antibody structure and function
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Antibody structure and function. Parham – Chapter 2. Outline. Antibody structure Antigens Antigen-antibody interactions Generation of antibody diversity Isotype switching Applications - immunoassays. Immunoglobulins – membrane-bound and soluble receptors. - PowerPoint PPT PresentationTRANSCRIPT
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H. HogenEsch, 2005
Antibody structure and function
Parham – Chapter 2
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H. HogenEsch, 2005
Outline
• Antibody structure• Antigens• Antigen-antibody interactions• Generation of antibody diversity• Isotype switching• Applications - immunoassays
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H. HogenEsch, 2005
Immunoglobulins – membrane-bound and soluble receptors
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Basic structure of immunoglobulins
Fig. 2.2
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Basic structure of immunoglobulins
Fig. 2.2Fig. 2.2
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Antigen-binding Fragment
Crystallizable Fragment
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Immunoglobulin classes (isotypes)
H:
L-chain: or
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Structure of immunoglobulins
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Structure of immunoglobulins
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Hypervariable and framework regions
CDR = complementarity-determining region
Fig. 2.7Fig. 2.7
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Differences between immunoglobulins
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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
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Immunoglobulin epitopes are usually located at the antigen’s surface.
Fig. 2.8
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Conformational vs. linear epitopes
Fig. 2.11
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Epitopes
Conformational epitopes - destroyed by denaturationLinear epitopes - unaffected by denaturation
heat, acid
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Epitope recognition
Conformational epitope
Linear epitope
B cell
T cell
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Haptens
Small molecules that are not immunogenic by themselves, but can bind immunoglobulins or TCRs. Haptens can induce an immune response when linkedto a larger protein.
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Hapten
ParhamFig. 10.25
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Hapten
ParhamFig. 10.26
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Antibody-antigen interaction
Fig. 2.10
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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
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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
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Crossreactivity
A B
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Immunoglobulin genes
Fig. 2.13
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Somatic recombination – light chain
Fig. 2.14
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Somatic recombination – Heavy chain
Fig. 2.14
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Number of gene segments
Fig. 2.15
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Recombination Signal Sequences
Fig. 2.16
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Recombination
V(D)J – recombinase
Fig. 2.17
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5’ // 3’
V
1 2 3 4 5 n 1 2 3 4 5
J C
5’ 3’
5’ 3’
V C
germline DNA
B cell DNA
primary RNA transcript
mRNA
chain polypeptide
rearrangement
transcription
splicing
translation
V2J3
V2J3C
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Generation of diversity
chain: 40 V x 5 J = 200 V chain: 30 V x 4 J = 120 VH chain: 65 V x 27D x 6 J = 10,530 VH
(200 + 120) x 10,530 = 3.4 x 106 combinations
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Mechanisms for additional diversity in immunoglobulins
• Imprecise joining of gene segments• Random nucleotide addition at joining regions
– terminal deoxynucleotidyl transferase (TdT)
Fig. 2.17
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Generation of diversity• Multiple gene segments:
chain: 40 V x 5 J = 200 V
chain: 30 V x 4 J = 120 V
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
> 108 different specificities
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Organization of CH genes
Fig. 2.19
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Naïve mature B cells express IgM and IgD
Fig. 2.20
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Allelic exclusionAllelic exclusion ensures that the B lymphocyte expressesimmunoglobulin molecules with only one specificity.Mechanism: Successful rearrangement of immunoglobulin gene segmentsone allele shuts down the rearrangement processof the other allele.
16 6 12
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B cell receptor complex
Fig. 2.21
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Changes in B cells after activation by antigen
• Somatic mutation – additional diversity
• Isotype switching
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Somatic hypermutation
Fig. 2.24
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Hypervariable and framework regions
CDR = complementarity-determining region
Fig. 2.7
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Isotype switching
IgM+/IgD+
IgG3
IgA2
IgE
IgG1IgG2
IgG4
IgA1
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Organization of CH genes
Fig. 2.19
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Isotype switching
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Physical properties of immunoglobulins
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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.
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IgG
• Highest concentration in serum.• Four subclasses: IgG1 - 4• Activates complement• Binds to Fc -receptors on neutrophils, macrophages
and NK cells
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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
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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 Fc receptor on mast cells and basophils.
Cross-linking results in degranulation and release of pro-inflammatory mediators.
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IgD
• Very low concentration in serum• Primarily found with IgM on naïve mature B cells• Function is unknown
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Functions of immunoglobulins
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Functions of immunoglobulins
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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
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Monoclonal antibodies
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Immunoassays
• Precipitation assay• Agglutination assay• Enzyme-linked immunosorbent assay (ELISA)• Radioimmunoassay (RIA)• Western blotting• Immunofluorescence• Flow cytometry
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Sensitivity of immunoassays
precipitation - 30 g/ml
agglutination - 1 g/ml
radioimmunoassays, ELISA - 1 pg/ml
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Precipitation reactionAggregates formed by interaction of multivalent antibodies and multivalentmacromolecular antigens.
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Antigens have multiple epitopes
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Hemagglutination
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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.
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Rhesus factor
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Principle of ELISA/RIAEnzyme-linked immunosorbent assay (ELISA)
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Western blottingWestern blot
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Immunofluorescence
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Flow cytometry