Chapter 5Chapter 5

ImmunoglobulinImmunoglobulin

Chapter 5Chapter 5

ImmunoglobulinImmunoglobulin

ContentsIntroductionSectionⅠ Molecular Structure of IgSectionⅡ Characteristics and Functions of the 5 Classes of IgSectionⅢ Fc Receptors for Ab MoleculesSectionⅣ Biological Activity of AbSectionⅤ Immunogenicity of IgSectionⅥ Artificial Ab

Concepts Antibody (Ab): Glycoprotein molecule

s 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 activ

ity of Ab or show a similar structure to Ab

• Therefore, All Abs are Igs, but not all Igs possess the functions of Abs

Other Conceptsγ- Globulin

Antiserum Humoral Immunity sIg and mIg(BCR)

SectionⅠ Molecular Structure of Ig

Ⅰ. Basic structure Ig is composed of four polypeptide chains j

oined 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~ IgG4IgA1, 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)(3) Hinge regionHinge region

(2) Variable(2) Variable regionregion (V) (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 chain

(2) Variable region ( V ): Hypervariable region(HVR) There are three highly diversity stret

ches 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 bi

nding 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 proteolys

is in extra secretory liquid.

IgA

Ⅱ. Domains of Ig

1. Domain :

Polypeptide chains of Ig are folded into a globul

ar 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 betw

een 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 hypersensitivit

y

Ⅱ. IgM 1. Highest MW ： pentamer （ 90 KD ） ,10 valence

s

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 recept

ors(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 hypersens

itivity

ⅢⅢ. . IgAIgA

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. IgD1. 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. IgE1.Concerntration of IgE in serum is the lo

west 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---FcR: FcRⅠ(CD64)---phagocyte FcRⅡ(CD32)---immune complex FcRⅢ(CD16)---NK,macrophage,T cell

IgE---FcR: FcRⅠ--- mast cell, basophil FcRⅡ--- macrophage, B cell

IgA---FcαR(CD89)---phagocyte, neutrophil

SectionⅣ Biological Activity of Ab

1. Recognize and bind to antigen specifically 2. Fix complement3. 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(FcRⅠ)

FcRI

degranulation

IgE

allergen

inflammationinflammation

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 specifi

cities and affinities

Generate in a natural response or artificial immunization

Cross reaction

Cross-reactivity:if two antigens share an epitopean antibody recognizes an unrelate

d,but chemically similar, epitope

2. Monoclonal Ab (mAb) Ab produced by single clone (or one hy

bridomas 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 techniqueCell fusion technique ：：Cell fusion techniqueCell fusion technique ：：

SplenocyteSplenocyte(B cell)(B cell)

Myeloma cellMyeloma cell

Hybridoma cellHybridoma cell

No AbNo AbLong lifeLong life

Secrete AbSecrete AbShort lifeShort life

Secrete AbSecrete AbLong lifeLong life

Köhler and Milstein, 1975Köhler and Milstein, 1975

1984, Nobel Prize1984, Nobel Prize

Screening of hybridoma cell:Screening of hybridoma cell:Screening of hybridoma cell:Screening of hybridoma cell:

SplenocyteSplenocyte(B cell)(B cell)

Myeloma cellMyeloma cell

Hybridoma cellHybridoma cell S/S/SS M/M/MM Myeloma cellMyeloma cellsplenocytesplenocyte

YesYesShort lifeShort lifeShort lifeShort life Short lifeShort lifeShort lifeShort life

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 Screening with HAT selective medium:medium:Screening with HAT selective Screening with HAT selective medium:medium:

H, 次黄嘌呤 ; A, 氨基喋呤 ; T, 胸腺嘧啶

DNADNADNADNAIntrinsic pathway(major)Intrinsic pathway(major)

谷氨酰胺 谷氨酰胺 oror单磷酸尿苷酸单磷酸尿苷酸

二氢叶酸还原酶二氢叶酸还原酶

AA

HHHH

TTTT

Extrinsic pathway(alternative)Extrinsic pathway(alternative)

((HHypoxanthine ypoxanthine gguznine uznine pphosphohosphorribosyl ibosyl ttransferase)ransferase) 次嘌呤鸟嘌呤磷酸核糖转移酶次嘌呤鸟嘌呤磷酸核糖转移酶 ( ( HGPRTHGPRT ))

胸腺嘧啶激酶胸腺嘧啶激酶 ( ( TK TK ))((TThymidine hymidine kkinase)inase)

BB cell cell ： ： HGPRTHGPRT++ ，， TKTK++

Myeloma cellMyeloma cell ： ： HGPRTHGPRT-- ，， TKTK--

livelive

diedie

Extrinsic pathway(minor)Extrinsic pathway(minor)

PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY

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

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 r

ecombination

• 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

DenmarkBasel Institute for Immunology Basel, Switzerland

Argentina MRC Laboratory of Molecular Biology Cambridge,