Immunoglobulin

Chapter 5
Immunoglobulin
Contents
Introduction
SectionⅠ Molecular Structure of Ig
SectionⅡ Characteristics and Functions of the 5 Classes of Ig
SectionⅢ Fc Receptors for Ab Molecules
SectionⅣ Biological Activity of Ab
SectionⅤ Immunogenicity of Ig
SectionⅥ Artificial Ab
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
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
Other Concepts
- Globulin
Antiserum
Humoral Immunity
sIg and mIg(BCR)
SectionⅠ Molecular Structure of Ig
Ⅰ. Basic structure

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.
(four polypeptide chains)
1. H and L chain:


. Heavy chains (H):
450 ~ 550 aa,
50 ~ 75 KD

. Light chains (L):
214 aa, 25 KD
Two terminal ends for each peptide chain


“N” terminal end
“C” terminal end

L chains attach to H chains
from “N” end

“N”
“C”
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
(2) According to the differences of L chains
Two types of L chain: , 

 :  20:1 (in mice); 2:1 (in human)
subtypes
 1~  4
3. Two regions of each peptide chain
(1) Constant region (C)
(3) Hinge region
(2) Variable region (V)


(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
3. Two regions of each peptide chain
(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
Ag-binding sites
Complementarity determining regions(CDR)
(2) Variable region (V)
Complementarity determining regions(CDR)
L: CDR1, CDR2, CDR3
H: CDR1, CDR2, CDR3
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
In fact:
HVR
CDR
Idiotype
are in the same sites of Ig
(3) Hinge region:
Flexible and suitable for CDR of Ig binding to antigenic determinants.
Sensitive to proteolytic enzyme
IgM, IgE
Other structures of Ig

Joining chain(J)
Secretory piece(SP)
Joining chain(J ) :

Produced by plasma cells
Functions:linker, to compose dimer、pentamer or polymer(IgA, IgM)
Secretory piece( SP):
. Produced by mucosa epithelial cells
. Secretory IgA (sIgA)
. Functions: protect sIgA, resist proteolysis in extra secretory liquid.
IgA
Ⅱ. Domains of Ig


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
2.   Domains of Ig

L chain(2) : VL, CL
H chain(4~5):
VH,
CH1, CH2, CH3
CH4(in IgM,IgE)
hinge region
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
Ⅲ. Hydrolytic fragments of Ig
Ig can be digested by papain and pepsin
Position
Fragments
Function
1. Digested by papain
Position:
near the S-S bonds of H inter-chains fromthe N end

Fragments:
2Fab:fragment antigen-binding
Fc:fragment crystallizable

Function:
Fab: recognize and bind Ag
Fc:
(1) fix complement
(2) crossing the placenta
(3) bind to FcR in different cells








Ag:Ab ratio


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
Significance
 Elucidating the relationships between the structure and function of Igs

Decrease the immunogenicity of Ig for clinical treatment
SectionⅡ Characteristics and Functions of the 5 Classes of Igs
Ⅰ. IgG
1. Highest concentration in serum(75% of total Ig)


2. Four subclasses: IgG1, IgG2, IgG3, IgG4
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
6. Functions of IgG:
Against bacteria and virus,neutralize toxin
Combine with the Fc receptor(FcγR)
Activate complement
Combine with SPA
Some belong to the auto-antibodies
Take part in type Ⅱ and Ⅲ hypersensitivity
Ⅱ. IgM
1. Highest MW：pentamer（90 KD）,10 valences

2. Half-life is shorter(4~5 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)
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 Ⅱ and Ⅲ hypersensitivity
Ⅲ. IgA

1. Two types
Serum type ：monomer
Secretary type（sIgA）: dimer，trimer or polymer

2. Two subclasses：IgA1，IgA2
3. To be produced at 4 months after birth

4. Exist in almost all body fluid

6. Local mucosal immunity

Immune barrier
Neutralize virus/toxin
Rich in colostrum
Activate C by alternative pathway
Take part in type Ⅲ hypersensitivity
Ⅳ. 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
Ⅴ. IgE
1.Concerntration of IgE in serum is the lowest in normal individual, but is very high in some patients.

2.Related to typeⅠpersensitivity
FcεRⅠ: mast cell, basophil
SectionⅢ Fc Receptors for
Ab Molecules
IgG---FcR: FcRⅠ(CD64)---phagocyte
FcRⅡ(CD32)---immune complex
FcRⅢ(CD16)---NK,macrophage,T cell

IgE---FcR: FcRⅠ--- mast cell, basophil
FcRⅡ--- macrophage, B cell

IgA---FcαR(CD89)---phagocyte, neutrophil
SectionⅣ Biological Activity of Ab
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)
Affinity and Avidity
Neutralization
IgM,IgG1~3: classical pathway
IgA,IgG4,IgE:
alternative pathway
MAC
(1) Opsonization(IgG, IgM):
Enhance the phagocytosis of MΦ
(2) ADCC( antibody dependent cell mediated cytotoxicity)
(3) Hypersensitivity typeⅠ
- mast cell, basophil(FcRⅠ)
SectionⅤ Immunogenicity of Ig
Isotype: CH, CL　
Allotype：CH, CL
Idiotype: VH, VL
Anti-idiotype antibody
SectionⅥ Artificial Ab
Polyclonal Ab
Monoclonal Ab
Gene engineering Ab
1. Polyclonal Ab

A mixture Ab with different specificities and affinities

Generate in a natural response or artificial immunization
Cross reaction
Cross-reactivity:
if two antigens share an epitope
an antibody recognizes an unrelated,
but chemically similar, epitope
2. Monoclonal Ab (mAb)

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


mAb / McAb
Prepared by hybridomas technique:

Immunized spleen cells(B) hybride with myeloma cells----hybridomas
Artificial antibodies
Derived from different B Lymphocytes cell lines
POLYCLONAL.
MONOCLONAL.
Derived from a single B cell clone
Batch to Batch variation affecting Ab reactivity & titre
mAb offer Reproducible, Predictable & Potentially inexhaustible supply of Ab with exquisite specificity
Enable the development of secure immunoassay systems.
NOT Powerful tools for clinical diagnostic tests
Cell fusion technique：
Hybridoma cell
No Ab
Long life
Secrete Ab
Short life
Secrete Ab
Long life
Köhler and Milstein, 1975
1984, Nobel Prize
Screening of hybridoma cell:
Hybridoma cell
S/S
M/M
Myeloma cell
splenocyte
HAT selective medium:
次黄嘌呤（hypoxanthine,H)
氨甲喋呤（aminopterin,A):叶酸拮抗剂
胸腺嘧啶核苷（thymidine,T)
Screening with HAT selective medium:
H, 次黄嘌呤; A, 氨基喋呤; T, 胸腺嘧啶
A
(Hypoxanthine guznine phosphoribosyl transferase)
次嘌呤鸟嘌呤磷酸核糖转移酶 ( HGPRT )
胸腺嘧啶激酶 ( TK )
(Thymidine kinase)
B cell： HGPRT+， TK+
Myeloma cell： HGPRT-， TK-
live
die
Extrinsic pathway(minor)
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Applications of Monoclonal Antibodies
Diagnostic Applications
Biosensors & Microarrays
Therapeutic Applications
Transplant rejection Muronomab-CD3
Cardiovascular disease Abciximab
Cancer Rituximab
Infectious Diseases Palivizumab
Inflammatory disease Infliximab
Clinical Applications
Purification of drugs, Imaging the target
Future Applications
Fight against Bioterrorism
EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC
DNA SPLICING / GENE KNOCK OUT
2. LIBRARIES
a.BACTERIOPHAGE
b. mRNA
c. Cell Surface
3.Gene engineering Ab
Abs prepared by the method of gene recombination

Chimeric Ab:human Fc bind with mice Fab
Recombinant single chain Ab:VH-linker-VL






Human-mouse chimeric Ab
Niels K Jerne J.Kohler S.Milstein
Germany
Basel Institute for Immunology
Basel, Switzerland

Denmark
Basel Institute for Immunology
Basel, Switzerland
Argentina
MRC Laboratory of
Molecular Biology
Cambridge,