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Immunoglobulins

Faisal Khatib MD; PhD Faculty of Medicine, University of Jordan

Immune system
Detects and inactivates foreign molecules, viruses, bacteria and microorganisms Two components with 2 strategies B Lymphocytes (humoral immune response):
Soluble antibodies (immunoglobulins) Secreted by plasma cells Recognize and bind to foreign molecules.

T Lymphocytes (cellular immune response):


Killer cells that display foreign substances on their surface Use specific receptors on their surface

Immunoglobulins
The binding to the foreign molecules is:
Specific High affinity

Effector functions: biological effects that result as consequence of antigen binding like
Inactivation, degradation, lysis

Can recognize and interact with foreign molecules even if not encountered before Huge number of different kinds (~108)

Synthesis is stimulated by invasion of the body

Formation of immunoglobulins (Ig)


Antigen
- Foreign molecules to which Ig binds - Can elicit antibody formation ( immounogen) - Macromolecule; Protein, polysaccharide, nucleic acid

Epitope
Particular site on antigen to which antibody binds Antigenic determinant

Hapten
small molecule, not an antigen Antigen if attached to macromolecule

Formation of antibodies against small molecules


OH

Attach DNP to Bovine Serum Albumin

Dinitrophenol
a small molecule

Formation of antibodies against DNP after injection of DNP-BSA to rabbit

Isolation of antibodies (AB) against DNP from rabbit


Antisera: The serum obtained which contains the antibodies (AB) AB formed can bind free and attached hapten AB can be purified by affinity chromatography AB obtained are NOT one kind of molecules but a mixture of antibodies with the same specificity

Monoclonal antibodies
One kind of antibodies Produced from clone of one single cell

Principles of preparation of monoclonal antibodies


Multiple Myeloma: Malignant growth of a single kind of B lymphocytes in human. Large amounts of one kind of antibodies are produced ( but unknown specificity). Mice strains with multiple myeloma are available Can be transplanted from one mouse to another.
Same kind of antibody is produced. Cells can be cultured (cell line)

Antibodies produced

Monoclonal

Large amounts

Desired specificity

Structure of antibodies
Immunoglobulin G ( IgG ) is the major class of antibodies in the plasma. 150 kDa Tetramer of
Two heavy chains 50 kDa Two light chains 25 kDa Connected by disulfide bonds

light chain

Heavy chain

IgG can bind and cross link antigens with multi binding sites

IgG is not a rigid molecule It has segmental flexibility

Properties of immunoglobulin classes


Class IgG IgA IgM IgD IgE Concent. Mass mg/dl kDa 1000 200 120 3 0.05 150 170-500 900 180 190 Heavy chain Chain structure Monomer Dimer or trimer Pentamer Monomer Monomer

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Classes of immunoglobulins

IgG

IgA

IgM

IgD

IgE

Properties of immunoglobulin classes


Class IgG IgA IgM IgD IgE The highest in concentration in the serum The major class in external secretions The first class to appear in serum after exposure to antigen Unknown role A role in conferring protection against parasites Allergic reactions

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Cleavage of IgG by papain (Limited proteolysis)


Fab 50 kDa Binds antigen Cant cross-link
Fc 50 kDa Readily crystalyzes Cant bind antigen

Amino acid sequence of antibodies


2 L chains 25 kDa 214 AA 2 H chains 50 kDa 446 AA AA sequence L chain (multiple myeloma patients) 1- 110 vary from one patient to another 111 214 same in many patients

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Amino acid sequence of antibodies

AA sequence H chain (multiple myeloma patients) 1- 113 vary from one patient to another 114 446 same in many patients

Variable and constant regions of light and heavy chains 3 stretches (7-12 amino acids) hypervariable VL VH CL VL CH 3 VH

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Immunoglobulin fold
Pair of sheets
Each built of antiparallel strands Connected by disulfide Three loops at one end of the structure Hypervariable stretches The binding surface complementarity determining regions (CDR)

The molecular basis of Antibodies diversity


The Variable and constant regions Multiple V variable genes are separate from single C gene in embryonic DNA embryonic cell V1, V2, V3 ..Vn + C

differentiated cell

VC

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Dreyer - Bennett hypothesis


Single germline C gene separate from multiple V genes

V V

V V

V V V V

V V

V V

V C

Rearranging V and C genes

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The molecular basis of Antibodies diversity


Immune system can generate > 108 antibodies proteins Human genome contains ~ 40,000 genes !

V genes in embryonic cells do not encode the entire variable gene


V genes encode the first 97 amino acids
Array of 40 segments

J genes encode the last 13 amino acids


Array of 5 segments near the C gene

V 1 , V2 ,

. V40 VJ

J1, J2,

J5

VJC

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Genes encoding heavy chains are present on chromosome 14 The variable gene is assembled from 3 segments

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~27

D join to JH then V is joined to DJH

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Number of possible combinations


For For For H 40 * 5 = 200 30 * 4 = 120 51 * 6 *27 = 8262 2.6 * 106

Combination of 320 L and 8262

Somatic mutations increases the diversity

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Class switching
Proper immune response: Secreted antibodies must have appropriate effector function IgM IgG or IgA ... Specificity unchanged
Light chain unchanged VH unchanged CH is changed

Gene rearrangement: movement of VDG from a site near one C gene to a site near another C gene Shifting of the recognition domain different effector function unchanged binding specificity

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Immune system can distinguish between self and nonself


Protection from invasion by foreign organisms is the primary function of immune system. Attacking host cells is avoided. How can immune system distinguish Cells that react strongly with self- antigens are killed early in the development of immune system

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C-Reacrive Protein
Undetectable in healthy individuals In patients with diverse inflammatory diseases
Acute rheumatic fever, bacterial infection, gout .. Tissue damage

Reacts with C substance ( polysaccharide in Pnumococci and molecular groups on a wide variety of bacteria

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