Antibody Structure and Function

Question 1

Antibody Overview

Answer 1

• Proteins that recognize and bind to foreign substances
• Comprise immunoglobulins - glycoproteins
• Present in circulation and on B-cells

Question 2

B-cell Overview

Answer 2

• B-cells rearrange DNA to produce Ig receptors
  
  • Once receptor expressed the B-cell is committed to that Ag
• Humoral immunity mediated by Ab and B-cells

Question 3

Antigen Classes

Answer 3

Ab able to recognize a wide variety of antigens:

• Microbial surface Ag
  
  • capsular components
  • viral envelops
• Internal components
  
  • capsid
  • transcriptase from virus
  • dsRNA
• Soluble microbial mediators
  
  • secreted toxins

Question 4

Ab & B-cell

Summary

Answer 4

Question 5

Ig Electrophoresis

Answer 5

• Humans have a broad gamma peak (hence gamma globulin) due to Ab diversity
• Individuals with multiple myeloma produce a single Ab in blood
  
  • Also have Bence Jones proteins in urine
    
    • dimers of Ig kappa or Ig lambda light chains

Question 6

Antibody Structure

Answer 6

• Comprised of two heavy chains and two light chains
  
  • Held together by disulfide and non-covalent bonds
• N-terminal variable region
  
  • _​_Includes 2 antigen binding sites
    
    • Formed by combination of the light and heavy chains
• Remaining domains termed c_onstant region_
  
  • Hinge region joins Ag binding domain to Fc region
  • Flexibility allows binding at different angles

Question 7

Ab Structure Summary

Answer 7

Question 8

Ab Genes

Answer 8

• Light chain
  
  • Kappa chain on chromosome 2
  • Lamda chain on chromosome 22
  • Each B cell expresses one or the other
    
    • Predominance of one type suggests malignancy
• Heavy chain
  
  • Single gene locus on chromosome 14
  • Determines Ab class
    
    • Which gene segment is closest to the variable region on the genome determines isotype production
      
      • μ (mu) = IgM
      • γ (gamma) = IgG
      • α (alpha) = IgA
      • ε (epsilon) = IgE
      • δ (delta) = IgD
  • Heavy chain determines effector activities and tissue distribution

Question 9

Ig Class Summary

Answer 9

Question 10

Ab Opsonization

Answer 10

• Ab coats the surface of a pathogen to promote phagocytosis
• IgG potent opsonin through binding of Fc region with Fc receptors on phagocytes

Question 11

Ab Complement Fixation

Answer 11

• IgG and IgM can activate the classicle complement pathway
• C1q binds to Fc region
• Activation leads to:
  
  • MAC formation
  • C3b binding and phagocytosis
  • Release of proinflammatory mediators
    
    • C5a
    • C3a

Question 12

Antibody-Dependent Cellular Cytotoxicity

(ADCC)

Answer 12

• Innate effector cell selectively kills a target cell coated with Ag-specific Ig
• Mostly NK cells but also neutrophils, macrophages, eosinophils, and basophils
  
  • Cells express Fc receptors that bind to Ig
• If phagocytes are unable to take in the pathogen they are programmed to self-destruct on its surface leading to tissue damage

Question 13

Neutralization

Answer 13

Ab defends a cell or the host from Ag or pathogen.

Ab binds to pathogen or toxin and sterically hinders their binding to cell surface receptors.

Ex. IgA prevents attachment and colonization of muscosal sites.

Induced through a number of vaccines to prevent the effects of microbial released toxins.

Question 14

Agglutination

Answer 14

Ab binds to and promotes clumping of particulate Ag.

Ab have at least 2 Ag-binding sites - one Ab can bind two epitopes on different Ag and hold them together.

Increases likelihood of phagocytosis.

Question 15

Mast Cell Degranulation

Answer 15

Antigen cross-links IgE already bound to high affinity FcεRI receptors on mast cells and basophils.

Question 16

IgM

Answer 16

• Monomeric IgM found on the surface of B-cells as the BCR
• Plasma IgM found as pentameric structure
  
  • 5 monomers linked by a single J-chain
  • 10 potential Ag binding sites but usually can only bind 5 due to steric hinderance
  • First Ab made following activation of naive B-cells
    
    • Found early in primary immune response
  • Adopts a planar conformation normally
    
    • After binding Ag takes on a “staple” or “crab” formation which exposes binding sites on Fc region

Question 17

IgG

Answer 17

• 75% of blood Ig
• Major Ab isotype found in interstitial fluids
  
  • 25% of IgG pool exchanges between intravascular and extravascular sites daily
  • Allows tissue to be constantly bathed in IgG containing plasma
• Four subclasses
• Made later during primary immune response
• Major Ab during secondary immune responses

Question 18

IgA

Answer 18

• Found in the blood and predominant Ab in seromucous secretions
• Two subclasses - IgA₁ and IgA₂
• Monomers linked by a J-chain
• Monomeric and dimeric IgA ~ 15-20% of blood Ig
• Secretory IgA (sIgA) is the most made Ig
  
  • Actively secreted through the cells of the mucosal epitheilum into secretions
    
    • Saliva
    • Tears
    • Colostrum
    • Bronchial/genitourinary/intestinal tracts
    • Found in breast milk and passed to infant
  • Secretory component made by epithelial cells
    
    • Facilitates transport of sIga across epithelium
    • Protects and extends half-life

Question 19

IgE

Answer 19

• Monomer with very short half-life in the blood
• Can remain bound to high affinity Fcε receptors on mast cells and basophils for weeks to months
  
  • Only instance where Ig remain bound to Fc receptors for a long time in absence of Ag
  • Binds as a monomer
  • Dimerization by Ag results in degranulation

Question 20

IgD

Answer 20

• Monomer
• Synthesized as BCR on naive B-cells
• No secreted form - only found in blood after it falls of B-cell
• No known biological role

Question 21

Antibody Distribution

Answer 21

• Found in blood plasma, perfused tissues, and most secretions.
• IgM and IgG most in blood
• IgG constantly bathes tissues
• sIgA in secretions
  
  • Provides barrier to microbial adhesion and colonization of mucosal surfaces
• IgE found in mucosal tissues and connective tissues

Question 22

Immunoglobulin Classes Summary Table

Answer 22

HIGH YIELD

Question 23

Antigenic Epitopes

Answer 23

• Component of the Ab which is actually bound by Ab
• Dependent onf the 3-D structure
  
  • Can be linear or conformational
• Includes:
  
  • proteins
  • polysaccharides
  • lipids
  • nucleic acids
  • many small chemicals

Question 24

Paratope

Answer 24

Tertiary structure of the Ab formed by a combination of the light and heavy chains which binds the 3-D structure of the epitope.

Lock and key fit.

Potential to bind soluble Ag, integral membrane components, or degraded components.

Allows Ab to bind to just about any potential threat.

Question 25

Ab-Ag Binding Summary

Answer 25

Question 26

Ab Specificity

Answer 26

Describes the degree to which the Ab differentiates Ag and is able to bind to one Ag over another. Ag specificity essential in targeting the immune response towards a foregin entity.

Question 27

Cross-Reactivity

Answer 27

* Refers to the ability of an individual Ab to react with more than one antigenic determinant or the ability of a population of Ab molecules to react with more than one Ag * Thought to occur when Abs react with two molecules that share similar epitopes but are otherwise dissimilar * Results in failure of immune system to distinguish self from nonself resulting in tissue damage or autoimmunity * Some pathogens attempt to mimic host tissues to evade detection = **molecular mimicry**

Question 28

Ab:Ag Chemical Interactions

Answer 28

Binding of Ag to Ab takes place due to formation of multiple non-covalent interactions. Individual interactions weak but multiplicity of the bonds leads to considerable binding energy.

Question 29

Affinity

Answer 29

The strength of a single antigen-antibody bond. Produced by summation of attractive and repulsive forces.

Question 30

Avidity

Answer 30

Strength with which a multivalent antibody binds a multivalent antigen. Ab have the potential to bind more than one epitope on multimeric Ag. Binding energy of multimeric interactions considerably greater than individual sites since all Ab-Ag interactions must be broken simultaneously before dissociation occurs. Dependent on the underlying affinity of the interactions.

Question 31

Affinity vs Avidity Summary

Answer 31

Question 32

Primary vs Secondary Immune Responses

Answer 32

* Lag time = time between contact with Ag and appearance of specific Ab * Much greater for primary responses * Ab titer much greater for secondary responses * Affinity of Ab for epitope generally improves with each subsequent Ag exposure = affinity maturation * IgM predominant in primary response * IgG predominant in secondary response * sIgA still major isotype in secretions

Question 33

Antigenic

Answer 33

Question 34

Immunogenic

Answer 34

Question 35

Haptens

Answer 35

Small antigenic molecules that are not immunogenic by themselves. Frequently covalently conjugated to large carrier proteins. Allows them to cross-link Ig receptors and induce an immune response.

Question 36

Ag Features Affecting Immunogenicity

Answer 36

1. Foreigness * Molecules must be recognized as non-self to generate an immune response * More foreign = more immunogenic 2. Chemical and structural complexity * epitope diversity contributes to the degree of immunogenicity * the more varied the epitope composition, the more likely different individual immune responses will be induced 3. Molecular size * the larger the molecule the better the immunogen 4. Genetic constitution of the host * species or individuals may respond differently to the same antigen * age, sex, immuno-competency influences response 5. Mode of administration * dose, route, timing important in optimizing immunogenicity 6. Adjuvants * a substance that enhances the body's immune response to an antigen * can promote a robust immune response through non-specific stimulation

Question 37

Papain

Answer 37

Digestion of IgG by papain cleaves at the hing region. Results in 3 fragments: two Fab fragments (antigen binding) Fc fragment

Question 38

Pepsin

Answer 38

Digestion of IgG by Pepsin cleaves below the hinge region. Generates one major fragment F(ab') that contains both antigen-binding regions.