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Antibody structure and function

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Immortalization of a single clone of antibody-secreting cells ... Produced by immortalized plasma cells, usually mouse origin. Single specificity and affinity ... – PowerPoint PPT presentation

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Title: Antibody structure and function


1
Antibody structure and function
  • Parham Chapter 4

2
Outline
  • Antigens
  • Antibody structure
  • Antigen-antibody interactions
  • Applications - immunoassays
  • Generation of antibody diversity
  • Isotype switching

3
Immunoglobulins membrane-bound and soluble
receptors
4
Epitopes
  • Epitope (antigenic determinant) is the part of an
    antigen to which an antibody binds.
  • Most antigens have multiple epitopes
    (multivalent)
  • Usually carbohydrate or peptide.

Fig. 2.9
http//micro.magnet.fsu.edu/cells/viruses/influenz
avirus.html
5
Epitopes recognized by antibodies are usually
located at the antigens surface.
Fig. 2.8
6
Conformational vs. linear epitopes
7
Epitopes
heat, acid
Conformational epitopes - destroyed by
denaturation Linear epitopes - unaffected by
denaturation
8
Epitope recognition
9
Haptens
Small molecules that are not immunogenic by
themselves, but can bind immunoglobulins or
TCRs. Haptens can induce an immune response when
linked to a larger protein (carrier).
10
Hapten
Parham Fig. 10.25
Fig. 12.26
11
Hapten
Parham Fig. 10.26
Fig. 12.27
12
Basic structure of immunoglobulins
  • 2 light chains
  • lambda (?)
  • kappa (?)

5 heavy chains - mu (µ) - gamma (?) - alpha (a) -
epsilon (e) - delta (d)
Fig. 4.2
13
Basic structure of immunoglobulins
Fig. 4.2
14
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15
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16
Hinge region provides flexibility to
antigen-binding sites
17
Structure of immunoglobulins
18
Structure of immunoglobulins
19
Hypervariable and framework regions
HV CDR complementarity -determining region
Fig. 2.7
20
Immunoglobulin classes (isotypes)
L-chain k or l
21
Differences between immunoglobulins
22
Allotypes
  • Small differences (few base pairs/amino acids)
  • May affect half life
  • May affect subclass distribution
  • Mendelian inheritance autosomal dominant
  • Different distributions among ethnic groups
  • Associated with susceptibility to infectious
    diseases and autoimmune diseases

23
Antibody-antigen interaction
Fig. 2.10
24
Antibody-antigen interaction
  • Non-covalent binding
  • Electrostatic
  • Hydrogen bonds
  • Van der Waals forces
  • Hydrophobic forces
  • Affinity Strength of interaction between epitope
    and one antigen-binding site
  • Avidity Strength of the sum of interactions
    between antibody and antigen

Short range
25
Crossreactivity
Antiserum raised against antigen A reacts also
with antigen B
Antigen A and B share epitopes
Antigen A and B have similar (but not identical)
epitopes
26
Crossreactivity
H2N2
H2N3
Influenza virus
27
Monoclonal antibodies
  • Immortalization of a single clone of
    antibody-secreting cells
  • Fusion of B cells with neoplastic plasma
    (myeloma) cells

28
Monoclonal antibodies
29
Polyclonal vs. monoclonal antibodies
  • Polyclonal antibodies
  • purified from serum of immunized animals, often
    goats or rabbits.
  • Multiple specificities and affinities
  • Variation from batch to batch
  • Monoclonal antibodies
  • Produced by immortalized plasma cells, usually
    mouse origin.
  • Single specificity and affinity
  • Unlimited supply of identical antibody molecules

30
Examples of monoclonal antibodies as therapeutics
Name Specificity Diseases
Tysabri Integrin (a4b1) Multiple sclerosis
Trastuzumab (Herceptin) Her2 Breast cancer
Rituximab CD20 (B cells) Non-Hodgkin lymphoma
Adalimumab (Humibra) TNF Rheumatoid arthritis
Omalizumab (Xolair) IgE Asthma
Motavizumab Respiratory syncytial virus bronchopneumonia
31
Types of therapeutic monoclonal antibodies
32
Rituximab in autoimmune disease (pemphigus
vulgaris)
Rituximab IVGG
Ahmed et al. NEJM, 355, 1772, 2006
Approximate cost 3,976 per infusion or 15,904
for a four-dose course
33
Immunoassays
  • Precipitation assay
  • Agglutination assay
  • Enzyme-linked immunosorbent assay (ELISA)
  • Radioimmunoassay (RIA)
  • Western blotting
  • Immunofluorescence
  • Flow cytometry

34
Sensitivity of immunoassays
precipitation - 30 mg/ml agglutination - 1
mg/ml radioimmunoassays, ELISA - 1 pg/ml
35
Precipitation reaction
Aggregates formed by interaction of multivalent
antibodies and multivalent macromolecular
antigens.
36
Antigens have multiple epitopes
37
Hemagglutination
38
Coombs test
  • Direct Add anti-human immunoglobulin antibodies
    (Coombs reagent) to red blood cells.
    Agglutination occurs if the red blood cells are
    coated with antibodies.
  • Indirect Incubate test serum with red blood
    cells. Wash red blood cells. Add anti-human
    immunoglobulin antibodies.

39
Rhesus factor
40
Enzyme-linked immunosorbent assay (ELISA)
Principle of ELISA/RIA
  • Coat wells with antigen
  • Add serum sample
  • Add enzyme-labeled
  • anti-human IgG
  • Add substrate

41
Western blot
Western blotting
42
Immunofluorescence
43
Flow cytometry
44
Flow cytometry for CD4 T cells
45
Monitoring CD4 T cells in HIV infection
46
Immunoglobulin genes
47
V-region domains are constructed from gene
segments
48
VL and VH-region domains are constructed from
gene segments
49
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50
Recombination Signal Sequences
51
Recombination
V(D)J recombinase
52
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53
Generation of diversity
k chain 40 V x 5 J 200 Vk l chain 30 V x 4
J 120 Vl H chain 65 V x 27D x 6 J 10,530
VH (200 120) x 10,530 3.4 x 106
combinations
  • Imprecise joining
  • N-nucleotide addition (TdT)


54
Rearrangement leads to functional heavy-chain gene
55
Naïve mature B cells co-express IgM and IgD
56
B cell receptor complex
57
Changes in B cells after activation by antigen
  • Antibody secretion
  • Somatic mutation additional diversity
  • Isotype switching

58
Alternative RNA processing underlies the change
from membrane to secreted IgM
59
Somatic hypermutation
Mediated by activation-induced cytidine deaminase
(cytosine ? uracil)
60
Hypervariable and framework regions
CDR complementarity -determining region
Fig. 2.7
61
Isotype switching
Isotype switching results in immunoglobulins with
different C regions (different biological
function) but identical antigen specificity.
62
Isotype switching
63
Isotype switching
IgG1
IgG2
IgG3
IgG4
IgM/IgD
IgA1
IgA2
IgE
64
Effector functions of antibodies
  • Neutralization
  • Opsonization
  • Complement activation

65
Physical properties of immunoglobulins
66
IgM
  • Membrane-bound monomer and secreted pentamer.
  • First immunoglobulin to be synthesized during
    ontogeny and in the immune response.
  • Activates complement pathway agglutination.
  • Can be transported into mucosal secretions.

67
IgG
  • Highest concentration in serum.
  • Four subclasses IgG1 - 4
  • Activates complement
  • Binds to Fcg -receptors on neutrophils,
    macrophages and NK cells

68
IgA
  • Usually dimer
  • Secretory IgA is a dimer with a secretory
    component.
  • Two subclasses IgA1 and IgA2
  • Major immunoglobulin in mucosal secretions
  • Neutralization Prevents binding of
    micro-organisms to receptors
  • Not effective activator of complement

69
IgE
  • Very low serum concentration in healthy
    individuals.
  • Concentration is higher in patients with helminth
    infections and often in patients with allergies.
  • Lacks hinge region extra CH domain
  • Binds to Fce receptor on mast cells and
    basophils. Cross-linking results in degranulation
    and release of pro-inflammatory mediators.

70
IgD
  • Very low concentration in serum
  • Primarily found with IgM on naïve mature B cells
  • Function is unknown

71
Functions of immunoglobulins
72
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