Antibody

Antibodies

Antibodies (Ab)

Glycoproteins that bind antigens with high specificity and affinity. (specific – react with only one antigen ) • Produced in B cells in response to an antigen : Activated B cells ⇒ Plasma cells ⇒ secrete antibodies • Found in plasma.Originally identified in the serum. • Sometimes are called gamma globulins or immunoglobulins (Ig). • Five classes: IgG, IgM, IgA, IgE & IgD

Basic Structure 4 polypeptide chain unit (Y shaped) • 2 heavy chains (five kinds: α , γ , µ , δ , εDisulfide ) bond

2 light chains (two types: λ ,

κ )

Carbohydrate

• Disulfide bonds – Inter-chain – Intra-chain

CL VL CH2

CH1 VH

Hinge Region

CH3

Basic Structure • Variable & Constant Regions – VL & VH: Ag binding site – CL & CH : determine the fate of Ag

• Hinge Region: rich in proline easy to bend and be hydrolyzed by some enzymes

Disulfide bond

• Structure of the V Region – Hypervariable (HVR) or complimentarity determining regions (CDR) – Framework regions

Carbohydrate

CL VL CH2

CH1 VH

Hinge Region

CH3

Antibody – another view - variable regions of the light chain (grey) and the heavy chain (yellow) form the antigen binding site

- light chain constant region is blue while heavy chain constant region is red. The two chains are joined by carbohydrate (purple).

• Domains

Basic Structure

– VL & CL – VH & CH1, CH2 ,CH3 (or CH4 )

Disulfide bond

Carbohydrate

• Functional properties: – VL , VH :Bind to Ag – CL , CH1 : genetic marker – CH2 : complement binding site;

CL VL

placental transfer (IgG) complement binding site(IgM)

CH2

CH1

– CH3 / CH4 : binding to FcR VH

Hinge Region

CH3

Valence and avidity • Valence:

the maximum number of antigenic determinants with which antibody can react. e.g. IgG: 2, Fab:1

• Avidity : the firmness of association between a multideterminant antigen and antibodies produced against it. Disulfide bond

Having multiple binding sites for antigen means multi-valence, and can increase its avidity to antigens on particles such as bacteria or virus.VL

Carbohydrate

CL CH2

CH1 VH

Hinge Region

CH3

Papain

Hydrolytic Fragments:

Pepsin

Fc Peptides

Fc Fab

papain • 1IgG 2Fab + 1Fc • Fab(fragment antigen binding) valence = 1 • Fc(fragment crystallizable) – Effector functions

F(ab’)2

pepsin • 1IgG 1F(ab’)2 + pFc’ • F(ab’)2: valence=2 • pFc’(no biological activities)

Human Ab Classes • IgG - Gamma (γ ) heavy chains 4 subclasses (IgG1, IgG2, IgG3, IgG4) have slightly different sequences in H-chain and corresponding differences in functional activities

• IgM - Mu (µ )heavy chains • IgA - Alpha (α ) heavy chains 2 subclasses (IgA1, IgA2)

• IgD - Delta (δ ) heavy chains • IgE - Epsilon (ε ) heavy chains

IgG • Structure – Monomer

IgG1, IgG2 and IgG4

IgG3

IgG • Structure • Properties – Found in vascular and extravascular spaces, secretions – Major serum Ig: 70-75%, provide immunity to most blood borne infectious agents – Major antibody of the secondary immune response – the only Ab class that can cross placenta to provide passive humoral immunity to fetus

• Structure

IgA

– Serum • monomer

– Secretions (secretory IgA , sIgA) • Dimer • J chain (joining chain) • SP (secretory piece) (involved in the transepithelial transport of exocrine IgA and stabilizes IgA against proteolytic degradation)

Secretory Piece

J Chain

IgA • Structure • Properties – Found mainly in serum, external secretions (such as: colostrum, milk, saliva) – 2nd highest serum Ig – Major secretory Ig – A first line of defence against microbial invaders at mucosal surfaces

IgM J Chain

• Structure – Pentamer – Extra domain (CH4) – J chain (joining chain)

Tail Piece

CH4

IgM • Structure • Properties – – – –

Found primarily in plasma, the surface of B cell 3rd highest serum Ig, <10% of serum Ig First Ab produced in an immune responce B cell surface Ig: serves as a membrane receptor

B Cell Antigen Receptor (BcR)

Ig- Ig-

IgD • Structure – Monomer – Tail piece

• Properties – 4th highest serum Ig – B cell surface Ig: serves as an antigen receptor

Tail Piece

IgE • Structure – Monomer – Extra domain (CH4 )

• Properties – Least common serum Ig – Associated with acute inflammation, parasitic infections and allergic reactions(such as asthma)

CH4

Biological properties of Antibody • Combine with antigen: Agglutinating bacteria, neutralizing toxins, inactivating viruses.

• Fixing and activating complement: IgG and IgM – Lead to complement mediated lysis of the microbes or the target cells; also lead to attraction of immune cells, and to opsonization and phagocytosis of the target cells.

• Placental transfer: IgG4

Biological properties of Antibody(continued) • Binding to Fc receptor: – ADCC(antibody-dependent cellular cytotoxicity) ： NK cells can kill Ab coated target cells directly.

– Opsonization ： Binding to FcR of phagocytes can enhance phagocytosis.

– Causes type I allergy ： Binding IgE Fc to FcR of mast cells/ basophils, can trigger degranulation and enhance the acute inflammatory response. Over-stimulation of mast cells/ basophils by this mechanism leads to type I allergy.

Monoclonal antibodies (mAb) • Polyclonal antibodies • Monoclonal antibodies: – monospeific – are standard research reagents and have siginificant clinical utility.

• Immune animals or humans • Fusion of an immortal cell with a specific predetermined Ab-producing cell • The hybridoma cell is immortal and synthesizes homogeneous, specific mAb which can be made in large quantities