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ANTIBODY © Universities Press (India) Private Limited Universities Press ANTIBODY • Humoral basis of immunity • Secreted by plasma cell • Present on B cell membrane • React with antigen - specifically observable • Antibody - phagocytosis, phagocytosis, ADCC • Sera - high antibody levels - ‘immune sera’ © Universities Press (India) Private Limited Universities Press ANTIBODY • Fractionation of immune sera - soluble albumin, insoluble globulin • Globulins - water soluble pseudoglobulin, insoluble globulin • Most antibodies - globulins • Tiselius (1937) - serum proteins - albumin, alpha, beta, gamma globulin • Antibody activity – gamma globulin fraction © Universities Press (India) Private Limited Universities Press ANTIBODY Serum electrophoresis showing γ globulin nature of antibodies © Universities Press (India) Private Limited Universities Press ANTIBODY • 1964 – WHO - immunoglobulin • Protein - known antibody activity and other proteins related to them by chemical structure • Includes - abnormal proteins – myeloma • Macroglobulinemia • Cryoglobulinemia © Universities Press (India) Private Limited Universities Press ANTIBODY • Immunoglobulins - synthesised by plasma cells and by lymphocytes • Immunoglobulins - structural and chemical concept • Antibody - functional and biological concept • All antibodies - immunoglobulins • All immunoglobulins - may not be antibodies © Universities Press (India) Private Limited Universities Press ANTIBODY - STRUCTURE ENZYME DIGESTION Rabbit lgG antibody - to egg albumin digested by papain split - two fractions Insoluble fraction Soluble fraction Crystallised in cold Bound with egg albumin Fc (crystallisable) Fab (antigen binding) © Universities Press (India) Private Limited Universities Press ANTIBODY STRUCTURE Each molecule of immunoglobulin Split by papain - three parts One Fc - Two Fab When treated with pepsin Two Fab fragments - held together Bivalent - F(ab)2 Fc portion – digested - small fragments © Universities Press (India) Private Limited Universities Press ANTIBODY STRUCTURE Basic structure of an immunoglobulin molecule and the fragments obtained by cleavage by papain and pepsin © Universities Press (India) Private Limited Universities Press IMMUNOGLOBULIN CHAINS • Each molecule - 4 polypeptide chains • Smaller chain - Light (L) chain • Longer chain - Heavy (H) chain • ‘L’ chain attached to ‘H’ chain by disulphide bond • Two ‘H’ chains - joined together by 1-5 S-S bond © Universities Press (India) Private Limited Universities Press IMMUNOGLOBULIN CHAINS H chain-structurally, antigenically distinct Designated by Greek letter Immunoglobulin class H chain lg G (gamma) lg A α (alpha) lg M (mu) lg D (delta) lg E (epsilon) © Universities Press (India) Private Limited Universities Press IMMUNOGLOBULIN CHAINS • L chains - similar in all classes of immunoglobulins • Two varieties - kappa (K), lambda () • A molecule of immunoglobulin may have either kappa or lambda, never both • Kappa andlambda named after Korngold and Lapari • Each light chain is bound to a heavy chain by interchain and intrachain disulphide bonds © Universities Press (India) Private Limited Universities Press IMMUNOGLOBULIN CLASSES • Antigen combining site - at amino terminus - composed of both ‘H’ and ‘L’ chains • Amino terminal - amino acid sequencevariable – variable region - antigen recognition • Carboxy terminal - constant regioneffector functions • Infinite range - antibody specificity © Universities Press (India) Private Limited Universities Press IMMUNOGLOBULIN Structure of immunoglobulin molecule © Universities Press (India) Private Limited Universities Press HYPERVARIABLE REGION • Amino acid sequence of variable region of L and H chains • Consist of relatively invariable and highly variable zones • Highly variable - three in L four in H • Hypervariable - ‘hot spots’ sites on hypervariable regions that make contact with epitope ‘complementary determining region’ or CDR © Universities Press (India) Private Limited Universities Press IMMUNOGLOBULIN CLASSES • IgG • IgA • IgM • IgD • IgE © Universities Press (India) Private Limited Universities Press IgG • Major serum immunoglobulin 80% of total • Mol wt - 150,000(7S) • Half-life - 23 days • Level raised - chronic malaria, kala azar, myeloma • lgG - transported across placenta • Natural passive immunity - newborn © Universities Press (India) Private Limited Universities Press IgG • lgG - binds microorganism - enhances their phagocytosis • Extracellular killing - target cell - coated with lgG antibody • Mediated through recognition of surface of Fc fragment • lgG complex - platelet Fc receptor • Aggregation of vasoactive amines © Universities Press (India) Private Limited Universities Press IgG Structure of immunoglobulin molecule © Universities Press (India) Private Limited Universities Press IgG • Participates in - complement fixation, precipitation, neutralisation of toxin and viruses • General purpose antibody • Protective against infections agents • Passively administered - suppress homologous antibody • IgG late antibody, initial immune response IgM • Subclasses - lgG1, lgG2, lgG3, lgG4 © Universities Press (India) Private Limited Universities Press IgA • Seondmost abundant class • 10-13% of serum immunoglobulin • Half-life of 6-8 days • Major immunoglobulin – colostrum, saliva, tears © Universities Press (India) Private Limited Universities Press IgA • Two forms - Serum lgA, Secretory lgA • Serum lgA - monomer • IgA on mucosa, in secretions - dimer • Two monomeric units - together at the carboxy terminal by glycopeptide - J chain (joining) • Dimeric secretory lgA - synthesised by plasma cells • J chain also synthesised by plasma cells © Universities Press (India) Private Limited Universities Press IgA Secretory IgA molecule © Universities Press (India) Private Limited Universities Press SIgA • Secretory lgA - contains glycine-rich polypeptide, called the secretory component or secretory piece • Produced by mucosal/glandular epithelial cells • Dimeric lgA - binds receptor on epithelial cell, endocytosed transported to luminal surface © Universities Press (India) Private Limited Universities Press SIgA • During the process - part of receptor remains attached to lgA dimer • Secretory component • Secretory piece - protects lgA from denaturation - by bacterial proteases in the intestinal mucosa • SlgA much larger than serum lgA © Universities Press (India) Private Limited Universities Press SIgA • Selectively concentrated in secretions and on mucus surfaces • Role in local immunity against respiratory and intestinal pathogens • Resistant to digestive enzymes • Inhibit adherence of organism to mucosa activates - alternate complement pathway • Promotes phagocytosis and intracellular killing of microorganisms © Universities Press (India) Private Limited Universities Press IgM • 5-8% of serum immunoglobulin • Half-life of 5 days • Heavy molecule - millionaire molecule • lgM – polymer - five peptide subunits • Polymerisation of subunit depends on J chain • Most lgM - intravascular • Earliest immunoglobulin to be synthesised © Universities Press (India) Private Limited Universities Press IgM IgM molecule © Universities Press (India) Private Limited Universities Press IgM • Not transported across placenta • lgM in fetus/newborn - indicates intrauterine infections • Short lived, disappear earlier than lgG • Presence in serum - recent infection © Universities Press (India) Private Limited Universities Press IgM • Unique structural features - suited to biological role • Protect - microorganism and other large antigens • Function lgM lgG • Immune hemolysis single molecule 1000 molecule © Universities Press (India) Private Limited Universities Press IgM • Opsonisation 500-1000 times more effective than lgG • Bactericidal action 100 times more • Bacterial agglutination 20 times more • Neutralisation of toxin, virus-less active than lgG © Universities Press (India) Private Limited Universities Press IgD • Resembles lgG structurally • Concentration-3 mg/100 ml serum intravascular • Surface of unstimulated B lymphocyte • Recognition receptor for antigen © Universities Press (India) Private Limited Universities Press IgE • 1966 Ishizaka - atopic reagin molecule • Structurally resemble lgG • Affinity for surface of tissue cells (mast cells) • Mediates- Prausnitz-Kustner reaction • Does not cross placenta • Does not fix complement • Elevated levels – asthma, hay fever, eczema © Universities Press (India) Private Limited Universities Press IgE • Produced in lining of respiratory and intestinal tract • Responsible for anaphylactic type of hypersensitivity • Protection by mast cell degranulation and release of inflammatory mediators © Universities Press (India) Private Limited Universities Press ABNORMAL IMMUNOGLOBULIN • Structurally similar proteins - pathological processes • Multiple myeloma – 1847, Bence-Jonesmultiple myeloma light chain immunoglobulin - kappa or lambda • In single patient - kappa/lambda, never both © Universities Press (India) Private Limited Universities Press ABNORMAL IMMUNOGLOBULIN • Myeloma - plasma cell dyscrasia • Unchecked proliferation of one clone of plasma cells - monoclonal • Multiple myeloma may affect plasma cell synthesis of lgG, lgA, lgD, lgE © Universities Press (India) Private Limited Universities Press ABNORMAL IMMUNOGLOBULIN • Waldenstrom’s microgloblinemia lgM-producing plasma cells • Heavy chain disease - lymphoid neoplasia over production of Fc part of heavy chain • Cryoglobulinemia – gel or precipitate forms on cooling serum © Universities Press (India) Private Limited Universities Press