Chapter 5 Immunoglobulin

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Chapter 5Immunoglobulin
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Contents

• Introduction
• Section? Molecular Structure of Ig
• Section? Characteristics and Functions of the 5
  Classes of Ig
• Section? Fc Receptors for Ab Molecules
• Section? Biological Activity of Ab
• Section? Immunogenicity of Ig
• Section? Artificial Ab

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Concepts

• Antibody (Ab) Glycoprotein molecules that are
  produced by plasma cells and can combine with the
  corresponding Ag specifically are called Ab.
• Ab is produced by B cells in the response to a
  stimulation of Ag.
• Ab possesses a high degree of specificity and
  affinity

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• Immunoglobulin(Ig)
• It refers to all globulins that possess the
  activity of Ab or show a similar structure to Ab
• Therefore, All Abs are Igs, but not all Igs
  possess the functions of Abs

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Other Concepts

• - Globulin
• Antiserum
• Humoral Immunity
• sIg and mIg(BCR)

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Section? Molecular Structure of Ig
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?. Basic structure
(four polypeptide chains)

• Ig is composed of four polypeptide chains joined
  by S-S bonds.
• inter-chain disulfide bonds (S-S)
• intra-chain disulfide bonds (S-S)
• It shows T or Y shape.

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1. H and L chain

• . Heavy chains (H)
• 450 550 aa,
• 50 75 KD
• . Light chains (L)
• 214 aa, 25 KD

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• Two terminal ends for each peptide chain
• N terminal end
• C terminal end
• L chains attach to H chains
• from N end

N
C
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2. classes and types of Ig

• (1) According to the differences of H chains
• (amino acid composition, sequence)
• Igs can be divided into 5 classes
• Five classes of H Chain ? ? ? ? ?
• Five classes of Igs IgG IgA IgM IgD IgE

subclasses
IgG1 IgG4
IgA1, IgA2
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• (2) According to the differences of L chains
• Two types of L chain ?, ?
• ? ? 201 (in mice) 21 (in human)

subtypes
? 1 ? 4
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3. Two regions of each peptide chain
(1) Constant region (C)
(2) Variable region (V)
(3) Hinge region
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3. Two regions of each peptide chain

• (1) Constant region ( C )
• CH 3/4 or 4/5 (?,?) of H chain from the c end
• CL 1/2 of L chain from the c end
• (2) Variable region ( V )
• CH 1/4 or 1/5 (?,?) of H chain from the N end
• CL 1/2 of L chain from the N end

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(2) Variable region ( V )

• Hypervariable region(HVR)
• There are three highly diversity stretches
  within the V egion,
• they are called HVR.
• Framework region(FR)
• FR1-FR4

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Ag-binding sites
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Complementarity determining regions(CDR)
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(2) Variable region (V)

• Complementarity determining regions(CDR)
• L CDR1, CDR2, CDR3
  
  
• H CDR1, CDR2, CDR3

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Idiotype of Ig

• Igs produced by different B cells possess
  unique structure respectively in hypervariable
  region (HVR), the unique structure of Ig is
  called idiotype or idiotypic determinant

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• In fact
• HVR
• CDR
• Idiotype
• are in the same sites of Ig

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• (3) Hinge region
• Flexible and suitable for CDR of Ig binding to
  antigenic determinants.
• Sensitive to proteolytic enzyme
• IgM, IgE

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Other structures of Ig

• Joining chain(J)

• Secretory piece(SP)

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Joining chain(J )

• Produced by plasma cells
• Functionslinker, to compose dimer?pentamer or
  polymer(IgA, IgM)

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Secretory piece( SP)

• . Produced by mucosa epithelial cells
• . Secretory IgA (sIgA)
• . Functions protect sIgA, resist
  proteolysis in extra secretory liquid.

IgA
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?. Domains of Ig

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1. Domain

• Polypeptide chains of Ig are folded into a
  globular structure by intra chain s-s bond within
  each 110aa region which is called a domain

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2.   Domains of Ig

• L chain(2) VL, CL
• H chain(45)
• VH,
• CH1, CH2, CH3
• CH4(in IgM,IgE)
• hinge region

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3. Function of each domain

• VH, VL antigen-binding site
• CH1, CL allogeneic marker
• CH2/CH3 complement-fixing site,
• permeate placenta(IgG)
  
• CH3/CH4 cell-binding site
• Hinge region

flexible and suitable for CDR of Ig binding to
antigenic determinants
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?. Hydrolytic fragments of Ig

• Ig can be digested by papain and pepsin
• Position
• Fragments
• Function

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1. Digested by papain

• Position
• near the S-S bonds of H inter-chains fromthe
  N end
• Fragments
• 2Fabfragment antigen-binding
• Fcfragment crystallizable
• Function
• Fab recognize and bind Ag Fc
• (1) fix complement
• (2) crossing the placenta
• (3) bind to FcR in different cells

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AgAb ratio
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• 2. Digested by pepsin
• Position
• near the S-S bond of H inter-chains from the C
  end
• Fragments and function
• F(ab')2 bind antigen(2 valence)
• pFc' no function

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Significance

•  Elucidating the relationships between the
  structure and function of Igs
• Decrease the immunogenicity of Ig for clinical
  treatment

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• Section? Characteristics and Functions of the 5
  Classes of Igs

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?. IgG

• 1. Highest concentration in serum(75 of total
  Ig)

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• 2. Four subclasses IgG1, IgG2, IgG3, IgG4

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• 3. Unique Ig that can pass through placenta
• 4. Half-life is longer( 16-24 days )
• 5. Starts to be produced at 2-3 month after birth
  and reach the level of adult at 5 years old

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• 6. Functions of IgG
• Against bacteria and virus,neutralize toxin
• Combine with the Fc receptor(Fc?R)
• Activate complement
• Combine with SPA
• Some belong to the auto-antibodies
• Take part in type ? and ? hypersensitivity

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1. Highest MWpentamer(90 KD),10 valences
?. IgM
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• 2. Half-life is shorter(45 days)
• 3. The first Ig to be synthesized
• Appear in the early stage after infection
• Be produced during fetus
• The first mIg of the B cells, act as the antigen
  receptors(BCR)

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4. Functions

• IgM is more effective in binding Ag and
  activating C, and play an important role in
  anti-infection
• Natural Ab for blood-type antigen
• Auto-antibody rheumatoid factor(RF)
• Take part in type ? and ? hypersensitivity

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?. IgA 1. Two types Serum type
monomer Secretary type(sIgA) dimer,trimer
or polymer 2. Two subclassesIgA1,IgA2
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• 3. To be produced at 4 months after birth
• 4. Exist in almost all body fluid

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6. Local mucosal immunity

• Immune barrier
• Neutralize virus/toxin
• Rich in colostrum
• Activate C by alternative pathway
• Take part in type ? hypersensitivity

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?. IgD

• 1. The concentration in serum is low and
  sensitive to proteinase
• 2. Act as the antigen receptor on B cells (mIgD)
• Regulate the differentiation of B cells

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?. IgE

• 1.Concerntration of IgE in serum is the lowest in
  normal individual, but is very high in some
  patients.
• 2.Related to type?persensitivity
• FceR? mast cell, basophil

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Section? Fc Receptors for
Ab Molecules
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• IgG---Fc?R Fc?R?(CD64)---phagocyte
• Fc?R?(CD32)---immune complex
• Fc?R?(CD16)---NK,macrophage,T
  cell
• IgE---Fc?R Fc?R?--- mast cell, basophil
• Fc?R?--- macrophage, B cell
• IgA---FcaR(CD89)---phagocyte, neutrophil

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Section? Biological Activity of Ab
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• 1. Recognize and bind to antigen specifically
• 2. Fix complement
• 3. Bind to Fc receptor on some cells
• 4. Transfer selectively
• .Planceta transfer (IgG)
• .Mucosa transfer (sIgA)

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Affinity and Avidity
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Neutralization
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IgM,IgG13 classical pathway IgA,IgG4,IgE altern
ative pathway
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MAC
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• (1) Opsonization(IgG, IgM)
• Enhance the phagocytosis of MF

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(2) ADCC( antibody dependent cell mediated
cytotoxicity)
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• (3) Hypersensitivity type?
• - mast cell, basophil(Fc?R?)

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Section? Immunogenicity of Ig
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Isotype CH, CL
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AllotypeCH, CL
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Idiotype VH, VL Anti-idiotype antibody
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Section? Artificial Ab

• Polyclonal Ab
• Monoclonal Ab
• Gene engineering Ab

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1. Polyclonal Ab

• A mixture Ab with different specificities and
  affinities
• Generate in a natural response or artificial
  immunization
• Cross reaction

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Cross-reactivity if two antigens share an
epitope an antibody recognizes an
unrelated, but chemically similar, epitope
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2. Monoclonal Ab (mAb)

• Ab produced by single clone (or one hybridomas
  clone ) and having a single specificity

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mAb / McAb

• Prepared by hybridomas technique
• Immunized spleen cells(B) hybride with myeloma
  cells----hybridomas

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Artificial antibodies
POLYCLONAL.
MONOCLONAL.
Derived from different B Lymphocytes cell lines
Derived from a single B cell clone
mAb offer Reproducible, Predictable Potentially
inexhaustible supply of Ab with exquisite
specificity
Batch to Batch variation affecting Ab reactivity
titre
Enable the development of secure immunoassay
systems.
NOT Powerful tools for clinical diagnostic tests
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Cell fusion technique
Secrete Ab Short life
No Ab Long life
Secrete Ab Long life
Hybridoma cell
Köhler and Milstein, 1975
1984, Nobel Prize
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Screening of hybridoma cell
Hybridoma cell
M/?M
S/?S
splenocyte
Myeloma cell
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• HAT selective medium
• ????(hypoxanthine,H)
• ????(aminopterin,A)?????
• ??????(thymidine,T)

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Screening with HAT selective medium
H, ???? A, ???? T, ????
(Hypoxanthine guznine phosphoribosyl transferase)
????????????? ( HGPRT )
A
Extrinsic pathway(minor)
?????? ( TK ) (Thymidine kinase)
B cell HGPRT, TK
live
die
Myeloma cell HGPRT-, TK-
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PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
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Applications of Monoclonal Antibodies

• Diagnostic ApplicationsBiosensors Microarrays
• Therapeutic ApplicationsTransplant rejection
  Muronomab-CD3Cardiovascular disease Abciximab
  Cancer RituximabInfectious Diseases
  PalivizumabInflammatory disease Infliximab
• Clinical ApplicationsPurification of drugs,
  Imaging the target
• Future Applications Fight against Bioterrorism

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EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC DNA SPLICING / GENE KNOCK OUT
2. LIBRARIES a.BACTERIOPHAGE b. mRNA c. Cell
Surface
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3.Gene engineering Ab

• Abs prepared by the method of gene recombination
  
• Chimeric Abhuman Fc bind with mice Fab
• Recombinant single chain AbVH-linker-VL

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Human-mouse chimeric Ab


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Niels K Jerne J.Kohler
S.Milstein
Denmark Basel Institute for Immunology Basel,
Switzerland
Germany Basel Institute for Immunology Basel,
Switzerland
Argentina MRC Laboratory of Molecular Biology
Cambridge,