Department of Immunology Room 225, Building of Basic Medicine
Chapter 4 Immunoglobulin (Antibody) Xing-cheng WEI () Tel (office) Department of Immunology Room 225, Building of Basic Medicine Introduction von Behring (German) Found Ab [1890] Passive immunization
Nobel prize [1901] 1890Koch, BehringKitasato,,antibody.1891BehringBehring1901 von Behring (German) Found Ab [1890] Passive immunization
-Throughout history, diphtheria was a leading cause of death among children, and it was once referred to as the strangling angel of children. 173517401080% -If diphtheria has become a rare disease today, we owe it to the efforts of Emil von Behring, who discovered the antitoxin to cure diphtheria. Found Ab [1890] Passive immunization Nobel prize [1901] 1890Koch, BehringKitasato,,antibody.1891BehringBehring1901 Bacillus diphtheria and the disease
1890Koch, BehringKitasato,,antibody.1891BehringBehring1901 Difficulty of breathing with diphtheria In 1940, USA, elementary school children Against diphtheria (A) goat anti-rabbit as the secondary antibody (green),
*cryptocidin (epithelia ofintestineantimicrobial peptides. To sterilize lumen (the killing mechanisms not known). Immunocytochemistry analysis of HeLa cells detected withSTAT6 Recombinant Rabbit MAb and using (A) goat anti-rabbit as the secondary antibody (green), (B) the nuclear stain (blue) (C) stain actin (red). (D) A composite image of cells shows cytoplasmic localization of STAT6. Antibody (Ab) A group of structurally related glycoproteins produced by B cells, whichspecificallybindto their correspondingAgsandmediate specific humoral immune responsesto eliminate the bound Ags. *cryptocidin (epithelia ofintestineantimicrobial peptides. To sterilize lumen (the killing mechanisms not known). Immunoglobulin (Ig) *Ig:
The molecules that perform Ab activity or that no Ab function but possess the similar structure with an Ab. *Ig: -Ab (functional, in blood & other fluids) -BCR (B Cell Receptor), mIg (Membrane Immunoglobulin) -sIgA (Secretory Immunoglobulin) *cryptocidin (epithelia ofintestineantimicrobial peptides. To sterilize lumen (the killing mechanisms not known). Immunoglobulins Plasma cells Ab-Ag BCR/mIg-Ag BCR/mIg Memory B Sec.1 Molecular Structure of Ig I. Basic Structure of Ig 1. Heavy Chain And Light Chain (1) H Chain: -450-550 amino acid residues.
(2) L Chain : -214 amino acid residues. H-L linked by disulfide bond. N Linked by S-S Linked by S-S *cryptocidin (epithelia ofintestineantimicrobial peptides. To sterilize lumen (the killing mechanisms not known). C (3) Domain of Ig Structure of IgG revealed by X-ray crystallography
[Ig folding] -A globular motif. -2 Anti-parallel b sheet. -110AA. -IgSF: any molecule that contains the Ig folding. Domain=functional area
[L Chain] VLCL [H Chain] (IgGIgAIgD) VHCH1CH2 CH3 (IgMIgE) CH3CH4 CH4 (4) Class IgA IgD IgE IgG IgM a a d d g g e e m m
-Class is distinguished according to the structure of H chain CH of Ig molecules. -There are 5 different H chain (g, a, m, d, e) in a body, witch make up 5 classes of Ig molecule (IgA, IgD, IgE, IgG, IgM). 5 Classes of Ig Processes of B Cell Mediated Immune Responses
Activation Proliferation Differentiation (5) Subclass -IgG1 -IgG2 -IgG3 -IgG4 IgG -IgA1 IgA -IgA2 Ig
-IgG and IgA are divided into subclass. -According to the difference of H chain constant region (CH). -IgG1 -IgG2 -IgG3 -IgG4 IgG -IgA1 -IgA2 IgA Ig (6) Type k k l l Type of the Ig is k Type of the Ig is l
-According to the difference of CL, L chains are divided into 2 kinds (k or l). -The L chain of a Ig decides its type. k k l l Type of the Ig is k Type of the Ig is l Class Subclass CH CL IgG IgG1-4 k/l IgA IgA1-2 IgM - IgD IgE
Type CH CL IgG IgG1-4 k/l IgA IgA1-2 IgM - IgD IgE 2. V and C Region -Ag-binding specificity of a Ab is determined
1 2 3 4 -Ag-binding specificity of a Ab is determined by Ag-binding sites of the Ab molecule, witch complementary bind to Epitopes. -Basic structures of Abs are similar, but the amino acid sequences that compose Ag-binding sites are different. -Thus in a individual, Abs are mixture with different structures. (1) V regions: The regions of variability in
amino acidsequence. (2) C regions: The regions of constancy in amino acid sequence. VH: H chain 1/4 from N end VL: L chain 1/2 from N end CL: L chain 1/2 from C end CH: H chain 3/4 or 4/5 from C end *cryptocidin (epithelia ofintestineantimicrobial peptides. To sterilize lumen (the killing mechanisms not known). VAAIg Ig The investigators who make clear the basic structure of the Ig
Rodney Porter Nobel Prize in 1972 Gerald Edelman Nobel Prize in 1972 (3) HVR / CDR: -The AA residues binding directly to an epitope.
-The most variable portion in a V region. -An epitope-binding HVR consists of 6 short AA sequences. *cryptocidin (epithelia ofintestineantimicrobial peptides. To sterilize lumen (the killing mechanisms not known). 3. Hinge Region Hinge Region -between CH1 and CH2, -a proline rich region.
-permitting flexibility to fit the structure of Ag. N *cryptocidin (epithelia ofintestineantimicrobial peptides. To sterilize lumen (the killing mechanisms not known). C II. Other Components of Ig 1. J chain (Joining chain)
-A polypeptide chain that links 5 IgM monomers to form a pentameric IgM. -A polypeptide chain that link 2 IgA monomers to form a dimeric IgA. Structure of IgM and IgA 2. SP (Secretory piece) -A polypeptide chain that bound to the CHs of a dimeric IgA to protect against proteolytic cleavage. -To form a SIgA (secretary IgA) that enters mucus layers to protects the mucosa. Structure of IgM and IgA Digesting Fragment of Ig
III. Digesting Fragment of Ig Rodney Porter Nobel Prize in 1972 1.Papain Digest Products
-2 Fab(fragment of antigen-binding) -1 Fc (fragment crystalizable) 2.Pepsin Digest Products -1F(ab)2 -n pFc 3.significance -Clarify the functions of Ig. -Prepare functional fragments for clinical or research usage. Sec.2 Functions of Ab Major Functions of Ab 1Neutralization 2Opsonization
3Complement activation 4Mediation of ADCC Activate Complement Bind to FcR
AgBinding Activate Complement Bind to FcR FcR Immune Cell Mechanisms of Neutralization CDR/HVR of a Ab binds to:
-Microbial surface molecules to inhibit the microbe to invade into host cell. -Microbial toxins to inhibit the toxins to interact with host cell. Mechanisms of Ab-Mediated
Opsonization -CDR/HVR of Ab specifically bind to the Ag of microbe. -Fc of Ab(IgG) bind to FcR on Phagocytes (Mf/Neutrophil). -The Abs enhance phagocytosis efficiently. -The Abs mediate a innate immune cell to kill the microbe specifically. Ab Microbe Phagocyte Mechanisms for Abs to Activate Complement
After an Ab binding to an Ag, the CH changes theconformationthatcanbeboundby C1,so as toinitiatecomplementactivation. Complementactivationgeneratesimportant effectors of both innate and adaptive immune responses. Mechanisms of ADCC (Ab-Dependent Cell-Mediated Cytotoxicity)
-CDR/HVR of Ab specifically bind to Ag of target cell (e.g. virus infected cell). -Fc of the Ab binds to FcR on NK cell. -The Abs enhance NK to kill the target cell efficiently and specifically. NK Cell FcR Mf or NK Catch Target Cell
by FcR-Ab-Ag Binding Phagocyte or NK cell Sec.3 Features & Functions of Each Ig Class Major Characteristics
Class Serum conc [mg/ml] Half life [day] Major Characteristics IgA 3.5 6 SIgA is the only Ab that is secreted into mucus layer to protect mucosa (mediate mucosal immune) -External secretion alimentary tract respiratory tract lacteal gland salivary gland tear gland -Colostrum (neonate) Generation & Secretion
of SIgA Secreted IgA is transported through
epithelial cells by the poly-Ig receptor SIgA is transported into the intestinal lumen by an IgA specific Fc receptor Characteristics Class Serum conc [mg/ml] Half life [day] IgD trace 3
mIg (BCR) Marker of mature B cell -IgM+IgD+ B cell. IgE 0.05 2 Mediate type I Hypersensitivity BCRs on mature B Cell Consist of both IgM and IgD Characteristics Class Serum conc [mg/ml] Half life [day] IgG 13.5 23
Most rich in blood. Longest half life. Major Ab in 2nd immune response. Highest Affinity. Pass placenta. Primary and Secondary Humoral Responses ()
Log 100000 10000 1000 100 10 1 IgG IgM Placental barrier Placental barrier FcRn transmit IgG through placental or intestinal cells
*FcRn: FcgR, intracellular trafficking receptor n: neonate Pentamer is strong in: The marker of immature B cell
IgM 1.7 10 Produced earliest in immune responses -1st line defense against infection. -Early diagnosis of infection. Produced earliest in ontogenesis -diagnosis of intra-uterine infection (Cord Blood) Pentamer is strong in: -Ag binding ability. -Complement activation. The marker of immature B cell -IgD-IgM+ B is a immature cell that resides in bone marrow. Primary and Secondary Humoral Responses
IgM Log - - 100000 10000 1000 100 10 1 IgG IgM Only 1 IgM can initiate complement Activating reaction
10 Ag-binding sites of 1 IgM Only 1 IgM can initiate complement Activating reaction Pentamer IgM is strong in Ag binding and complement activation The Cell that Only Expresses IgM on surface is a Immature B Cell
IgD has not yet been expressed on the cell surface Sec.4 Preparation of Ab -Polyclonal Ab [pAb] -Monoclonal Ab [mAb] -Genetic engineering Ab mAb [Hybridomas] -Proliferation in vitro or in vivo
By cell fusion between a normal B cell and a myeloma cell to get the hybridomas that secret specific Abs to one kind of epitopes. [Hybridomas] -Proliferation in vitro or in vivo -Secret Ab in vitro or in vivo [mAb] Single epitope specific Uniform High purity Low cost of preparation Genetic Engineering Antibody
The Abs expressed by recombined Ig gene that was deleted, spliced or revised before transferred into cells. -human-mouse chimeric antibody. - -reshaped humanized antibody. -small molecular antibody Fab fragment Fcfragment Fvfragment -bispecific antibody -Ig-fusion protein Ig- -antibody-directed enzyme [The main points] 1.Concept: (1) Monoclonal Ab;
(2) Genetic engineering Ab; 2.Functions of Ab (Ig). 3.Characteristics of: (1) IgA (2) IgD (3) IgE (4) IgG (5) IgM