Antibodies

LECTURE FOR MBBS, IMMUNOLOGY
ANTIBODY

React specifically & in some observable manner with Ag which stimulated their production
 globulins produced in response to antigenic stimulation

1. ANTIBODIES DR. ROMA GOYAL ASSISTANT PROFESSOR DEPARTMENT OF MICROBIOLOGY

2. A n tibody Gamma globulin proteins that react specifically with antigen that stimulated their production 20% of plasma protein F i v e classe s of antib o dies: IgG, IgM, IgA, IgD, and IgE (ba s ed on di f f e r en c es in h e a v y cha i ns) 2

3. A n tibodies 3 After stimulation, B cells differentiate into plasma cells, & secrete antibodies, (immunoglobulins), mediators of humoral immunity

4. Antibodies Characteristics 4 D i v ersity R esp o n d t o di f f e r ent anti g ens Long memory Respond many years after initial exposure due to memory T cells and B cells

5. Antibodies Characteristics 5 Specificity Actions specifically directed against antigen that initiated response Inflammatory response : Combined effect of cells (e.g., T cells, B cells, macrophages & neutrophils) & proteins (e.g., interleukins, antibodies & complement)

6. Functions of antibodies 6 N eu t r a l i z e t o xins and viru s es Opsonize microbes to be easily phagocytosed A ct i v a t e C omple m ent, a n d p r e v ent a t tachmen t of microbes to mucosal surfaces Catalytic : Antibody can act as an enzyme to catalyze synthesis of ozone (O3) that has microbicidal activity.

7. Structure of immunoglobulin 7 Simplest antibody molecule has Y shape C o n sists of f our p o l y p eptide chai n s: Two H chains and two L chains F our chain s lin k ed b y disul f id e b o n d s Antibody molecule always consists of identical H chains and identical L chains

8. Structure of immunoglobulin 8 L and H chains subdivided into variable and constant regions. Regions composed of three-dimensionally folded, repeating segments called domains Each domain is about 110 amino acids long ⚫ globular in shape stabilized by intrachain disulphide bonds Antigen binding sites located in variable domains Amino terminus Variable region Carboxy terminus Constant region Complement fixing

9. Structure of immunoglobulin 9 Variable regions of light and heavy chain responsible for antigen-binding Constant region of heavy chain responsible for biologic functions (e.g., complement activation and b indi n g t o c ell surfa c e r e c e p t o r s)

10. Structure of immunoglobulin 10 Light chain : One variable region and one constant region L chain attached to H chain by disulphide & non- covalent bonds

11. Structure of immunoglobulin 11 L chains belong to one of two types, κ ( kappa) or λ (lambda), due to amino acid differences in their constant regions Both types occur in all classes of immunoglobulins (IgG, IgM, etc.), but any one immunoglobulin molecul e c o n tain s o n l y o n e typ e of L chain

12. Structure of immunoglobulin 12 Heavy chain consists of a variable region and a constant region divided into three domains: CH1, CH2, and CH3 Each domain 110 amino acids long CH2 domain contains complement-binding site CH3 domain is site of attachment of IgG to receptors on neutrophils and macrophages H chains structurally & antigenically distinct f or each class H chains are distinct for each of five immunoglobulin classes and are designated γ, α, μ, ε, and δ

13. Structure of immunoglobulin 13 Variable regions of L and H chains have three extremely variable( hypervariable) amino acid sequences at amino-terminal end that form antigen- binding site Speci f ic i t y of antib o die s i s du e t o h y p e r v ar i able regions

14. Structure of immunoglobulin 14 Amino-terminal portion of each L & H chain participates in antigen-binding site Carboxy terminal forms Fc fragment, which has biologic activities

15. Heavy chain 15 H chain designated by Greek letter. Ig class H chain Ig G  Ig M  Ig A  Ig D  Ig E 

16. Structure of immunoglobulin 16

17. Structure of immunoglobulin 17 Antibody molecule treated with a proteolytic enzyme s papain,break peptide bonds in “hinge” region , producing two identical Fab fragments, which carry antigen-binding sites, and one Fc fragment, involved in placental transfer, complement fixation, attachment si t e f or v ar i ous c ell s ,& other bi o log i c act i vities

18. Enzymatic digestion – generates various fragments 15 Papain digestion Cleave Ig above disulfide bridge of hinge region Results in 3 fragments each Two Fab fragments – soluble fragments which bind to Ag One Fc fragment – insoluble, crystallised in cold

19. PAPAIN CLEAVAGE

20. Enzymatic digestion – generates various fragments Pepsin digestion Cleaves Ig molecule at point below disulfide bridge of hinge region One F(ab’) 2 fragment; 2 Fab subunits bound together Many smaller fragments

21. PEPSIN DIGESTION

22. 3D ANTIBODY STRUCTURE

23. Classification of antibodies 23 Immunoglobulin A (IgA) Immunoglobulin G (IgG) Immunoglobulin M (IgM) Immunoglobulin D (IgD) Immunoglobulin E (IgE) Based on structural differences in constant regions of heavy chains Classe s h a v e spe c ial i z ed ef f ec t or fun c t i ons

24. Ig M 24 5-8 % of serum immunoglobulins Short lived Pentameric structure Predominant Ab in primary immune response . Earliest Ab synthesized by fetus Confined to intravascular pool due to large size Not transported across placenta Presence of IgM in newborn indicates intra uterine infection Useful in the diagnosis of congenital infections like syphilis, rubella, HIV, toxoplasmosis etc.

25. Distribution Class of Immuno- globulin (Antibody) IgM (pe n tamer) J chain First Ig class produced after initial exposure to antigen; then its concentration in the blood declines 25 Promotes neutraliza- tion and cross- linking of antigens; very effective in complement system activation Function

26. 75% of total immunoglobulins 4 subclasses – IgG1, IgG2, IgG3 & IgG4 Each having a distinct type of gamma chain Major Antibody of secondary response , found in serum & body fluids Only maternal Ig to be transported across placenta – natural passive immunity in newborn Participates in complement fixation, precipitation & neutralization of viruses & toxins I mmunoglobulin G ( IgG ) 26

27. Distribution 27 Function Class of Immuno- globulin (Antibody) IgG (monomer) Most abundant Ig class in blood; also present in tissue fluids Promotes opsoniza- tion, neutralization, and cross-linking of antigens; less effec- tive in activation of complement system than IgM Only Ig class that crosses placenta, thus conferring passive immunity on fetus

28. Ig A 28 2 nd most abundant 10-13 % Major Ig in colostrum, saliva, tears & other body fluids. Two forms : IgA1 & IgA2. Secretory IgA in dimeric form – composed of 2 basic chain units, a J chain & the secretory component. Secretory component helps to transport dimer from submucosa to mucosal cell surface. Secretory component protects IgA from proteolytic digestion and denaturation.

29. Distribution Function Class of Immuno- globulin (Antibody) IgA (di m er) J chain Secretory component Present in secretions such as tears, saliva, mucus, and breast milk 29 Provides localized defense of mucous membranes by cross-linking and neutralization of antigens Presence in breast milk confers passive immunity on nursing infant

30. Ig E 30 Low levels in serum On surface of mast cells & basophils which have specific receptors for Fc portion of IgE Produced in linings of respiratory & intestinal tracts Causes anaphylactic type of hypersensitivity Defense against parasitic infections

31. Distribution 31 Function Class of Immuno- globulin (Antibody) IgE (mono m er) Present in blood at low concen- trations Triggers release from mast cells and basophils of hista- mine and other chemicals that cause allergic reactions

32. Ig D 32 Resembles Ig G structurally Present with Ig M on B cell surface Susceptible to proteolytic attack

33. Distribution Function Class of Immuno- globulin (Antibody) IgD ( m o n o m er) Trans- membrane region Present primarily on surface of B cells that have not been exposed to antigens Acts as antigen receptor in the antige n -sti m ulated proliferation and differentiation of B cells (clonal selection) 33

34. Primary & Secondary antibody response 34 Primary Response Following exposure to an antigen, there is a slow rise in IgM followed by a slow rise in IgG Secondary Response Following exposure to previously encountered antigen, there is a rapid rise in IgG and slow or no rise in IgM Memory or anamnestic response

35. Abnormal Immunoglobulins Bence Jones proteins Produced in neoplastic condition of plasma cells called Multiple Myeloma Also called light chain disease Cancerous plasma cells produce excess light chain (BJP) accumulated in pt serum and urine Waldenstrom’s Macroglobulinemia B cell lymphoma, produce excess IgM Heavy chain disease Cryoglobulinemia Blood contains cryoglobulin Associated with Multiple Myeloma and Hepatitis C infection

36. 37 Antibodies produced in response to antigens are he t e r o g eneou s , f ormed b y di f f e r ent clo n es of pl a sma cells ( polyclonal) An tibo d ies that ari s e f r om a sin g l e clon e of c ells (e.g., in a plasma cell tumor [myeloma])are homogeneous ( monoclonal)

37. Hybridoma Monoclonal antibodies made in laboratory by fusing a myeloma cell with an antibody-producing cell are called hybridoma 38

38. Hybridoma 39 Hybridoma cells made as following A mouse immunized with antigen of interest Spleen cells from this mouse grown in a culture dish i n th e p r esen c e of mous e m y eloma c ells Myeloma cells grow indefinitely in culture, & do not produce immunoglobulins

39. Hybridoma 40 3)Fusion of cells by adding certain chemicals (e. g ., p o l y et hy lene g l y c ol) Cells grown in a special culture medium(HAT medium) that supports growth of fused, hybrid cells but not of “parental” cells Resulting clones of cells screened for production of antibody to antigen of interest

40. Production of monoclonal antibodies 41

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