DEPARTMENT OF IMMUNOBIOLOGY Antibody (Ab) Xiaowu Hong Department of Immunology Shanghai Medical College of Fudan University

Emil von Behring, 1901, antitoxins Georeges Kohler and Cesar Milstein, 1984, monoclonal antibody Susumu Tonegama,1987, structure of Ig gene Gerald Edelman and Rodney Porter, 1972, structure of antibody Paul Ehrlich, 1908, production of antibody Nobel Prize winners

Emil von Behring ( ) Emil von Behring, 1901, antitoxins

I Definition of antibody II Structure of antibody III Function of antibody IV Biological characteristics of different antibodies Contents

Antibody (Ab) A globulin which is produced by plasma cell as a result of the introduction of an antigen and which has the ability to combine with the antigen that stimulated its production.

Immunoglobulins (Ig) Globulins composed of H and L chains, or globulins function as antibody. Immunoglobulins Antibody-containing serum is place in an electrical field Antibodies migrated with the globular proteins.

All antibodies are immunoglobulins, but it is not certain that all immunoglobulins have antibody function. The relationship between Ab & Ig

Antibody molecules are found in serum on the surface (account for approximately 20% of the total plasma protein ), in extravascular fluids, in exocrine secretions, and on the surface of some lymphocytes. Distribution of antibody

Section 1 Structure of Ig

* A four polypeptide chains: two identical light chains two identical heavy chains, held by disulfide bonds. ** Y-shape structure, symmetric. *** –NH2 terminal, - COOH terminal. **** variable & constant regions. ***** domains 1 Basic four chain structure

(1)Heavy chain (H): ① Composed of about 500 aa, oligosaccharide(+) ② Class : heavy chain , , , , . immunoglobulin(Ig) IgA, IgG, IgM IgD, IgE (2) Lght chain (L): ① Composed of about 214 aa, oligosaccharide(-) ② Type: ,

2 Variable (V) and Constant (C) regions

(1) V region ① N-terminal 1/2L+1/4(1/5)H; VL, VH

(2) Constant region (C-terminal 1/2L+3/4(4/5)H) CL CH1 CH2 CH3 (CH4)

( complimentarity determining region, CDR ) : formation of the Ag binding site Framework region （ FR ) : maintaining the 3- dimensional configuration (3) hypervarible region (HVR)

( complimentarity determining region,) CDR

purple : HV CDR ( in both the ribbon and ball and stick views) green : antigen HV sequences contact the antigen. antibody antigen antigen-antibody complex:

Representation of the disassociation of an antibody (top) and antigen (botton) molecule. CDR Epitope antibody antigen

(4) Hinge region VHVH CH1CH1 CH2CH2 CH3CH3 CH4CH4 VLVL CLCL Hinge region COO – NH 3 + Properties: 1) Flexible 2) Rich in proline Function: 1) Facilitating the interaction between Ag and Ab 2) Facilitating complement fixation

(IgM CH3 ， IgG CH2) IgG Molecule Conformational Changes Induced by Antigen Binding Fab Fc PREBINDING C H1 C H2 Barricaded C1q-binding site POSTBINDING Exposed C1q-binding site

Flexibility of immunoglobulins

domains: polypeptide chains folded by disulfide bonds into globular regions.

domains: Fc  R binding (mast, basophil) (IgE)CH2+CH3 C1q binding (IgM)CH3 Fc  R binding (MC, M , B,NK) (IgG)CH3 C1q binding(IgG)CH2 Allotypic markerCH1+CL a Ag- binding siteVH+VL

( 胃蛋白酶 ) Fab papain: Fab (Ag-binding Fragment ) Fc (crystallizable Fragment ) Complement fixation FcR 3 Enzymatically generated Ab fragments papain

Figure 3-3

Figure 3-3 part 1 of 2 3 Enzymatically generated Ab fragments (1) Papain : Fab (Ag-binding Fragment ) Fc (Crystallizable Fragment ) : complement fixation, FcR

Figure 3-3 part 2 of 2 ( 胃蛋白酶 ) (2) Pepsin ( Fab’ ) 2 pFc ( peptides of Fc )

Section 2 Biological functions of antibodies

1 Antibody function in the absence of other factors V region: Ag binding Neutralizing toxin & virus Agglutination microbes, Prevention adhesion

Neutralization By Antitoxin Antibodies

Neutralization By Antiviral Antibodies

Bacterial ‘ Neutralization ’ By Ab

C region: Fixation of complement 2 Role of antibodies in complement activation

C region: Binding cells Opsonization Mediating ADCC 3 Role of antibodies binding to effector cells

(1) Opsonization : The process of attaching opsonins, such as IgG or complement fragments, to microbial surfaces to target the microbes for phagocytosis.

antibody complement CD16 （ Fc  R III ） CD16 （ Fc  R III ） CD11b/CD18 CD25 CD28 CD32 （ Fc  R II ） CD35 （ CR1 ） CD64 （ Fc  R I ） CD71 B7-2 IL-2 antibody Recognition of microbes by neutrophils and macrophages complement Lactoferrin Surface receptor on macrophage

Adherence of bacteria via receptors

Opsonin. A macromolecule that becomes attached to the surface of a microbe and can be recognized by surface receptors of neutrophils and macrophages and that increase the efficiency of phagocytosis of the microbe. Opsonins include IgG antibodies, which are recognized by the Fc  receptor on phagocytes, and fragment of complement proteins, which are recognized by CR1.

Fc  R and Complement Receptors Cooperate To Induce Greater Phagocytosis

Fc  RIII CD16 Antibody Marks Target Cells For NK Cell Attack (ADCC) (2) ADCC

Figure 1-24

Figure 1-24 part 1 of 3

Figure 1-24 part 2 of 3

Figure 1-24 part 3 of 3

Section 5 Biological characteristics of different antibodies

interchain disulfide bonds. 1 IgG

(1) Properties (A) IgG is the major Ig in serum - 75% (B) The longest half life (t ½ =23days) (C) IgG is the major Ig in extravascular spaces (D) Placental transfer (E) Fixation complement – (F) Binding to cells –Opsonization mediating ADCC Immunity is transferred from mother to fetus through placental transfer of IgG.

(1) Structure Secreted IgM (sIgM): pentamer Membrane-bound IgM (mIgM): monomer J chain Ig  Ig  Ig  Ig  2 IgM

(1)Chemical nature: polypeptide chain secreted by plasma cell (2) Presence: polymeric Igs such as IgM (pentamer), sIgA (dimer). Joining chain J CHAIN IgM IgA Secrete piece Joining chain

(2) Properties (A) The first Ig made by fetus and B cells (B) Fixation complement –classical pathway (C) The largest size of molecule blood type (D) Natural blood type antibody (E) Binding to cells – Opsonization. mediating ADCC

3 IgA (1) Structure

Secretory Piece ( SC ) synthesized by nonmotible epithelial cells near the mucosal membrane Function: i. Enabling IgA to be transported across mucosal tissues into secretions. ii. Protecting sIgA from being proteolytic attack. IgA dimer Secrete piece Joining chain

(2) Properties (A) The major Ig in secretions. (secretory IgA, sIgA. 5-15g/d) (B) sIgA :transferred to the newborn through colostrum (C) The important antibody against mucosal infections (Local (Mucosal )immunity)

4 IgE (1) Structure

A) The least common Ig B) Binds to basophils & mast cells (Fc  R) (2) Properties

Involved in allergic reactions ( hypersensitivity I)

(1) Structure 5 IgD

(2) Properties a. On the surface of mature B cell serving as BCR mature marker of B cell b. In serum (uncertain Ab activity)

Immunoglobulin Isotypes Are Distributed To Different Parts Of The Body IgM – Blood IgG – Tissues IgA – Mucosa IgE - Surfaces

Master the concepts of Ig and Ab; Master the relationship between structure and functions of Ab; Master properties and biological activities of five classes of Igs. Emphases

School of Medicine Fudan University