ANTIBODY and its immunology with pharmac

ANTIBODY and its immunology with pharmac

1. ANTIBODY

2. Definition Antibodies are substance which are formed in the serum and tissue fluids in response to an antigen and react with that antigen specifically and in some observable manner. Take part in AMI Various designations: agglutains , precipitins etc. INTRODUCTION

3. Based on electrophoretic mobility Tiselius and Kabat (1938) separated Serum proteins Albumin alpha, beta and gamma globulins. Also he showed that antibody activity was associated with the gamma globulin fraction. Chemical nature of antibodies is globulin hence they named as immunoglobulins.

4. Serum globulins could be separated into pseudoglobulins (water soluble) euglobulins (water insoluble) Most antibodies are found to be euglobulins .

5. IMMUNOGLOBULIN Proteins of animal origin enclosed with known antibody activity. Term also used for other proteins related by chemical structure. Besides antibody globulins, the abnormal proteins found in myeloma, macroglobulinemia , cryoglobulinemia . All antibodies are immunoglobulins but all immunoglobulins may not be antibodies.

6. Immunoglobulins constitute about 20-25% of the total serum protein. They are mainly synthesized by plasma cells. Based on physiochemical and antigenic differences 5 classes of immunoglobulins have been recognized. Immunoglobulin G ( IgG ) Immunoglobulin A ( IgA ) Immunoglobulin M ( IgM ) Immunoglobulin D ( IgD ) Immunoglobulin E ( IgE )

7. Antibodies are wide range of functions and uses Elimination (removal) of foreign antigen from the circulation. Recruitment and enhancement of host effector circulation. Measurement of specific antipathogen antibody levels in diagnosis. Passive administration of pooled antibodies for host therapy FUNCTIONS

8. Antibody or immunoglobulins are glycoprotein molecule consists of 2 identical heavy and light chains. Both types of chains are polypeptide in nature. STRUCTURE OF IMMUNOGLOBULIN

9. Heavy chains are structurally and antigenically distinct in different classes of immunoglobulins Light chain are similar in all classes of immunoglobulins They are present in 2 form kappa(K) and lambda(L ) Class of immunoglobulins Heavy chain IgG g amma ( λ ) IgM mu ( μ ) IgA alpha ( α ) IgD d elta ( δ ) IgE epsilon (€ )

10. Porter, edelman and Nisonoff developed a technique for cleavage of immunoglobulin molecules. EFFECT OF ENZYMES PAPAIN DIGESTION . Porter and colleagues split rabbit IgG antibody to egg albumin. Papain digest immunoglobulin in the presence of cysteine , splits in to 2 fractions. Structure of immunoglobulin

11. One Insoluble fraction Fc fragments ( Fc crystallisable ) Two soluble fraction Fab fragments(fragment antigen binding) Fab fragment posses antigen binding sites Fc fragment determines the biological properties of imunoglobulin molecules such as complement fixation, placental transfer, skin fixation and catabolic rate.

12. PEPSIN DIGESTION . Pepsin cleaves at a different point of immunoglobulin molecules and give rise to Fc portion and two Fab fragament held in position. Fab fragment is bivalent and can still precipitate with antigen. This fragment is called F( ab )2 Also degrades the Fc portion into smaller fragments

13. Light chain contains 210-230 amino acids and M.W is 25,000 Heavy chain contains 420-460 amino acids and M.W is 50,000. Both consists of two portions each, Variable region – are present at aminoterminus Constant region – are present at carboxyterminus AMINOACID SEQUENCES

14. In variable region of the H and L domains, Maximum sequence variation is concentrated in a few discrete regions called as hypervariable regions Form the antigen binding site of the molecules which is complementary to the structure of the epitope are called complementarity determining regions. Hypervariable and framework regions

15. Fab fragment has six CDRs ( 3 in H and 3 in L chain) Less variable stretches are termed Framework regions Acts as a scaffold support to six CDR loops in each Fab fragment

16. Immunoglobulins are folded to form globular variable and constant domains. Each heavy chain consists of 4 domains 1 in variable region(VH) 3 in constant regions (CH1,CH2,CH3 ). Light chain consists of 2 domains 1 domain in variable region 1 in constant regions Immunological domains

17. Each domain has separate function Variable region domains ( VL and VH) are responsible for the formation of a specific antigen binding site. Constant region domains mediate secondary biological functions. CH2 region in IgG binds C1q in the classical complement pathway, CH3 mediates adherence to the monocyte surface.

18. Hinge Region ( Area between CH1 and CH2 domains) Rich in proline residues (flexible) Hinge found in IgG , IgA and IgD IgM and IgE lack hinge region Proline residues are target for proteolytic digestion ( papain and pepsin) Rich in cysteine residues (disulfide bonds) They instead have extra C H 4 Domain

19. Immunoglobulins are glycoprotein and can act as immunogens when inoculated into a foreign species. Differences in amino acid sequences between immunoglobulin classes, subclasses and types determine their antigenic specificity. There are 3 major types of immunoglobulin antigen determinants. 1. ISOTYPES – Determinants are shared by all members of the same species. On the basis of isotypes markers on H chains, different classes of immunoglobulins are differentiated. Immunoglobulin antigen determinants

20. 2. ALLOTYPES – These are individual specific determinants within the species. 3. IDIOTYPES – Idiotype markers are located in hypervariable regions of immunoglobulin molecule. specific for each antibody. Antibodies are generated are known as anti- idiotypes antibodies. By immunisation with Fab fragments, antiidiotypic antibodies can be produced.

21. It the major serum immunoglobulin ( about 80% of the total amount). Distributed equally between the intravascular and extravascular comportments. Normal serum concentration (8-16mg/ml) Molecular weight is 150.000 Half life - 23 days ( longest) Only immunoglobulin that transported through placenta and provides natural passive immunity to newborn. Immunoglobulin G ( IgG )

22. IgG appear late but persists for longer period Catabolism of IgG is unique in that it depends on the Serum IgG concentration when level of IgG is raised as in myeloma or kala-azar , the synthesis of IgG against that particular antigen is catabolised rapidly and may result in deficiency of that particular antibody. In hypogammaglobulinaemia IgG antibody given for therapeutic purpose will be catabolised slowly. Four subclasses - IgG1, IgG2, IgG3, IgG4

23. Binds to organisms and enhances phagocytosis. Participates in precipitation, complement fixation and neutralization of toxin and viruses. Protective against infectious agents which are active in blood and tissues. Passively administered IgG suppresses the homologous antibody synthesis by feed back mechanism. Based on this property, isoimmunisation of women is done by administration of anti- RhD IgG during delivery. FUNCTIONS

24. Second major serum immunoglobulin (about 10-13% of serum immunoglobulin) Normal serum concentration is 0.6-4.2mg/ml Half life - 6-8 days Principal immunoglobulin present in secretions such as milk, saliva, tears, sweat, nasal fluids, colostrum and in secretions of respiratory, intestinal and genital systems. IgA occurs in two forms Serum IgA and Secretory IgA Immunoglobulin A ( IgA )

25. 1. Serum IgA – monomeric MW 160,000 2. Secretory IgA Dimer M W 400,000 Dimer formed by two monomer units joined together by glycoprotein named J chain J chain produced by plasma cells situated near mucosal or glandular epithelium.

26. Secretory IgA contains another glycine rich polypeptide called secretory piece. Protect IgA from denaturation by bacterial proteases in sites such as the instestinal mucosa which is rich in bacterial flora. IgA found in mucosal surfaces and in secretions It form antibody paste on mucosa.(local immunity) Covers the microorganisms to inhibits their adherence to mucosal surfaces. Two subclasses of IgA (IgA1 and IgA2 )

27. Inhibits adherence of organisms to mucosal cells; and therefore entry into body tissues Activates alternate complement pathway. Promotes phagocytosis of microorganisms. FUNCTIONS

28. Constitutes about 5-8 % of total serum immunoglobulins Normal level in serum is 0.5 -2 mg/ml. Half life – 5 days. It is a heavy molecules (M.W – 900,000 to 1,000,000 hence called as Millionaire molecules ). Mainly distributed intravascularly (80%) Immunoglobulin M ( IgM )

29. Pentamer Each bearing an extra CH domain Polymerization of the subunits depends on the presence of the J chain. Structure of IgM

30. Earliest synthesized immunoglobulin by foetus in 20 weeks of age. Not cross the placenta (presence of IgM in fetus or newborn indicates intrauterine infection) Demonstration in serum indicates recent infection. Treatment of serum with 0.12 μ 2-mercaptoethanol destroy IgM Functions Provides protection against blood invasion by microorganisms

31. Resembles IgG structurally Serum concentration is 3mg/100ml Mostly intravascular in distribution. M.W is 1,80,000 Half life – 3 days Functions Serves as a recognition molecule on the surface of unstimulated B lymphocytes. Immunoglobulin D ( IgD )

32. Resembles IgG structure Serum contains only traces (few nanograms /ml) increased serum concentration in asthma, eczema, hayfever and parasitic infections. M.W is 1,90,000 Half life - 2-3 days Produced in the lining of respiratory and intestinal tracts. Mostly extravascularly Immunoglobulin E ( IgE )

33. Can not cross the placental barrier or fix the complement. Unique properties. Heat labile ( inactivated at 56 C in 1hr) Affinity for surface of tissue cells particularly mast cells Mediates type I hypersensitivity. Functions Responsible for anaphylactic type of hypersensitivity. Defense against helminthic infections.

34. IgG – protects the body fluid IgA – protects the body surfaces IgM – protects the blood stream IgE – mediates reaginic hypersensitivity IgD – recognition molecules on the surface of B lymphocytes. Role of different immunoglobulin classes.

35. 1. Multiple myeloma – It is plasma cell tumor in the bone marrow producing excess of a single class of immunoglobulin and their light chain. Myeloma may be occur with any of the five classes of immunoglobulins. ( 50-60% IgG type) Waldenstrons’s macroglobulinemia (involving IgM producing cells) light chain of immunoglobulin - Bence Jones protein ABNORMAL IMMUNOGLOBULINS

36. 2. Heavy chain disease – Abnormal heavy chain are produced in excess. Due to lymphoid neoplasia . 3. Cryglobulinemia It is condition in which formation of precipitate on cooling the serum, precipitate redissolves on warming. Due presence of cryoglobulins (made of IgG , IgM or mixture of two) in blood. Found in macroglobulimeia , SLE and myeloma.

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