H. HogenEsch, 2005 Antibody structure and function Parham – Chapter 2

H. HogenEsch, 2005

Antibody structure and function

Parham – Chapter 2

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Outline

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

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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

IgG1

IgG2

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