosely referred to as affinity. (attraction to form bonds)

The strength of the interaction between a multivalent Ag and a multivalent Ab is referred to as avidity. (number of bonds)

The avidity of an Ab for its Ag is dependent on the affinities of the individual Ag combining sites.

• ex. IgM has a greater Avidity than IgG due to its pentameric structure, and ability to multivalently bind at ten different points, rather than just two.

Question 26

Hapten/Carrier Effect

Answer 26

• Basis for many drug allergies.
• Drugs alone are poor stimulators of immune responses due to their simple structure and low molecular weight.
• Drugs can fulfill the requirement for multivalency and cause an immune response by forming multivalent hapten-carrier complexes.
• The hapten is the drug, the carrier is a protein that is not immunogenic in free form.
• Penicillins and other b-lactam antibiotics bind covalently to proteins. Other types of drugs may become noncovalently associated with proteins.

Question 27

Monoclonal Antibodies

Answer 27

• mAB = Identical antibodies produced by one immune cell either in a mouse or in vitro. They are clones of one single parent
  ex. Yervoy = ipilimumab = blocking Ab for CTLA-4: Melanoma drug to extend life
  ex. ELISAS: mAB’s are basis for Strep Cassetes, pregnancy test, HIV tests, etc.
  ex. Flow Cytometry: can tell the population of CD4 to CD8 populations of B cells based on their CD markers

*** RECOMBINANT AB’s

Question 28

SUMMARY SLIDES

Answer 28

• Circulating antibodies are soluble glycoproteins that recognize and bind antigens present in serum, tissue fluids or on cell membranes.
• Abs also function as membrane-bound antigen receptors on B cells, and play key roles in B cell differentiation.
• All Ab isotypes, with the exception of IgD, are bifunctional.
• Ab are made up of a unit with four polypeptide chains : two identical light chains and two identical heavy chains.
• There are five classes of Abs in mammals : IgG, IgA, IgM, IgD, and IgE.
• Each Ab isotype mediates a distinct set of effector functions.
• Receptors for Ab constant regions (Fc receptors) are expressed by mononuclear cells, neutrophils, natural killer cells, eosinophils, basophils, and mast cells.
• Ags are foreign molecules that bind to an antibody or TCR.
• Ags can be divided into several subgroups: endogenous, exogenous, T-dependent, T-independent, mitogens, super ags, etc.
• Route, dosing, chemical compostion and other charateristics determine immunogenicity of Ags.
• Ag-binding sites of Abs are specific for the three-dimensional shape (conformation) of their target, the epitope. Ag-binding of T-cell is specific for linear peptides.
• Antibody affinity is a measure of the strength of the interaction between an antibody combining site and its epitope.
• The avidity of an antibody depends on its number of binding sites and its ability to engage multiple epitopes on the antigen.