Understanding Antibody Structure and Immunoglobulin Classes

1. ANTIBODY/IMMUNOGLOBULIN

2. Antibody structure • Antibodies belong to a class of proteins (glycoprotein) • called immunoglobulins 2. Antibody molecules belong to one of five classes i.e. IgG, IgM, IgA, IgD & IgE 3. Immunoglobulins are “Y” shaped proteins. The “arms” of the “Y” bind antigens. The tail of the “Y” is responsible for biological activity eg. C’ activity or binding to cells 4. Ability of immunoglobulins to bind antigen determined by AA sequence in variable region

3. Antibody Structure N Terminal end Carboxy End

4. Antibody Structure • Antibodies Are Made Up Of: • 2 Light Chains (identical/Shorter) ~25 Kda (214 AA) • 2 Heavy Chains (identical) ~50 Kda • Ig Consist of 2 region • 1. Constant region 2 . Variable region • Each Light Chain Bound To Heavy Chain By Disulfide (H-L) • Based on the Structure of AA sequence Lights chains are Grouped in to 2 Types – • Kappa (K)/ L-type • Lambda (ʎ ) • Approximately 60% Ig are K type • ,, ,, 40 % L type

5. Immunoglobulin Heavy chain H chains • Ig have 2 Heavy chain • They are Longer • Identical • Heavy chain made up of 450-700 AA • Heavy chains are 5 types IgG (Monomeric) g IgA (Dimeric) a IgM (Pentameric) m IgE (Monomeric) e IgD (Monomeric) d

6. Ig Fragments Fab means Ag biding Fragment It consist 2 Identical Fragment Each Fab consist 2 Light 2 heavy chain They are MONOVALENT (Antigen binding site) Fc- Refers Crystallization It include 2 identical Fragment Made up of 2 Heavy chain Involve Complement Fixation Attachment to Phagocytosis

7. Functions of Ig • Agglutination of Ag ( Process of clumping of Particulate • Ag With AB) • Precipitation of Ag (Ig combines with soluble antigenic agent) • Neutralization of Ag (Ag cover the toxin sites of the antigenic • agent and thus NUTRALIZE them ( INACTIVE) • Lysis of Ag (Rupture the cells) • Opsonization ( Process of COATING the BACTERIA with Ab • and making SUSPECTIBLE for PHAGOCYTOSIS. • Activation of Mast cells and basophils • TissueFixation (Attach to the cells)

8. Monomer • Made up of 2 light chain 2 heavy chain (g) • Most abundant immunoglobin 80% of serum Ig ~10mg/mL • Again classified in to IgG1,2,3,4 • IgG1, IgG3 and IgG4 cross placenta in human • IgG3 Most effective complement activator • Responsible for protection of the newborn during the first months of life. Because of its relative abundance and excellent specificity toward antigens • Properties of IgG • Molecular weight: 150,000 • H-chain type (MW): gamma (53,000) • Percent of total immunoglobulin: 80% • Half life 25 days • Function: secondary response Ig G (Monomeric) (g)

9. Structure of Ig G

10. Ig A • Glycoprotein • Dimer • It have 4 Polypeptide chain (2 Light chain (K and lambda) and 2 Heavy chain (Alpha) ) • M.W 160,000 H-chain type (MW): alpha (55,000) • Normal serum level 0.6-4.2 mg/ml • Half life 6-8 days • Predominant Ig in secretions • Milk, saliva, tears, mucus • Important role in Mucosal Activity • 3-5 g of Ig A are secreted in to the intestinal every day • This accumulate 75% of the total immunoglobulin produced in the entire body • Ig A subclass are Ig A1, Ig A2

11. Function • Mucosal paint/ antiseptic paint • Neutralize –local toxin----Promote phagocytosis • Immunity against Tape Worms • Present in the CLOSTRUM protects the baby from intestinal pathogen.

12. Structure of Ig A

13. IgM • Largest Immunoglobulin • Pentamer Structure ( 5) • Made up of 2 light (Kappa, lambda) and • 2 Heavy chain (Mu) • Normally this called Natural Antibody • Multiple antigen binding site • 3rd highest serum Ig • Produced early in the primary response • It is activate complement pathways • Half life 5 days • Molecular weight: 900,000--- mu (65,000) • H-chain type (MW): mu (65,000) • Percent of total immunoglobulin: 5- 10%

14. Structure of Ig M

15. Ig E • Monomer • Made up of 2 light (K and Lamda) and 2 heavy • chain (Epsilon) • This is the only Igwithout hinge Region • M.W 190,000 epsilon (73,000) • Heat liable Ig, where as Ig G, IgA, IgM and IgD • Heat stable • Its found only trace amount in the serum • (0.2% of total Ig) • This induce Allergic Reaction • Half life 2-3 days

16. Structure of Ig E

17. Ig D • It’s a Monomer (Glycoprotein) • Found in Blood serum and Surface of B cell • Made up of 2 Light chain (K and Lamda) and 2 Heavy chain • ( Delta) • It s a primitive (ancient) Ab found in fishes to mammals • except Birds • Ig D slightly larger than Ig G --- M.W--- 180,000 (delta • (70,000) • Ig D limited to the Blood serum Average serum level -0.03 • mg/ml • Half life 2-3 days • Ig D binds to Basophils and mast cells and activates them to • secrete ANTIMICEROBIAL Factors

18. Structure of Ig D

19. Serum and plasma Serum Blood Clot Plasma Blood + anticoagulant Cells

20. Isolation & characterization Chromatography Molecular sieving Ion exhange Affinity Salting out/ Dehydration Ammonium sulfate Alcohol precipitation Ultracentrifugation Immunochemical

21. + + - - + + + + + Chromatography Molecular sieving Ion exchange Affinity

22. Salting out Ammonium sulfate (half saturated) Ethanol (90%)

23. Density gradient ultracentrifugation Centrifugation 60,000 - 100,00 X G Displacement = Svedberg

24. + - Albumin a b g Globulins Kabat & Tiselius (1939) -Discovered that hyperimmunizing rabbits resulted in increased g -Purification revealed antibody activity resided in this serum portion

25. + + + - Albumin a b g Globulins Electrophoretic analysis of serum Sample application Separation by charge - - Anode Cathode

26. Protein concentration - + Protein concentration Characterizing chains Albumin Broad peak of g = heterogenous proteins g b a2 a1 Need pure Ig to chemically analyze normal Narrow peak of g = homogenous proteins Bence-Jones proteins in urine of multiple myeloma pts. = dimers of immunoglobulin light chains, k or l - + multiple myeloma

27. Fab Fab S S S S S S Fc Porter (England) Treatment with proteolytic enzyme, papain, resulted in three approximately equal sized fragments: 2 capable of ag rx (fragment antigen binding) 1 could be crystallized (fragment- crystallizable) Fab specifically bind antigen, univalent (can’t ppt) one binding site each, identical to each other Fc crystallizable (thus homogenous) can’t bind ag responsible for biological activity of molecule after ag bound to Fab portions

28. SH HS Eddleman (USA) Treatment with mercaptoethanol = 4 chains: (mercaptoethanol breaks S-S bonds) HS SH SH HS 2 chains = 53,000 daltons 2 chains = 22,000 daltons All immunoglobulins basic unit consisting of 4 polypeptides i.e. 2 H, 2 L

29. N-terminus Fab Fab S S S S S S S S S S S S Proteolytic enzyme digestion reveals N- & C-terminal Papain digestion results in cleavage @ “N” terminus in proline hinge region @ disulfide bridge Fc Fab2 Pepsin digestion results in cleavage @ “C” terminal portion, resulting in a divalent fragment (Fab2) joins by S-S & several Fc fragments C-terminus

30. HS SH HS SH SH HS Fab Fab S S S S S S S S S S S S Cleavage of Ig 4 polypeptide chains Reduced w ME Papain 2 Fragment ag binding 1 Fragment crystallizable Pepsin 1 Fab2 Several small pieces Fc

31. Myeloma cells Plasma cell becomes tumorous = myeloma produces homogenous (monoclonal) Ig Myeloma Ig = myeloma “proteins” Myeloma proteins may be purified & structurally analyzed AA sequence in L & H chains Normal, non immune Normal, immune Myeloid myeloma

32. N-terminus Fab Fab S S S S S S C terminus Different sequences in N-termin. domain Variable (VL) Identical sequences in C-termin. domain Constant (CL) Light chain sequences N-terminus 214 AA in two domains Analysis from several myelomas C terminus

33. N-terminus Fab Fab S S S S S S C terminus Heavy-chain sequence N-terminal domain varies (HV) Heavy chain consists of 445 AA in 4 domains Other 3 domains constant (HC) Flexible Hinge Region

34. Sequence variability not distributed evenly in variable region % var Variation restricted to 3 regions CDR 1 CDR 3 Hypervariable regions (HV1,HV2,HV3) Location of antigen binding site AA sequence det. shape of ag binding site (paratope) CDR 2 Determine epitopes to which Ig binds = complementary- determining regions (CDR) CDR = 6-10 AA CDR 1 = 24 - 34 CDR 2 = 50 - 56 CDR 3 = 89 -97 % var Framework regions = regions where AA seq. is relatively constant (FR) Heavy chain CDR CDR 1 = 31-35 CDR 2 = 50-65 CDR 3 = 95-102 Wu & Kabat plot

35. Light chain C domain Light chain V domain N terminus CDR 3 Disulfide bonds CDR 1 CDR 2 C terminus B strands B strands Structure of variable & constant domains (X-ray crystallography) Two layers, linked by disulfide bonds Each layer formed several stretches conformation = b strand Layers = b sheet Order of b strands is characteristic for each sheet 3 D structure = Ig fold

36. C domain V domain Opening to reveal b strands comprising each b sheet Principal difference between C & V domains V domain has 2 more b strains forming extra loop unique strands Order & orientation characteristic for each domain b strands lettered sequentially according to occurrence in AA sequence in dom.

37. Location of CDR & FR in Ig L & H chains CDR 1 2 3 CL FR1 FR2 FR3 FR4 CH1 FR1 FR2 FR3 FR4 H 1 2 3 CDR

38. L & H chain folding to yield 3 CDR in each chain to form walls of ag binding groove

39. Immunoglobulin consists of 4 polypeptide chains N terminus Ag binding site Ag binding site Variable regions VH VL CH1 CL Disulfide bonds CH2 Constant region EM Rabbit Ig X 2,042,500 CH3 Light chain Heavy chain C terminus

40. Immunologic analysis of immunoglobulins Ig (like most other proteins) stimulate ab in other animal species All species have two major classes of L chains i.e. k,l individual of species produces both types; ratio of k : l varies by species (e.g. mouse 95%k ; human 60% k) in any Ig molecule, both L chains = either k or l, never one of each Ig of all species consist of 5 classes (isotypes) differ in structure of H chains H chains among isotypes differ serologically, CHO content, size & biological function

41. Isotypic (Class) structure of Ig L H L H IgG isotype = k2g2 or l2g2, but not k1l1g1a1 L H L H IgE isotype = k2e2 or l2e2 ….etc…...

42. Immunoglobulin isotype Heavy chain H chains H chain confers unique biologic properties of molecule e.g. 1/2 life, receptor binding , enzyme-, C’ activation with ag IgM m IgG g IgA a IgD d IgE e Individual of species produces all H chains, in proportion characteristic for species, but ab molecule H chains are identical ( i.e., 2e, 2d etc.)

43. Immunoglobulin consists of 4 polypeptide chains N terminus Ag binding site Ag binding site Variable regions VH VL CH1 CL Disulfide bonds CH2 Constant region EM Rabbit Ig X 2,042,500 CH3 Light chain Heavy chain C terminus

44. Isotypic (Class) structure of Ig L H L H IgG isotype = k2g2 or l2g2, but not k1l1g1a1 L H L H IgE isotype = k2e2 or l2e2 ….etc…...

45. Hinge region Consists of approx 12 AA between CH1 & CH2 No homology between hinge & other Heavy chain domains Angle = 900 AA sequence unique for each class & subclass Angle = 600 Angle = 0o

46. Light Chain Cys -Arg-Val-Glu-Pro-Lys-Ser- Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu- -Arg-Val-Glu-Pro-Lys-Ser- Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu- Cys Papain Fab region Fc region AA sequence in hinge region Hinge region Light Chain

47. Characteristics of hinge region Immunoglobulins (with possible exception of IgM & IgE) contain hinge between CH1 & CH2 No homology between AA sequence of hinge & heavy chains AA sequence differs with different classes Comprised of many cysteine and proline residues Cysteine involved in formation of interchain disulfide bonds Proline prevents folding in a globular structure, allowing flexibility between two Fab arms of the Y-shaped antibody; allows open & close to accommodate binding to two epitopes; because it is open, it can be cleaved by proteases (e.g. papain) to generate the Fab & Fc fragments

48. IgG1 IgG2 IgG3 IgG4 IgG Highest concentration in serum Plays major role in immune dfns. MW approx. 150 kDa Small size ppt in surfaces (e.g. cross placental barrier) Opsinize, aggl. & ppt ag Subclasses show close overall relation Only activates classical C’ p’way Heavy chains = g1,g2,g3,g4 All normal indiv. have all IgG1>IgG2>IgG3>IgG4 IgG3 has shortest 1/2 life, highest catabolic rate, highest # S-S IgG4 = monoval. no aggl ppt rx., autoab to clot fac Autoab to DNA = IgG1,IgG3

49. Variable HV Constant - + Structural features of IgG Two g H chains, Two L chains (either k or l but not both) Each H chain = 50 kD Each L chain = 25 kD 150 kD, 7S, “g globulin” Least anodic of all serum proteins Anode Cathode

50. S S S S S S S S S S S S S S S S S S J chain S S S S S S S S S S S S Structural features of IgM Pentamer (5) First Ig produced following immun. Macroglobulin (M) 900 kD, 19S Doesn’t have hinge region has additional H domain Has a J chain (one of 2 Ig isotypes) 15 kD