Antibody Structure and the Generation of B - Cell Diversity

Dr. Glorivee Rosario Pérez

BIOL 4056

Parham P. (2009). The Immune System. Third Edition. Garland Publishing, New York.

Introduction

Antibodies Proteins Produced by the B lymphocytes in response to

infection They circulate as a major component of the plasma in

blood and lymph Their function is to bind to pathogenic microorganisms

and their toxins (antigens) Are specific

Introduction cont.

Figure 3-1

Introduction cont.

Antibody production is the single effector function of the B lymphocytes of the immune system.

The structural basis of antibody diversity

Antibodies : Variable and Constant region

N-terminal

C-terminal

Antibodies : Variable and Constant region cont.

Antigen-binding site

Hypervariable regions (HV regions) Small regions of high amino-acid sequence diversity

within the variable regions of immunoglobulin and T-cell receptor.

They correspond to the complementarity-determining regions.

Complementarity-determining regions (CDRs)

Antibodies : Variable and Constant region cont.

Antibodies : Variable and Constant region cont.

Immunoglobulin chains

Immunoglobulin domain

Components of protein structure consisting of about 100 amino acids that fold into a sandwich of two β sheets held together by a disulfide bond.

Immunoglobulin heavy and light chain are made up of a series of immunoglobulin domains.

Immunoglobulin chains cont.

Antigen-binding site Hypervariable regions

(HV regions) Small regions of high

amino-acid sequence diversity within the variable regions of immunoglobulin and T-cell receptor.

They correspond to the complementarity-determining regions.

Antigen-binding site cont.

Complementarity-determining regions (CDRs)The localized regions of immunoglobulin and

T-cell receptor chains that determine the antigenic specificity and bind to the antigen.

The CDRs are the most variable parts of the variable domains and are also called hypervariable regions.

Antigen-binding sites

Antigenic determinant (epitope)The part of the antigen to which an antibody

binds.These structures are usually either

carbohydrate or protein, or both, because the surface molecules of pathogens are commonly glycoproteins, polysaccharides, glycolipids, and peptidoglycans.

Figure 3-8

Antigen-binding sites cont.

Multivalent antigen – any antigen that contains more than one epitope, or more than one copy of the same epitope.

Antigen-binding sites cont. The binding of antigens to antibodies is based on

noncovalent forces:

Generation of immunoglobulin diversity in B cells before encounter with antigen

Gene segments

Multiple short DNA sequences in the immunoglobulin and T-cell receptor genes.

These can be rearranged in many different

combinations to produce the vast diversity of immunoglobulin or T-cell receptor polypeptide chain.

The organization of the human immunoglobulin heavy- and light-chain loci

Figure 4-3

Gene segments (V)

Variable (V) gene segments DNA sequence in the immunoglobulin or T-cell

receptor genes that encodes the first 95 or so amino acids of the V domain.

There are multiple different V gene segments in the germINAL genome.

To produce a complete exon encoding a V domain, one V gene segment must be rearranged to join up with a J or a rearranged DJ gene segment.

Gene segments (J)

Joining (J) gene segmentsOne of the types of gene segment in

immunoglobulin and T-cell receptor genes that is rearranged to make functional variable-region exons.

Gene segments (D)

Diversity (D) gene segmentsShort DNA sequence present in

immunoglobulin heavy chain loci and in T-cell receptor β- and δ-chain loci.

In the rearranged functional genes at these loci, a D region connects the V and J region.

Mechanisms contributing to the diversity in V-region1. Random combination of different V and J segments

in light chains genes and of different V, D, and J segments in rearranged heavy chain genes.

2. Introduction of additional nucleotides at the connections between gene segments during the process of recombination.

3. Diversity in the antigen-binding sites of antibodies is the association of heavy and light chains in different combinations.

1. Random recombination of gene segments produces diversity in the antigen-binding sites of immunoglobulins

Figure 4-2

Figure 4-13

Naïve B cells

A mature B cell that has left the bone marrow but has not yet encountered its specific antigen.

IgM and IgD

Expression of IgM and IgD

Allelic exclusion

In a developing B cell, the process of immunoglobulin-gene rearrangement is controlled so that only one heavy chain and one light chain are finally expressed.

Membrane-bound immunoglobulins

Diversification of antibodies after B cells encounter antigen

Secreted antibodies

Mature cell (naïve cell)

IgM IgD(membrane-bound)

antigen

(secreted antibodies)↑↑↑IgM ↑IgD

antigen

The surface and secreted forms of an immunoglobulin

Somatic hypermutation

Mutation that occur at high frequency in the rearranged variable region DNA of immunoglobulin genes in activated B cells, resulting in the production of variant antibodies, some of which have a higher affinity for the antigen.

Isotype switching

The physical properties of the human immunoglobulin isotypes

Figure 4-18

Figure 4-23

Antibody function : IgM

Is the first antibody produced in an immune response.

It is made principally by plasma cells resident in:Lymph nodesSpleen (BASO)Bone marrow

Circulates in blood and lymph.

Antibody function : IgG

Is the most abundant antibody in the internal body fluids, including blood and lymph.

It is made in the: Lymph nodes Spleen Bone marrow

Is smaller and more flexible than IgM, properties that give it easier access to antigens in the extracellular spaces of damaged and infected tissues.

Antibody function : IgA

Is made by plasma cells in: Lymph nodes Spleen(HIGADO) Bone marrow

Is secreted into the bloodstream. Dimeric IgA is made in the lymphoid tissues

fundamental mucosal surfaces. Is the antibody that is secreted into the lumen of

the gut. Principal antibody of milk, saliva, sweat, and

tears.

Antibody function : IgE

Is highly specialized towards the activation of mast cells, which are present in epithelial tissues.

The major impact of IgE is in the allergies that result when it is produced against antigens.

Mechanisms by which antibodies combat infection

Functions and properties of immunoglobulin

Applications: Antigen-Antibody

Figure A-1

Figure A-5

Figure A-6

Figure A-7

Figure A-8

Figure A-9

Figure A-10

Figure A-13

Monoclonal antibodies

The traditional method for making antibodies of desired specificity is to immunize animals with the appropriate antigen and then prepare antisuero from their blood.

antisera

Monoclonal antibodies cont.

Monoclonal antibodies cont.

Figure A-15

Radioimmunoassay (RIA)

FIGURA 18.37

Figure A-18Radioimmunoassay (RIA) cont.

Figure A-20

Figure A-22

Figure A-23

Figure A-26

Figure A-29

Figure A-40

Figure A-41

Figure A-16Immunofluorescence

Figure A-17Immunofluorescence cont.

FIGURA 18.35

Immunofluorescence cont.

FIGURA 18.38

Immunofluorescence (ELISA) cont.

FIGURA 18.39

Immunofluorescence (Western Blot) cont.

Flow cytometry

Figure A-25 part 2 of 2Flow cytometry cont.