Microsoft PowerPoint - ANTIBODY

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1 Antibodies: Structure and Function IgM, the 1 st responder

2 Basic structure of Abs

3 Electrophoresis of serum proteins (Tiselius & Kabat, 1939) Immune sera Removed by Ag

4 globulin ( G) Immunoglobulin (Ig): IgG, IgM, IgA, IgE, IgD Antibody (Ab)

5 Basic structure of Ab molecules (H) (L) IgG: IgM: IgA: IgD: IgE: V: variable C: constant

6 Papain digestion of an IgG molecule (Porter, 1950s) mw 150 kda Fab fragment: antigen binding mw 45 kda Fc fragment: crystallizable mw 50 kda

7 Pepsin digestion of an IgG molecule (Nisonoff, 1950s) mw 100 kda (multiple fragments)

8 Mercaptoethanol reduction of an IgG molecule 2 x mw 25 kda mw 50 kda

10 Antibody to the Fab fragment could react with both the H and L chains, whereas antibody to the Fc fragment reacted only with the H chain. Fab consists of portions of an H and a L chain. Fc contains only H chain components.

11 Obstacles to Ab sequencing

13 Ig fine structure

14 An Ab molecule 1 heavy chain 1 heavy chain 2 light chains

15 Heavy and light chains are folded into Domains (IgG, IgD, IgA) (IgM, IgE)

16 Immunoglobulin Domains - each contains about 110 a.a. residues and an interchain disulfide bond that forms a loop of 60 a.a. Variable-Region Domains - hypervariable (HV) regions: (15% - 20% of the variable domain) = complementarity-determining regions (CDR) CDR1, CDR2, CDR3 - framework regions (FR) Constant-Region Domains Hinge Region

17 Associations between domains of an Ab molecule

18 Complementarity Determing Regions Complementarity- Determining Regions

19 Variability of a.a. residues in the V H and V L domains

20 Ab-mediated effector functions A

21 - Antibodies generally do not kill or remove pathogens solely by binding to them. - While V regions bind to Ag, the C H region is responsible for a variety of collaborative interactions with other proteins, cells, and tissues that result in the effector functions of the humoral responses.

22 Ab-Mediated Effector Functions - Abs activate complement ( 補體 ) (Chapter 13) - Opsonization ( 調理作用 ) is promoted by Ab. - Antibody-dependent cell-mediated cytotoxicity (ADCC) kills cells (Chapter 14). - Some Abs can cross epithelial layers by transcytosis

23 Activation of Complement (C) Complement Components: 1. More than 30 soluble and cell-bound proteins 2. Participate in both innate and acquired immunity 3. Produced by hepatocytes, monocytes and epithelial cells of the gastrointestinal and genitourinary tracts 4. Constitute 5% (by weight) of the serum globulin fraction 5. Circulate in the serum in functionally inactive forms 6. Many components are proenzymes, which are inactive until proteolytic cleavage, which removes an inhibitory fragment and exposes the active site.

24 Functions of Complement (Figure 13-1)

25 Opsonization - the promotion of phagocytosis of Ags by phagocytes - Ab promotes opsonization through a binding to Fc receptors (FcR)

26 Opsonization by Ab and complement (Figure 13-12)

27 Antibody-dependent cell-mediated cytotoxicity (ADCC) (Figure 14-15)

28 Transcytosis - movement of Ab across epithelial layer - delivery of IgA to the mucosal surfaces of the respiratory, gastrointestinal, and urogenital tracts, as well as its export to breast milk - transfer of IgG from mother to fetus (passive immunization)

29 Formation of Secretory IgA Transcytosis

30 Secretory IgA in Breast Milk Bind to microbes in baby s digestive tract and thereby prevent their attachment to the walls of the gut and their subsequent passage into the body s tissues.

31 Ab classes and biological activities

32 5 major classes of secreted antibody

33 IgG - most abundant in serum - 80% of total serum Ig - 4 IgG subclasses

34 4 subclasses of human IgG - size of the hinge region - no. & position of the interchain -S-S- bond - IgG1>IgG2>IgG3>IgG4 in serum conc. - 90% - 95% homologous in DNA sequences

35 IgM Pentamer - monomer on the membrane & pentamer in secretion - 5% - 10% serum Ig - 1 st Ab in neonates - 1 st Ab in primary response - more efficient in agglutination & C fixation - J (joining) chain allows IgM to transport across the epithelium. - also present in secretions

37 IgA Dimer - 10% - 15% of total serum Ig - monomers, dimers, trimers and tetramers in serum - predominant in external secretions, e.g., breast milk, saliva, tears, and mucus of the bronchial, genitourinary, and digestive tracts

38 Secretory IgA Dimers and tetramers in secretion with a secretory component

40 Formation of Secretory IgA Transcytosis

41 IgE - potent biological activity - extremely low conc. in serum - mediates the immediate hypersensitivity reactions - responsible for the symptoms of hay fever, asthma, hives, and anaphylactic shock

42 Allergen cross-linkage of receptorbound IgE on mast cells - induces degranulation, causing release of substances that mediate allergic manifestations

43 IgD - 0.2% of total serum Ig - together with IgM, is the major membrane-bound Ig on mature B cells - thought to function in the activation of B cells - no biological effector function has been identified

45 Active and Passive Immunity

46 Passive Ab therapy Behring & Kitasato (1890): IVIG: i.p. injection of anti-tetanus immune serum in mice can protect animals from tetanus infection. (Behring: Nobel prize in Medicine 1901) intravenous immune globulin

48 Passive immunity Source of antibody 破傷風 Serum Large animals Human

49 Antigens Recognition

50 Antigenic determinants on Igs

51 3 Antigenic Determinants of Immunoglobulins Isotypes Allotypes Idiotypes

52 Isotypic Determinants - constant-region determinants that collectively define each H-chain class and subclass, and each L-chain type and subtype within a species

53 Allotypic Determinants - differences in amino acids in C regions, which occur in some, but not all, members of a species

54 Idiotypic Determinants - The unique amino acid sequence of the V regions of a given Ab. - In some cases an idiotype is the actual antigen-binding site.

56 抗體種類 1. 單株抗體 ( monoclonal antibody) 2. 多株抗體 (polyclonal antibody)

57 Monoclonal Abs

58 BA LB/c 小白鼠免疫流程 wk BALB/c Antigen (50 mg/mouse) Emulsified in 0.5 ml Freund's Complete Adjuvant At least three booster shots, same dose in 0.5 ml Freund's Incomplete Adjuvant Booster shots might be reduced if TiterMax is use as adjuvant Trial Bleeding Final booster (soluble antigen) Cell fusion 加佐劑製成乳劑 Titer Determination 試採血

59 免疫後取出脾臟取出脾臟

60 取出脾臟內細胞脾臟為 B 細胞集中地

61 以化學試劑將細胞融合加入 PEG 細胞融合 2 min

63 骨髓癌細胞 (NS-1): NS-1 是穩定的小白鼠 BALB/c 癌細胞株, 除了能在培養基中永續生長外, 尚有一重要特性, 其細胞缺乏兩種核酸代謝的重要酵素 (TK, thymidine kinase 及 HGPRT, hypoxanthine-guanine phospho ribosyltransferase) 一般細胞內的核酸合成路徑有兩條, 當核酸的正常代謝途若被 aminopterin 阻礙, 可由救急途徑 (salvage pathway) 取用 thymidine 及 hypoxanthine 來合成 DNA 但 NS-1 缺乏 TK 及 HGPRT 兩種酵素, 因此在 aminopterin 存在下, NS-1 無法生長 ; 正常細胞 ( 如 B 細胞 ) 則可經由救急途徑繼續生長 HAT 培養基含有 aminopterin, thymidine 及 hypoxanthine,ns-1 在 HAT 中無法生長, 除非經由細胞融合導入 TK 及 HGPRT 兩酵素的基因 ( 可由正常脾臟細胞得來 )

64 1) 若只含 NS-1 (N, N-N...), 則在 HAT 中會因 DNA 合成抑制而死亡 2) 若只含脾細胞 (B, B-B, B-B-B...), 則在體外培養會漸漸死去 3) 可能活下去的細胞 (B-N, B-B-N, N-N-B...) 含有來自兩方的染色體, 同源染色體可能發生基因重組, 互換遺傳訊息 ; 接下去的細胞分裂會排除多餘的染色體, 一直到只剩一組染色體時才告穩定在初期的分裂增殖過程中, 失去含有重要基因染色體的子細胞可能會死去成功穩定的融合細胞株會經由染色體的重組, 得兼有雙方的特性 : 即可分泌抗體, 亦可在培養基 HAT 中生長下去

65 剛融合完成融合後的細胞生長情形一個癌細胞與兩個脾細胞 National Geographic 經過數次分裂後穩定的細胞群落

66 酵素免疫分析法 ELISA Enzyme-Linked Immunosorbent Assay EIA Enzyme Immunoassay 酵素免疫分析法可偵測專一性抗原或抗體

67 96 槽細胞培養盤把融合後的細胞均分在培養盤的小槽中

69 Clonal Selection of B Lymphocytes

70 Production of monoclonal antibodies (mab)

71 Immunotoxins

72 How does toxin or immunotoxin function?

73 Alternatives to Ag-Ab reactions Protein A: a cell-wall protein from Staphylococcus aureus ( 葡萄球菌 ) Protein G: a cell-wall protein in group C and G Streptococcus ( 鏈球菌 ) Avidin: an egg white protein Streptavidin: a protein made by Streptomyces avidinii - Protein A & protein G can bind to the Fc region of IgG molecules with high affinity (Kd ~ 10-8 M). - Avidin (or strptavidin) binds biotin (a vitamin that is essential for fat synthesis) with Kd ~ M.

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<B2A1D4ADC9FACEEFD3EBC3E2D2DF2E733932> 学习目标瞯掌握抗体免疫球蛋白的基本概念及免疫球蛋白的基本结构瞯熟悉抗体的功能及五类免疫球蛋白的特性瞯了解人工制备的抗体类型抗体 (antibody,ab) 是 B 细胞识别抗原后增殖分化为浆细胞, 由浆细胞产生的一类能与相应