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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 (~10 8 )

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
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

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

chromatography • AB obtained are NOT one kind of molecules but a mixture of antibodies with
5
5

Monoclonal

antibodies

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

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

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
light chain
Heavy
chain
IgG can bind and cross link antigens with multi binding sites

IgG can bind and cross link antigens with multi binding sites

IgG is not a rigid molecule It has segmental flexibility 9

IgG is not a rigid molecule It has segmental flexibility

IgG is not a rigid molecule It has segmental flexibility
Properties of immunoglobulin classes Class Concent. Mass Heavy Chain mg/dl kDa chain

Properties of immunoglobulin classes

Class

Concent.

Mass

Heavy

Chain

mg/dl

kDa

chain

structure

IgG

1000

150

γ

γ

Monomer

IgA

200

170-500

 

α Dimer or

trimer

IgM

120

900

 

µ Pentamer

IgD

3

180

 

δ Monomer

IgE

0.05

190

 

ε Monomer

Classes of immunoglobulins

IgD IgE IgG IgA IgM
IgD
IgE
IgG
IgA
IgM

Properties of immunoglobulin classes

Class

 

IgG

The highest in concentration in the serum

IgA

The major class in external secretions

IgM

The first class to appear in serum after exposure to antigen

IgD

Unknown role

IgE

A role in conferring protection against parasites Allergic reactions

Cleavage of IgG by papain (Limited proteolysis) F a b 50 kDa Binds antigen Can’t

Cleavage of IgG by papain (Limited proteolysis)

F ab 50 kDa

Binds antigen

Can’t cross-link

F c 50 kDa Readily crystalyzes Can’t bind antigen

Amino acid sequence of antibodies 2 L chains 25 kDa 214 AA 2 H chains

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

Amino acid sequence of antibodies AA sequence H chain (multiple myeloma patients) 1- 113 vary

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

Variable and constant regions of light and heavy chains

Variable and constant regions of light and heavy chains 3 stretches (7-12 amino acids) hypervariable V

3 stretches (7-12 amino acids) hypervariable

V L ≈ V H

C L ≈ V L

C H

3 V H

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14

Immunoglobulin fold

• Pair of β sheets

– Each built of antiparallel β 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

• Multiple V variable genes are separate from single C gene in embryonic DNA embryonic cell

differentiated cell

VC

V 1 , V 2 , V 3

V n + C

Dreyer - Bennett hypothesis Single germline C gene V V separate from multiple V genes

Dreyer - Bennett hypothesis

Single germline C gene V V separate from multiple V genes V V V V
Single germline C gene
V
V
separate from multiple V genes
V
V V
V
V
V V
C
V
V V
C Rearranging V and C genes
V
V

The molecular basis of Antibodies diversity

• Immune system can generate > 10 8 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 , V 2 , . V 40

VJ

J 1 , J 2 , J 5

acids – Array of 5 segments near the C gene • V 1 , V 2

VJC

C

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18
Genes encoding heavy chains are present on chromosome 14 The variable gene is assembled from

Genes encoding heavy chains are present on chromosome 14

The variable gene is assembled from 3 segments

51 ~27 6 D join to J H then V is joined to DJ H
51
~27
6
D
join to J H then
V
is joined to DJ H
Number of possible combinations • 40 * 5 = 200 For κ • 30 *

Number of possible combinations

• 40 * 5 = 200

For κ

• 30 * 4 = 120

For λ

• 51 * 6 *27 = 8262

For H

• Combination of 320 L and 8262 2.6 * 10 6

• Somatic mutations increases the diversity

Class switching

• Proper immune response: Secreted antibodies must have appropriate effector function

• IgM IgG or IgA

• Specificity unchanged

– Light chain unchanged

– V H unchanged

– C H 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

another C gene • Shifting of the recognition domain different effector function unchanged binding specificity 21
Immune system can distinguish between self and nonself • Protection from invasion by foreign organisms

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 –

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