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

H. HogenEsch, 2005 Outline Antibody structure Antigens Antigen-antibody interactions Generation of antibody diversity Isotype switching Applications - immunoassays

H. HogenEsch, 2005 Immunoglobulins – membrane-bound and soluble receptors

H. HogenEsch, 2005 Basic structure of immunoglobulins Fig. 2.2

H. HogenEsch, 2005 Basic structure of immunoglobulins Fig. 2.2

H. HogenEsch, 2005 Antigen-binding Fragment Crystallizable Fragment

H. HogenEsch, 2005

Immunoglobulin classes (isotypes) H:     L-chain:  or

H. HogenEsch, 2005 Structure of immunoglobulins

H. HogenEsch, 2005 Structure of immunoglobulins

H. HogenEsch, 2005 Hypervariable and framework regions CDR = complementarity -determining region Fig. 2.7

H. HogenEsch, 2005 Differences between immunoglobulins

H. HogenEsch, 2005 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

H. HogenEsch, 2005 Immunoglobulin epitopes are usually located at the antigen’s surface. Fig. 2.8

H. HogenEsch, 2005 Conformational vs. linear epitopes Fig. 2.11

H. HogenEsch, 2005 Epitopes Conformational epitopes - destroyed by denaturation Linear epitopes - unaffected by denaturation heat, acid

H. HogenEsch, 2005 Epitope recognition

H. HogenEsch, 2005 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.

H. HogenEsch, 2005 Hapten Parham Fig

H. HogenEsch, 2005 Hapten Parham Fig

H. HogenEsch, 2005 Antibody-antigen interaction Fig. 2.10

H. HogenEsch, 2005 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

H. HogenEsch, 2005 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

H. HogenEsch, 2005 Crossreactivity AB

H. HogenEsch, 2005 Immunoglobulin genes Fig. 2.13

H. HogenEsch, 2005 Somatic recombination – light chain Fig. 2.14

H. HogenEsch, 2005 Somatic recombination – Heavy chain Fig. 2.14

H. HogenEsch, 2005 Number of gene segments Fig. 2.15

H. HogenEsch, 2005 Recombination Signal Sequences Fig. 2.16

H. HogenEsch, 2005 Recombination V(D)J – recombinase Fig. 2.17

H. HogenEsch, ’ // 3’ V  n12345 J  C  5’3’ 5’3’ V  C  germline DNA B cell DNA primary RNA transcript mRNA  chain polypeptide rearrangement transcription splicing translation V2J3V2J3 V2J3CV2J3C

H. HogenEsch, 2005 Generation of diversity  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 V H ( ) x 10,530 = 3.4 x 10 6 combinations

H. HogenEsch, 2005 Mechanisms for additional diversity in immunoglobulins Imprecise joining of gene segments Random nucleotide addition at joining regions –terminal deoxynucleotidyl transferase (TdT) Fig. 2.17

H. HogenEsch, 2005 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 V H Combination of H and L chain: ( ) x 10,530 = 3.4 x 10 6 combinations Imprecise joining and nucleotide addition > 10 8 different specificities

H. HogenEsch, 2005 Organization of C H genes Fig. 2.19

H. HogenEsch, 2005 Naïve mature B cells express IgM and IgD Fig. 2.20

H. HogenEsch, 2005 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 

H. HogenEsch, 2005 B cell receptor complex Fig. 2.21

H. HogenEsch, 2005 Changes in B cells after activation by antigen Somatic mutation – additional diversity Isotype switching

H. HogenEsch, 2005 Somatic hypermutation Fig. 2.24

H. HogenEsch, 2005 Hypervariable and framework regions CDR = complementarity -determining region Fig. 2.7

H. HogenEsch, 2005 Isotype switching IgM + /IgD + IgG3 IgA2 IgE IgG1 IgG2 IgG4 IgA1

H. HogenEsch, 2005 Organization of C H genes Fig. 2.19

H. HogenEsch, 2005 Isotype switching

H. HogenEsch, 2005 Physical properties of immunoglobulins

H. HogenEsch, 2005 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.

H. HogenEsch, 2005 IgG Highest concentration in serum. Four subclasses: IgG1 - 4 Activates complement Binds to Fc  -receptors on neutrophils, macrophages and NK cells

H. HogenEsch, 2005 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

H. HogenEsch, 2005 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 C H domain Binds to Fc  receptor on mast cells and basophils. Cross-linking results in degranulation and release of pro-inflammatory mediators.

H. HogenEsch, 2005 IgD Very low concentration in serum Primarily found with IgM on naïve mature B cells Function is unknown

H. HogenEsch, 2005 Functions of immunoglobulins

H. HogenEsch, 2005 Functions of immunoglobulins

H. HogenEsch, 2005

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

H. HogenEsch, 2005 Monoclonal antibodies

H. HogenEsch, 2005 Immunoassays Precipitation assay Agglutination assay Enzyme-linked immunosorbent assay (ELISA) Radioimmunoassay (RIA) Western blotting Immunofluorescence Flow cytometry

H. HogenEsch, 2005 Sensitivity of immunoassays precipitation - 30  g/ml agglutination - 1  g/ml radioimmunoassays, ELISA - 1 pg/ml

H. HogenEsch, 2005 Precipitation reaction Aggregates formed by interaction of multivalent antibodies and multivalent macromolecular antigens.

H. HogenEsch, 2005 Antigens have multiple epitopes

H. HogenEsch, 2005 Hemagglutination

H. HogenEsch, 2005 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.

H. HogenEsch, 2005 Rhesus factor

H. HogenEsch, 2005 Principle of ELISA/RIA Enzyme-linked immunosorbent assay (ELISA)

H. HogenEsch, 2005 Western blotting Western blot

H. HogenEsch, 2005 Immunofluorescence

H. HogenEsch, 2005 Flow cytometry