Function and Importance of Antibodies

Question 1

Antibodies

Answer 1

Also called immunoglobulins- part of the adaptive immune system and produced by B cells.

Question 2

Why are antibodies important?

Answer 2

1. Immunity
2. Research
3. Medicine

Question 3

Antibody structure

Answer 3

Y shaped, with 2 light chains and 2 heavy chains. These proteins come together through disulfide bonding to form a quaternary structure. The antigen binding site is located at the ends of the arms of the Y

Question 4

Variable and constant regions

Answer 4

The constant regions are mostly invariant, especially within the same antibody class. The variable region, which contains the antigen binding site, is located at the end of the arms of the Y

Question 5

Variable region

Answer 5

Composed of complementarity-determining regions (CDRs), which is the most variable portion of the antibody. This region is responsible for the specificity of the antibody, as it is complementary to the antigen it’s binding to. There are 3 CDRs per heavy chain and 3 per light chain. All 6 need to come together in the antibody structure, so the tertiary and quaternary structures are critical

Question 6

Antigen-binding site specificity

Answer 6

Antigens with a high specificity have high affinity binding, while antigens with broad specificity have low affinity binding. As the immune response develops and somatic hypermutation occurs, antigen binding sites will have a higher affinity

Question 7

Affinity

Answer 7

Strength of the antibody to antigen interaction. The affinity is independent of the number of antigen binding sites. Kd is the equilibrium dissociation constant and relates to the concentration of antibodies needed to promote binding to a specific antigen. A lower KD means you need less antibodies and have a better binding affinity

Question 8

V region vs framework region

Answer 8

The variable region is composed of some CDRs, but the remainder is called the framework region. The framework forms a scaffold on which the CDRs can be arranged to form an antigen binding site. They are important structurally, but not functionally.

Question 9

V-regions, C-regions, & Ig classes

Answer 9

There are 5 possible constant regions- A, G, D, M, or E on the heavy chain. There are also two possible constant regions on the light chain- kappa or lambda. The heavy chain constant regions correspond to the class of antibodies

Question 10

Avidity

Answer 10

Total binding strength of an antibody, which takes into account the number of antigen binding sites

Question 11

Immunoglobulin classes (5)

Answer 11

1. Serum antibodies- IgG, IgD, IgE, and IgA (when present in the blood). These antibodies have the classic Y shape
2. IgM- pentameric- 5 antibodies are joined with a J chain
3. Secretory (mucosal) IgA- dimeric. Formed by a J chain but stabilized by a secretory component protein that it acquires as it makes its way to the mucosa

Question 12

Fab fragments

Answer 12

Proteases, like papain or pepsin, can be used to cleave the hinge region of the antibody and generate antibody fragments. Cleavage generates 2 Fab fragments and one Fc. Fab stands for fragment antigen binding, and this is the arms of the Y. Contains the antigen binding site.

Question 13

Fc fragments

Answer 13

Fragment crystallizable- bottom of the Y. Pepsin will degrade the Fc fragment

Question 14

Antigens

Answer 14

Any foreign molecule- protein, lipid, or polysaccharide. If it’s different from molecules in the host, it will be recognized as foreign

Question 15

Epitope

Answer 15

The piece of antigen that antibodies bind to

Question 16

Continuous vs discontinuous epitopes

Answer 16

Continuous epitopes- if you were to denature an antigen and make it linear, the epitope would not be broken up. Discontinuous epitopes are the opposite- they only come together due to the way the antigen has folded. Denaturing the antigen would break up the epitope.

Question 17

Agglutination

Answer 17

Antibodies can clump antigens together in a variety of different ways. Usually when the antigens are clumped together, they are neutralized and will be destroyed

Question 18

Antibody hinge region

Answer 18

The hinge region is very flexible and helps the antibody to bind to epitopes

Question 19

Primary antibody repertoire

Answer 19

Antibodies that have already been made by B cells, regardless of the specific infection or whether we’ve been exposed to a pathogen. Once infection occurs, these B cells are activated and antibodies are secreted. They have a broad and very diverse specificity, so they are prepared to come into contact with a variety of pathogens

Question 20

Secondary antibody repertoire

Answer 20

Develops during an active infection. Antibodies are altered such that there is a great increase in their binding strength and the diversity of specificity. This process is known as affinity maturation

Question 21

V(D)J recombination

Answer 21

Generates a primary antibody repertoire, occurring during development of B cells in the bone marrow and before we have come into contact with any pathogen. Each type of Ab chain (κ & λ L-chains, H-chains) is encoded by separate locus on separate chromosome- each locus has large number of V gene segments. Site-specific recombination occurs and produces different combinations of V, D, and J segments to make many different antigen binding sites of different specificities

Question 22

L-chain V regions are encoded by

Answer 22

Encoded by DNA sequence from long V gene segment & short joining (J) gene segment

Question 23

H-chain V regions are encoded by

Answer 23

Encoded by DNA seq from long V gene segment, short J gene segment and diversity (D) gene segment

Question 24

V(D)J recombination – L-chain example

Answer 24

Recombination brings V3 (in this case) in close proximity with J3. Recombined DNA is transcribed to pre-RNA, and other J segments are spliced out during alternative splicing. Now, the antibody has its own unique variable region

Question 25

V(D)J recombination – H-chain

Answer 25

Only difference between heavy and light V(D)J recombination is the extra D gene segment. D encodes amino acids in CDRs

Question 26

Joining of gene segments in V(D)J recombination

Answer 26

The gene segments are joined by non-homologous end joining. A variable number of nucleotides may be lost or added. This increases the diversity of the variable regions

Question 27

Junctional diversification

Answer 27

There is a random loss and gain of nucleotides at joining sites. It increases the diversification of V regions enormously

Question 28

Why is V(D)J recombination considered site-specific?

Answer 28

There are conserved recombination signal sequences that flank each gene segment, and there are recognition sites for the joining process. This ensures that only appropriate gene segments recombine

Question 29

V(D)J recombinase

Answer 29

The enzyme complex that mediates joining of the gene segments. Encoded by the recombination activating genes (rag) 1 & 2, which encode RAG1 & RAG2

Question 30

V(D)J recombination process

Answer 30

1. The V/D/J segments are flanked by sequences that are recognized by VDJ recombinase (the RAG proteins)
2. That is going to cause curvature in the DNA, which brings 2 gene segments into close proximity
3. The VDJ recombinase cleaves the DNA
4. The 2 gene segments are joined through non-homologous end joining

Question 31

V(D)J recombination at the κ light chain locus

Answer 31

Any of the 40 V gene segments may join to any of the 5 J segments. Encodes 200 different κ-chain V regions

Question 32

V(D)J recombination at the heavy chain locus

Answer 32

Any of the 40 V gene segments may join to any of the 25 D & 6 J gene segments. Encodes 6000 different heavy chain V regions

Question 33

How many different antigen binding sites can be produced by V(D)J recombination?

Answer 33

Humans can produce 320 different VL regions (200 κ and 120 λ) and 6000 different VH regions. Could be combined to make 1.9 X 106 different Ag-binding sites

Question 34

Somatic hypermutation

Answer 34

Following stimulation, like infection or T cells, antibodies undergo mutations. This occurs long after the coding regions have been assembled. There is 1 mutation per V region coding sequence per generation. There is a 106 X greater than spontaneous mutation rate in other genes- not all mutations will result in an increased affinity for antigen

Question 35

How does somatic hypermutation occur?

Answer 35

Very few mutations will result in an increased affinity for antigen. The antigen will stimulate preferentially those few B cells that do make antibodies with increased affinity, and clones of those altered B cells will preferentially survive and proliferate. Most other B cells die by apoptosis

Question 36

Result of somatic hypermutation

Answer 36

Abs become of increasingly higher affinity as immune response progresses

Question 37

Activation-induced deaminase (AID)

Answer 37

An enzyme that is expressed specifically in activated B cells and carries out somatic hypermutation. It deaminates C to U in the V region gene segments, producing U:G mismatches in the double helix. Repair of mismatches may leads to various mutations. Some mutations will be corrected to the original, while others result in a diverse range of mutations

Question 38

Antibody class switch

Answer 38

As B cells are activated, antibodies typically undergo a class switch. Generally, the first antibodies released in an immune response will be IgM. With B cell activation, antibodies typically can switch from IgM to IgG. But they can switch to other isotypes as well, depending on the type of infection. IgA would be secreted for a mucosal infection

Question 39

Why are antibodies so important to the immune response? (5)

Answer 39

1. Specificity
2. Neutralization
3. Opsonization for phagocytosis
4. Complement recruitment
5. Lytic effects- C1q recruitment for MAC. Unique antibodies are bactericidal and fungicidal

Question 40

Antibody opsonization

Answer 40

When the surface of the pathogen is coated with antibody. This improves phagocytosis, as macrophages and other phagocytes have lots of Fc receptors that recognize the Fc portions of the antibody

Question 41

Complement functions (3)

Answer 41

Antibodies specifically recruit complement through the classical pathway. There are 3 possible outcomes- inflammatory cell recruitment, opsonization, and formation of the membrane attack complex, which will destroy the pathogen

Question 42

Complement pathways (3)

Answer 42

1. Classical pathway- binds antibody
2. Lectin pathway- binds sugars on pathogen surfaces
3. Alternative pathway- binds membranes

Question 43

C3 convertase

Answer 43

An enzyme formed by the classical pathway. Once formed, C3 convertase cleaves C3, creating C3b and C3a. C3b is a potent opsonin, and C3a is a potent inflammatory mediator

Question 44

C5 convertase

Answer 44

Formed by C3b binding to the C3 convertase. It produces C5a (inflammatory mediator) and C5b- it initiates polymerization reactions leading to MAC

Question 45

C1q recruitment

Answer 45

Part of the classical pathway. Antibodies bind to antigens on the pathogen surface and then recruit complement component C1q, which recruits other components that lead to the 3 pathogen outcomes

Question 46

Membrane attack complex (MAC)

Answer 46

Complements the binding activity of antibodies. C5b binds to C6 and C7, which then binds to the pathogen surface. It binds to C8, which inserts into the cell membrane. C9 molecules bind to the complex and polymerize, with 10-16 molecules binding to form a pore in the membrane. The MAC complements the binding activity of antibodies.

Question 47

Complement-independent bactericidal antibodies

Answer 47

These antibodies can kill bacteria without using the MAC

Question 48

How do antibodies have a bactericidal function?

Answer 48

They target antigens in lipid rafts. Many rafts aggregate together, creating a situation where there are large stretches of rafts and large stretches of non-raft regions. There is increased tension b/w LO and LD portions, which leads to membrane curvature. Membrane curvature leads to blebs

Question 49

Bleb formation

Answer 49

Lipid rafts participate in bleb formation. Cholesterol and lipid rafts are critical for the effects of complement-independent antibodies

Question 50

IgM functions (5)

Answer 50

1. BCR (monomer)
2. Early Ig responses
3. Neutralization (+),
4. C1q recruitment (+++) and formation of the MAC
5. Opsonization (+)

Question 51

IgG functions (4)

Answer 51

Subclasses: IgG1, IgG2, IgG3, IgG4.
1. Neutralization (++),
2. C1q recruitment (+++),
3. Opsonization (+++)
4. ADCC (++)

Question 52

IgA functions (2)

Answer 52

1. Neutralization (++)
2. Mucosal immunity (+++)

Question 53

IgD functions

Answer 53

BCR, no other known functions

Question 54

IgE functions

Answer 54

Mast cell degranulation, associated with allergic responses

Question 55

Secretory IgA transport

Answer 55

IgA comes from tissues underlying the mucosal tissues in a partial dimeric form. It is picked up by an Fc receptor on epithelial cells, and is then transcytosed to the mucosal surface. Following transcytosis, the Fc receptor remains bound to the IgA

Question 56

IgM C1q recruitment

Answer 56

IgM binds in a flat orientation on the surface of a pathogen

Question 57

What signals V(D)J recombination to stop occurring?

Answer 57

Once VDJ recombination has occurred, that will send signals in the B cell that turns off the RAG genes so that recombination will cease, and VDJ recombination will stop. Then, the B cell will make many antibodies, all with the same antigen binding sites- VDJ recombination results in antibodies with one specificity per B cell