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Antibody structure and function

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Most antigens have multiple epitopes (multivalent) Usually carbohydrate or peptide. ... Aggregates formed by interaction of multivalent antibodies and multivalent ... – PowerPoint PPT presentation

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Title: Antibody structure and function


1
Antibody structure and function
  • Parham Chapter 2

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

3
Immunoglobulins membrane-bound and soluble
receptors
4
Basic structure of immunoglobulins
Fig. 2.2
5
Basic structure of immunoglobulins
Fig. 2.2
Fig. 2.2
6
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7
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8
Immunoglobulin classes (isotypes)
L-chain k or l
9
Structure of immunoglobulins
10
Structure of immunoglobulins
11
Hypervariable and framework regions
CDR complementarity -determining region
Fig. 2.7
Fig. 2.7
12
Differences between immunoglobulins
13
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
14
Immunoglobulin epitopes are usually located at
the antigens surface.
Fig. 2.8
15
Conformational vs. linear epitopes
Fig. 2.11
16
Epitopes
heat, acid
Conformational epitopes - destroyed by
denaturation Linear epitopes - unaffected by
denaturation
17
Epitope recognition
18
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.
19
Hapten
Parham Fig. 10.25
20
Hapten
Parham Fig. 10.26
21
Antibody-antigen interaction
Fig. 2.10
22
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
23
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
24
Crossreactivity
25
Immunoglobulin genes
Fig. 2.13
26
Somatic recombination light chain
Fig. 2.14
27
Somatic recombination Heavy chain
Fig. 2.14
28
Number of gene segments
Fig. 2.15
29
Recombination Signal Sequences
Fig. 2.16
30
Recombination
V(D)J recombinase
Fig. 2.17
31
germline DNA
//
5
3
1
2
3
4
5
n
1
2
3
4
5
rearrangement
5
3
B cell DNA
V2J3
transcription
5
3
primary RNA transcript
splicing
mRNA
V2J3C
translation
32
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
33
Mechanisms for additional diversity in
immunoglobulins
  • Imprecise joining of gene segments
  • Random nucleotide addition at joining regions
  • terminal deoxynucleotidyl transferase (TdT)

Fig. 2.17
34
Generation of diversity
  • Multiple gene segments
  • - 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
  • Combination of H and L chain
  • (200 120) x 10,530 3.4 x 106 combinations
  • Imprecise joining and nucleotide addition

gt 108 different specificities
35
Organization of CH genes
Fig. 2.19
36
Naïve mature B cells express IgM and IgD
Fig. 2.20
37
Allelic exclusion
Allelic exclusion ensures that the B lymphocyte
expresses immunoglobulin molecules with only one
specificity. Mechanism Successful rearrangement
of immunoglobulin gene segmentsone allele shuts
down the rearrangement process of the other
allele.
l
k
H
16
6
12
38
B cell receptor complex
Fig. 2.21
39
Changes in B cells after activation by antigen
  • Somatic mutation additional diversity
  • Isotype switching

40
Somatic hypermutation
Fig. 2.24
41
Hypervariable and framework regions
CDR complementarity -determining region
Fig. 2.7
42
Isotype switching
IgG1
IgG2
IgG3
IgG4
IgM/IgD
IgA1
IgA2
IgE
43
Organization of CH genes
Fig. 2.19
44
Isotype switching
45
Physical properties of immunoglobulins
46
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.

47
IgG
  • Highest concentration in serum.
  • Four subclasses IgG1 - 4
  • Activates complement
  • Binds to Fcg -receptors on neutrophils,
    macrophages and NK cells

48
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

49
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.

50
IgD
  • Very low concentration in serum
  • Primarily found with IgM on naïve mature B cells
  • Function is unknown

51
Functions of immunoglobulins
52
Functions of immunoglobulins
53
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54
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

55
Monoclonal antibodies
56
Immunoassays
  • Precipitation assay
  • Agglutination assay
  • Enzyme-linked immunosorbent assay (ELISA)
  • Radioimmunoassay (RIA)
  • Western blotting
  • Immunofluorescence
  • Flow cytometry

57
Sensitivity of immunoassays
precipitation - 30 mg/ml agglutination - 1
mg/ml radioimmunoassays, ELISA - 1 pg/ml
58
Precipitation reaction
Aggregates formed by interaction of multivalent
antibodies and multivalent macromolecular
antigens.
59
Antigens have multiple epitopes
60
Hemagglutination
61
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.

62
Rhesus factor
63
Enzyme-linked immunosorbent assay (ELISA)
Principle of ELISA/RIA
64
Western blot
Western blotting
65
Immunofluorescence
66
Flow cytometry
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