医学免疫学英文教学课件：Chapter 5 Immunoglobulin

1、 Chapter 5 Immunoglobulin(Ig) ContentsIntroductionPart Molecular Structure of IgPart Immunogenicity of IgPart Biological Activity of AbPart Characteristics and Functions of the 5 Classes of IgPart Artificial AbIntroductionAntibody (Ab): Glycoprotein molecules that are produced by plasma cells and ca

2、n combine with the corresponding Ag specifically are called Ab. Ab is produced by B cells in the response to stimulation of Ag. Ab possesses a high degree of specificity and affinity Immunoglobulin(Ig): It refers to all globulins that possess the activity of Ab or show a similar structure to Ab. The

3、refore, All Abs are Igs, but not all Igs possess the functions of Abs.Other ConceptsAntiserum (immune serum) Humoral Immunity sIg(Ab) and mIg(BCR)Part Molecular Structure of Ig Vase. Basic structure Ig is composed of four polypeptide chains joined by disulfide bonds. inter-chain disulfide bonds (S-S

4、) 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 chains attach to H chains from “N” end “N” “C

5、”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 IgEsubclassesIgG1 IgG4IgA1, IgA2NNCSubclass: According to differences of the amino acid composit

6、ion and number or position of disulfide bonds in hinge region IgG-IgG1,IgG2,IgG3,IgG4 IgA-IgA1,IgA2 (2) According to the differences of L chains Two types of L chain: , : 20:1 (in mice); 2:1 (in human)subtypes 1 43. Two regions of each peptide chain(1) Constant region (C)(3) Hinge region(2) Variable

7、 region (V) (1) Constant region ( C )CH: (,) or 4/5 (,) of H chain from the c end CL: 1/2 of L chain from the c end (2) Variable region ( V )VH: 1/4 (,) or 1/5 (,) of H chain from the N end VL: 1/2 of L chain from the N end 3. Two regions of each peptide chain (2) Variable region ( V ): Hypervariabl

8、e region(HVR) three short stretches in the V region of H and L chains called HVR. Framework region(FR): FR1-FR4Ag-binding sitesComplementarity determining region(CDR)The three HVR of L chain and H chain can be brought together to form antigen-binding surface, so HVR is also called CDR.(2) Variable r

9、egion (V) Complementarity determining region(CDR) L: CDR1, CDR2, CDR3 H: CDR1, CDR2, CDR3 Idiotype of Ig Igs produced by different B cell clones possess unique structure respectively in hypervariable region (HVR), the unique structure of Ig is called idiotype or idiotypic determinant.In fact: HVR CD

10、R 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 enzymeOther structures of Ig Joining chain(J) Secretory piece(SP)Joining chain(J) : Produced by plasma cells Functions: linker, to compose dimer, pen

11、tamer or polymer (IgA, IgM)Secretory piece(SP): . Produced by mucosa epithelial cells . Secretory IgA (sIgA) . Functions: protect sIgA, resist proteolysis in extra secretory liquid. Domains of Ig 1. Domain: Polypeptide chains of Ig are folded into a globular structure by intra chain s-s bond within

12、each 110aa region which is called a domain -S-S-CL-S-S-CL-S-S-S-S-CL-S-S-CL-S-S-S-S-CL-S-S-CL-S-S-S-S-S-S-S-S-S-S-CL-S-S-CL-S-S- -S-S-V-S-S-V-S-S-CL-S-S-CL-S-S- -S-S-V-S-S-V-S-S-CL-S-S-CL-S-S-S-S-V-S-S-V-S-S-S-S-S-S-S-S-CLCL-S-S- -S-S-S-S-S-S-CLCL-S-S- -S-S-V-S-S-V-S-S-C2C2-S-S-S-S-C2-S-S-C2-S-S-C2-

13、S-S-C2-S-S-V-S-S-S-S-V-S-S-S-S-C2-S-S-V-S-S-V-S-S-V-S-S-VC2C2-S-S-S-S-C2C2-S-S-S-S-C2C2-S-S-S-S-C2-S-S-C2C2-S-S-S-S-C2C2C2C2C2C2C2-S-S-S-S-S-S-S-S-S-S-S-S-S-S-CD2LAF-3CD3CD4CD8VCAM-1ICAM-1ICAM-2IgR g d eMHC-IMHC-IIb2m （IgM）TCRIga/bIg Superfamily (IgSF)All molecules that contain Ig-like domain belong

14、 to IgSF.2. Domains of IgL chain(2) : VL, CLH chain(45): VH, CH1, CH2, CH3 CH4(IgM,IgE)3. Functions of each domain VH, VL: antigen-binding siteCH, CL: allogeneic markerCH2/CH3: complement-fixing site permeate placenta(IgG) CH3/CH4: cell-binding site . Hydrolytic fragments of IgIg can be digested by

15、papain and pepsinPosition Fragments Functionpapainpepsin1. Digested by papainPosition: near the S-S bonds of H inter-chains from the N end Fragments: 2Fab:fragment of antigen-binding Fc:fragment crystallizable Function: Fab: recognize and bind Ag Fc: (1) fix complement (2) crossing the placenta (3)

16、bind to FcR in different cells papainAg:Ab ratio 2. Digested by pepsinPosition: near the S-S bond of H inter-chains from the C endFragments and function : F(ab)2: bind antigen(2 valence) pFc: no function pepsinSignificance Elucidating the relationships between the structure and function of Igs. Decr

17、ease the immunogenicity of Ig for clinical treatment.Part Immunogenicity of IgIsotype: CH, CLIsotype of antibody:Immunize RabbitMouse IgG1Rabbit anti-mouse IgG1Secondary AbAllotype：CH, CLIdiotype: VH, VLAnti-idiotype antibodyPart Biological Activity of Ab1. Recognize and bind to antigen specifically

18、 2. Fix complement3. Bind to Fc receptor on some cells NeutralizationIgM,IgG13: classical pathwayIgA,IgG4,IgE:alternative pathwayIgG-FcR: FcR(CD64)-phagocyte FcR(CD32)-immune complex FcR(CD16)-NK,macrophage,T cellIgE-FcR: FcR- mast cell, basophil FcR- macrophage, B cellIgA-FcR(CD89)-phagocyte, neutr

19、ophil(1) Opsonization(IgG, IgM): Enhance the phagocytosis of M Opsonization: The process of attaching opsonins(such as IgG and complements) to microbial surfaces to target the microbes for phagocytosis.Opsonins: a macromolecule that attaches to the surface of a microbe and can be recognized by surfa

20、ce receptors of neutrophils and macrophages and that increases the efficiency of phagocytosis of the microbe.(2) ADCC(antibody dependent cell mediated cytotoxicity) ADCCAntibody dependent cell mediated cytotoxicity(ADCC): A process by which NK cells or other cells are targeted to Ig-coated cells, re

21、sulting in lysis of the antibody-coated cells. A specific receptor for the constant region of IgG, called FcR (CD16), is expressed on the NK cell membrane and mediates binding to the IgG. (3) Hypersensitivity type - mast cell, basophil(FcR) FceRIdegranulation IgEallergeninflammationhypersensitivity

22、type I- mast cell and basophils (4) Transfer selectively: .Planceta transfer (IgG) .Mucosa transfer (sIgA)Part Characteristics and Functions of the 5 Classes of Igs . IgG 1. Highest concentration in serum(75% of total Ig) 2. Four subclasses: IgG1, IgG2, IgG3, IgG43. Unique Ig that can pass through p

23、lacenta4. Starts to be produced at 2-3 month after birth and reach the level of adult at 5 years old5. Half-life is longer(16-24 days) 6. Functions of IgG:Against bacteria and virus,neutralize toxinCombine with the Fc receptor(FcR)Activate complementCombine with SPA (Staphylococcal Protein A）Some be

24、long to the auto-antibodiesTake part in type and hypersensitivity. IgM 1. Highest MW：pentamer（90 KD）,10 valences 2. Half-life is shorter(45 days)3. The first Ig to be synthesizedAppear in the early stage after infection Be produced during fetusThe first mIg of the B cells, act as the antigen recepto

25、rs(BCR)4. Functions:IgM is more effective in binding Ag and activating C, and play an important role in anti-infectionNatural Ab for blood-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

26、 or polymer2. Two subclasses：IgA1，IgA23. To be produced at 4 months after birth4. Exist in almost all body fluid, rich in colostrum. 6. Local mucosal immunity Immune barrier Neutralize virus/toxin Rich in colostrum (7 days) Activate C by alternative pathway Take part in type hypersensitivity . IgD1.

27、 The concentration in serum is low and sensitive to proteinase2. Act as the antigen receptor on B cells (mIgD): Regulate the differentiation of B cells, the marker of mature B cells. . IgE1.Concerntration of IgE in serum is the lowest in normal individual, but is very high in some patients.2.Related

28、 to typepersensitivity FcR: mast cell, basophil Part Artificial AbPolyclonal AbMonoclonal AbGene engineering Ab1. Polyclonal Ab A mixture Ab with different specificities and affinities Generate in a natural response or artificial immunizationCross reaction 2. Monoclonal Ab (mAb) Ab produced by singl

29、e clone (or one hybridomas clone ) and having a single specificity mAb / McAb Prepared by hybridomas technique: Immunized spleen cells(B) hybride with myeloma cells-hybridomasDefinition: All antibodies which are produced by one B cell clone and only bind to unique antigenic epitope are called monocl

30、onal Ab.Advantage: Identical structure,high fineness,specificity and titerDisadvantage: immunogenecity, cause human anti-mouse antibody(HAMA)Artificial antibodiesDerived from different B Lymphocytes cell linesPOLYCLONAL.MONOCLONAL.Derived from a single B cell cloneBatch to Batch variation affecting

31、Ab reactivity & titremAb offer Reproducible, Predictable & Potentially inexhaustible supply of Ab with exquisite specificityEnable the development of secure immunoassay systems.NOT Powerful tools for clinical diagnostic testsCell fusion technique：Splenocyte(B cell)Myeloma cellHybridoma cellNo AbLong

32、 lifeSecrete AbShort lifeSecrete AbLong lifeKhler and Milstein, 19751984, Nobel Prize1975年Khler和Milstein首先报道用细胞杂交技术将绵羊红细胞（SRBC)免疫的小鼠脾细胞与骨髓瘤细胞融合,建立起第一个B细胞杂交瘤细胞株,并成功地制得抗SRBC的单克隆抗体。 1984年获得诺贝尔奖杂交瘤细胞： 骨髓瘤细胞 无限增殖 B细胞 合成、分泌特异性抗体杂交瘤技术 ： HAT培养基：次黄嘌(H)、氨基蝶呤(A) 和胸腺嘧啶核苷(T)HGPR:次黄嘌呤磷酸核糖转化酶细胞DNA合成有两条途径:主途径是由糖和氨基酸合成核苷酸，进而合成DNA，叶酸作为重要的辅酶参与这一合成过程。另一辅助途径是在次黄嘌呤和胸腺嘧啶核苷存在的情况下，经次黄嘌呤磷酸核糖转化酶（HGPRT）和胸腺嘧啶核苷激酶（TK）的催化作用合成DNA。细胞融合的选择培养基中三种关键成分：次黄嘌呤（hypoxanthine，H）、氨甲蝶呤（aminopterin，A）和胸腺嘧啶核苷（thymidine，T），称为HAT培养基。氨甲蝶呤是叶酸的拮抗剂，可阻断瘤细胞利用正常途径合成DNA，而融合所用的瘤细胞是经毒性培养基选出的HGPRT细胞株