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1、Chapter 5 Immunoglobulin,Contents,Introduction Section Molecular Structure of Ig Section Characteristics and Functions of the 5 Classes of Ig Section Fc Receptors for Ab Molecules Section Biological Activity of Ab Section Immunogenicity of Ig Section Artificial Ab,Concepts,Antibody (Ab): Glycoprotei
2、n molecules 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
3、 activity of Ab or show a similar structure to Ab Therefore, All Abs are Igs, but not all Igs possess the functions of Abs,Other Concepts,- Globulin Antiserum Humoral Immunity sIg and mIg(BCR),Section Molecular Structure of Ig,. Basic structure,Ig is composed of four polypeptide chains joined by S-S
4、 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 c
5、hains 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 IgG4,IgA1, IgA2,(2) According t
6、o 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) Hinge region,(2) Variable region (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 th
7、e 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,(2) Variable region ( V ):,Hypervariable region(HVR) There are three highly diversity stretches within the V egion, they are called HVR. Framework reg
8、ion(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), th
9、e 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 binding to antigenic determinants. Sensitive to proteolytic enzyme IgM, IgE,Other structures of Ig,Joining chain(J),Secreto
10、ry 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 proteolysis in extra secretory liquid.,IgA,. Domains of Ig,1
11、. Domain :,Polypeptide chains of Ig are folded into a globular 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(45): VH, CH1, CH2, CH3 CH4(in IgM,IgE) hinge region,3. Function of each domain,VH, VL: antigen-binding site
12、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,. Hydrolytic fragments of Ig,Ig can be digested by papain and pepsin Position Fragments Function,1. Diges
13、ted 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 Po
14、sition: 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 between the structure and function of Igs Decrease the immunogenicity of Ig for clinical treatment,Section Characteristics a
15、nd Functions of the 5 Classes of Igs,. 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
16、5 years old,6. Functions of IgG: Against bacteria and virus,neutralize toxin Combine with the Fc receptor(FcR) Activate complement Combine with SPA Some belong to the auto-antibodies Take part in type and hypersensitivity,. IgM,1. Highest MW：pentamer（90 KD）,10 valences,2. Half-life is shorter(45 day
17、s) 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 receptors(BCR),4. Functions:,IgM is more effective in binding Ag and activating C, and play an important role in anti-infection Natural Ab for bloo
18、d-type antigen Auto-antibody: rheumatoid factor(RF) Take part in type and hypersensitivity,. IgA 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 imm
19、unity,Immune barrier Neutralize virus/toxin Rich in colostrum Activate C by alternative pathway Take part in type hypersensitivity,. IgD,1. 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,. IgE,1.
20、Concerntration of IgE in serum is the lowest in normal individual, but is very high in some patients. 2.Related to typepersensitivity FcR: mast cell, basophil,Section Fc Receptors for Ab Molecules,IgG-FcR: FcR(CD64)-phagocyte FcR(CD32)-immune complex FcR(CD16)-NK,macrophage,T cell IgE-FcR: FcR- mast
21、 cell, basophil FcR- macrophage, B cell IgA-FcR(CD89)-phagocyte, neutrophil,Section Biological Activity of Ab,1. Recognize and bind to antigen specifically 2. Fix complement 3. Bind to Fc receptor on some cells 4. Transfer selectively : .Planceta transfer (IgG) .Mucosa transfer (sIgA),Affinity and A
22、vidity,Neutralization,IgM,IgG13: classical pathway IgA,IgG4,IgE: alternative pathway,MAC,(1) Opsonization(IgG, IgM): Enhance the phagocytosis of M,(2) ADCC( antibody dependent cell mediated cytotoxicity),(3) Hypersensitivity type - mast cell, basophil(FcR),Section Immunogenicity of Ig,Isotype: CH, C
23、L,Allotype：CH, CL,Idiotype: VH, VL Anti-idiotype antibody,Section Artificial Ab,Polyclonal Ab Monoclonal Ab Gene engineering Ab,1. Polyclonal Ab,A mixture Ab with different specificities and affinities Generate in a natural response or artificial immunization Cross reaction,Cross-reactivity: if two
24、antigens share an epitope an antibody recognizes an unrelated, but chemically similar, epitope,2. Monoclonal Ab (mAb),Ab produced by single clone (or one hybridomas clone ) and having a single specificity,mAb / McAb,Prepared by hybridomas technique: Immunized spleen cells(B) hybride with myeloma cel
25、ls-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 specific
26、ity,Enable the development of secure immunoassay systems.,NOT Powerful tools for clinical diagnostic tests,Cell fusion technique：,Hybridoma cell,No Ab Long life,Secrete Ab Short life,Secrete Ab Long life,Khler and Milstein, 1975,1984, Nobel Prize,Screening of hybridoma cell:,Hybridoma cell,S/S,M/M,M
27、yeloma cell,splenocyte,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 medium:,H, 次黄嘌呤; A, 氨基喋呤; T, 胸腺嘧啶,A,(Hypoxanthine guznine phosphoribosyl transferase) 次嘌呤鸟嘌呤磷酸核糖转移酶 ( HGPRT ),胸腺嘧啶激酶 ( TK ) (Thymidine kinase),B cell： HGPRT+，
28、TK+,Myeloma cell： HGPRT-， TK-,live,die,Extrinsic pathway(minor),PRODUCTION OF MONOCLONAL ANTIBODY,HYBRIDOMA TECHNOLOGY,Applications of Monoclonal Antibodies,Diagnostic Applications Biosensors & Microarrays Therapeutic Applications Transplant rejection Muronomab-CD3 Cardiovascular disease Abciximab C
29、ancer Rituximab Infectious Diseases Palivizumab Inflammatory disease Infliximab Clinical Applications Purification 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.
30、mRNA c. Cell Surface,3.Gene engineering Ab,Abs prepared by the method of gene recombination 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,Denmark Basel Institute for Immunology Basel, Switzerland,Argentina MRC Laboratory of Molecular Biology Cambridge,