Immunoglobulin And Antibody Xiaojian wang, Ph.D. Institute of Immunology ZJU School of Medicine Tel: IMMUNOLOGY

immunoglobulin 1. Introduction 2. The structure of immunoglobulins 3. Heterogeneity of immunoglobulins 4. Functions of immunoglobulins 5. The structures and properties of immunoglobulin classes 6. Production of antibodies

immunoglobulin Antibody(Ab) Globulin produced by plasma cells in response to antigen and binds specifically to particular Ag. Ab is generally defined in terms of their specific binding to the immunizing Ag Immunoglobulins (Ig) The Igs are globulin which function as Abs or similar to Abs in chemical structure.

Not all of Ig have the functions of Ab, e.g. Patients with multiple myeloma have very high levels of Ig, but not Ab (to irrelevant things)) Secreted Ig (sIg)- mediated humoral immunity Membrane Ig (mIg): BCR B cell receptor immunoglobulin

Electrophoretic mobility of serum proteins

1. Heavy Chain and Light Chain heavy chain (H chain) 50-75Kd aa H chains: , , , ,  Isotypes: IgM, IgG, IgA, IgE, IgD Light chain (L chain) 25Kd 214aa Types of L chains: , immunoglobulin I. Basic structure of the immunoglobulin

In any given Ig, all the H chains are identical. An antibody molecule has either two  light chains or two light chains, but never one of each. In humans, about 60% of Ab molecules have  light chains, and 40% have light chains. Market changes in this ratio can occur in patients with monoclonal B cell tumors. immunoglobulin

2. Variable region and constant region 1) Variable region (V region) Light Chain - VL (110 aa) Heavy Chain - VH (110 aa) immunoglobulin

HVR (hypervariable region): 9-12aa, 3 HVRs CDR (complementarity-determining region): CDR1, CDR2, CDR3 FR (framework region): 15-30aa

immunoglobulin CDR binds with epitope of antigen Antigen peptide

2) Constant region (C region) Light chain - CL (110 aa) Heavy chain - CH ( aa) CH1, CH2, CH3, (CH4) immunoglobulin

3. Hinge Region - between the CH1 and CH2 region - rich in proline residues flexibility: allow the two Fab arms to bind epitope; be cleaved by proteases. - IgM and IgE have no hinge region

Structure of the immunoglobulins immunoglobulin

3D images of the Ig molecule show that it is not straight as depicted in Figure. It is folded into globular regions, each of which contains an intra-chain disulfide bond. These regions are called domains. Light Chain Domains - VL and CL Heavy Chain Domains -VH, CH1, CH2, CH3 IgA, IgD,IgG (CH4 in IgE and IgM) 4.Domains of immunoglobulin immunoglobulin

Downloaded from: StudentConsult (on 1 June :58 PM) © 2005 Elsevier Binding of an antigen by an antibody.

immunoglobulin Functions of the domains on Ig VH 和 VL: 抗原结合部位 CH1-3 和 CL: Ig 遗传标志 CH2(IgG) 、 CH3(IgM): 补体结合部 位 CH2-CH3(IgG): 结合并通过胎盘 CH3(IgG) CH4 (IgE) ： FcR 结合部 位 Fab Fc

II. Other components in the Ig 1. Joining chain J chain is a small acidic protein that is synthesized by all plasma cells that secrete polymeric Igs (i.e. IgM and sIgA). Join the momomer to polymer immunoglobulin

2. Secretory piece (SP) Secretory component (SC) is a single glycopeptide with MW of 70KD. Produced by mucosa epithelial cells Functions: protect sIgA, resist proteolysis in extra secretory liquid. immunoglobulin lumen

III. Immunoglobulin fragments Fab(fragment of antigen binding) Fc (fragment crystalizable)

immunoglobulin Heterogenecity of Ig Characterist of Ig 1. Binding to antigen 2. Immunogenicity

Isotype Isotypes are the antigenic features of a class of Ig H (e.g.  chain is different from  ). Isotypes of one species can induce immune response in another species. immunoglobulin Heterogenecity of Ig

Classes and subclasses of Ig IgG-  (gamma) IgA-  (alpha) IgM-  (Mu) IgD-  (delta) IgE-  (epsilon) subclass IgG1~IgG4, IgA1~IgA2,IgM1~Igm2 Type and subtype Type: κ(Kappa) λ(lambda) subtype: λ1~λ4 immunoglobulin

Allotype immunoglobulin This refers to genetic variation between individuals within a species. CH 和 CL 上的一个或数个氨基酸残基的差异 --- 遗传标志 Gm1-30

Idiotype (Id) immunoglobulin Variation in the V domain,particularly in CDR, produces idiotype.

Functions of immunoglobulins

Functions of V regions Recognition and binding to antigen - HVR (CDR) - neutralization of toxins (e.g. diphtheria, tetanus); - immobilization of microorganisms; - neutralization of viral activity Antibody: monomer(IgG,IgE)---2 价 dimer(sIgA) 价 Petamer(IgM) 价 immunoglobulin

Neutralization

neutralization of viral activity

Function of C regions (Fc portion) 1. Activation of complement: IgM, IgG Ab( IgM 、 IgG )+Ag--- C1q---- 补体经典途径 IgG4 、 IgA 和 IgE 的凝聚物 ---- 补体旁路途径 2. Binding to Fc receptor of cells  Opsonization: enhancement of Ag uptake by macrophages (M  )  Antibody-dependent cell-mediated cytotoxicity (ADCC): NK cells.  Participation in type I hypersensitivity: IgE 3. Passage through the placenta (IgG, CH2) and mucosa (sIgA) immunoglobulin

Opsonization of antibody

immunoglobulin Antibody-dependent cell-mediated cytotoxicity (ADCC)

passage through the placenta(IgG) and mucosal(SIgA) Passage through mucosal(SIgA)

Function of immunoglobulins immunoglobulin

Characteristic and function of Different class of Ig

(1) 单体分子； (2) 四个亚类； (3) 血清中含量最高 (75% Ig ) ； (4) 半衰期最长 (20 ～ 23 天 ) ； (5)3 ～ 5 岁达成人水平 (8.0 ～ 17 mg/ml ) ； (6) 可与 SPA 结合。 (7 ）唯一能通过胎盘的 Ig IgG immunoglobulin

IgG immunoglobulin 1.Fixes complement –IgG1 and IgG3 fix complement well; IgG4 does not fix complement. 2. Opsonization: enhancement of Ag uptake by macrophages (M  ) 3. Antibody-dependent cell-mediated cytotoxicity (ADCC): NK cells 3. Crosses the placenta IgG is the only class of Ig that can crosses the placenta and it confers a high degree of passive immunity to the newborn

IgM immunoglobulin 1. Penatmer 900kd Macroglobulin 2. IgM is the first Ig synthesized in life 3.IgM is the first Ig produced in primary immune response. 4. IgM is a good complement fixing Ig 5. IgM is also a good agglutinating Ig 6. IgM is mIgM ( IgD) of B cells : 7. Half time: 5 Days

IgA immunoglobulin 1.IgA : monomer and secretary serum IgA (IgA1) monomer secretory IgA (IgA2) dimer 2. sIgA exists in secretions such as tears, saliva, colostrum, mucus and has important role in local (mucosal) immunity. 3. IgA can be transmitted from mother to newborn

IgD immunoglobulin 1.IgD is found at low levels in serum; its role in serum remains unclear. 2.IgD is primarily found on naive B cell surfaces where it functions as a receptor for antigen. 3. Membrane IgD disappear in activated or memory B cell

IgE immunoglobulin 1.IgE is the least common serum Ig. It binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen. 2.Involved in allergic reactions. 3.IgE also helps to clear parasites (e.g. worms) by eosinophils.

Five Classes/Isotypes

Production of antibodies immunoglobulin 1) Polyclonal antibody 2) Monoclonal antibody 3) Genetic engineering antibody

bearing more than one epitopes Ag B cells Clonal proliferation Polyclonal antibody Immunizing mouse pAb

Georeges Kohler and Cesar Milstein, 1984, monoclonal antibody immunoglobulin

Monoclonal antibody production

Human Monoclonals? It would be very nice to have available human monoclonals - much more useful therapeutically Problems Hard to get antigen-primed B cells In vitro primed SCID mice Knock-out mice No good immortal partners Can sometimes transform antibody-secreting B cells (EBV) immunoglobulin

Genetically engineered antibody  Chimeric Ab Mouse V gene + human C gene vector expression  CDR-grafted Ab Mouse CDR gene human framework region  Bispecific antibody immunoglobulin

Chimeric Ab mouse Ab CDR-grafted Ab immunoglobulin

Being Used Now... Humanized antibodies, where mouse CDR regions are combined with human framework and constant regions Anti CD20 (B cell antigen) against non-Hodgkins Lymphoma Anti-HER2 (human epidermal growth factor receptor 2) against breast cancer - Herceptin More than 200 such drugs in clinical trials immunoglobulin

1. Which of the following antibodies contains Joining chain ( ) A. IgG1 B. IgG2 C. sIgA D. IgE E. IgD Sample questions 2. Which of the following immunoglobulins can cross the placenta in humans------( ) A.IgA B.IgM C.IgE D.IgG E.IgD Answer=D Answer=C 3. Describe the structure of an Ig molecule and how this structure enables it to bind to a specific antigen.