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ANTIBODIES DR.JAGADEESH (P.G) 21-2-2015 2013 IR BATCH ANTIBODIES • Antibodies are globulin proteins “IMMUNOGLOBULINS” that react specifically with the antigen that stimulated their production. • “All antibodies are Immunoglobulin’s but all Immunoglobulin’s may not be antibodies”. • They make up 20-25 % of proteins in blood plasma. • Blood contains 3 types of globulins based on their electrophoretic migration rates. • They are called as α, β and√ globulins. • The antibodies are gamma globulins and there are 5 antibody classes. • IgG, IgA, IgM, IgD and IgE. • Antibodies that arise in an animal in response to typical antigen are heterogeneous • As they are formed from a different clone of plasma cells, they are polyclonal. • If antibodies arise from a single clone of cells e.g. in a plasma cell tumor (myeloma) they are Homogeneous i.e. they are monoclonal. Function of antibody • Mainly to neutralize toxins and viruses. • To opsonize bacteria and make it easier to phagocytize. • They have a role in allergy e.g. hay fever and in auto immune diseases. Immunoglobulin structure • Immunoglobulin are glycoproteins made up of light (L) and heavy (H) polypeptide chains. • Light and heavy refer to molecular weight.L-25,000. H-50 to 70,000. • The simplest antibody molecules is “Y” shaped having four polypeptide chains- two ‘H’ chains and two light ‘L’ chains. • The 4 chains are linked by disulphide bonds. • Always individual Ab molecule has identical ‘H’ & ‘L’ chains. • This is mainly due to allelic exclusion and regulation within B cell which ensures the synthesis of either Kappa K or Lambda λ Light chains (L) but not both. • In humans the ratio of Ig having KL and λL chain is approx 2:1. • High resolution X-ray crystallography reveals that L & H chains are subdivided into Variable and constant regions. • The regions are composed of 3 dimensional folded, repeating segments called domains. • All ‘L’ chain consists of one variable [VL] and one constant [CL] domains. • Most ‘H’ chain consists of one variable [VH] and three constant [CH] domains. • IgG, IgA has 3 CH domains, IgM, IgE – have 4. • Each domain is apex 110 amino acid long. • The variable regions are responsible for antigen binding where as constant region are responsible for various biological functions e.g. complement activation, binding to cell surface receptors, activation of phagocytes. • Complement binding site is in the CH2 domain. • The variable regions of both ‘L’ & ‘H’ chain have 3 extremely variable a. acid sequences at the amino terminal and that forms the antigen binding site. • This is the hyper variable region – CDR’s (complementarities determining regions). • Only 5-10 a. acid in each hyper variable region for the antigen binding site. • The Ag- Ab binding involves electrostatic forces, van- der-Vaal forces and hydrogen and hydrophobic bonds rather than co valent bonds. • Remarkable specificity is due to the hyper variable region. • L chain belongs to either kappa K or Lambda λ. • Both types occur is all Ig classes but any one Ig molecule contain only one type of L chains. • The amino terminal portion of each L chains participate in the antigen binding site. • H chains are distinct for each Ig classes and are designated as √,α, µ, €,∂. • The amino terminal portion of each of H chain participate in the antigen binding site. • The carboxyterminal form the FC fragment – have biological activities. • If Ab molecule is treated with a proteolytic enzyme papain the peptide bonds in the ‘hinge’ region are broken producing 2 identical Fab fragments – which carry antigen binding site and one Fc fragment which is involved in placental transfer, CF, attachment site of various cells and other biological activities. • If pepsin acts on Fab – 2 fragments are formed. • Fc portion also has a Fd piece which carry carbohydrate moiety distinct for each class of Ig. Immunoglobulin classes: • IgG- each IgG has two L and two H chain linked with disulfide bonds [Mol.formula H2L2]. Because of 2 antigen binding site it is divalent. • There are four subclasses IgG1 – IgG4 based on antigenic differences is H chains as also on the no., location of disulfide bonds. IgG1 makes up to 65% of total IgG. IgG2 antibody is directed against polysaccharide antigen and is an important host defense against encapsulated bacteria. IgG is mainly serum Immunoglobulin. It is predominant Ig is secondary response and is defense against bacteria and viruses. • It is only antibody to cross the placenta. • Its Fc portion binds to the receptors on the placental cells surface. • So it is also the most abundant Ig is new borns. • It is one of the two Ig [other being IgM] which can activates complement. • IgG is immunoglobulin that opsonizes. • It can opsonize i.e. enhance phagocytosis, because there are receptors of √ H chains on surface of phagocytes. • IgM also opsonizes but indirectly by activating complement. i.e. produce C3b which is are opsonin having binding sites on surface of phagocytes. IgA – Main Ig is secretion such as colostrums, saliva, tears, respiratory tract, intestinal and genital tract secretions. • It prevents attachment of micro organisms. Eg. Bacteria and viruses to mucous membranes. • But bacteria having IgA protease can make it inactive and still cause disease. • Each secretory IgA consists of two H2L2 units plus one molecular each of J[joining] chain and secretory component. • Only IgA and IgM have J chains. • These two Ig exist as multimers i.e., dimer and pentamer. • The J chain initiates polymerization process and multimers are held together by disulphide bonds between their Fc regions. • The secretory component is a polypeptide synthesized by epithelial cells that provide the passage for IgA to the mucosal surface. • It also protects IgA from being degraded in intestinal tract. In serum some IgA exists as monomeric H2L2. • IgM :- Is main Ig produced early in primary response. It is present as monomer on surface of virtually all B cells. • Here it function as antigen binding receptor. • The surface monomer and serum IgM have µ heavy chains. But the heavy chain of surface IgM have a hydrophobic sequence that mediates binding in the cell membrane where as serum IgM does not. • In serum IgM is a pentamer, millionaire molecule having 5 H2L2 units plus one molecule of J chains. • Because it is pentamer it has 10 antigen binding sites. • It is the most efficient Ig in agglutination, CF [activation] and other antibody reactions and also is most important defense against bacteria and viruses. • It can be produced by fetus in certain infections. • So its detection from foetal blood indicates congenital infections. Why? It cannot cross the placenta. • It has the highest avidity of the Igs. Its interaction with antigen can involve all 10 binding sites. • 2-mercaptoethonol can destroy it. • • • • • • • • Ig D – Has no known Ab functions. But may function as an antigen receptor. It is present on surface of many B cells. It is in small amounts in serum, It may also the present on some lymphatic leukemia cell surface. IgE – It is medically important for 2 reasons It mediates immediate [anaphylactic] hypersensitivity. It participates in host defense against certain parasites eg. Helminths [worms] The Fc region of IgE binds to surface of mast cells and basophils. • Bound IgE serves as a receptor for allergens or antigen. • When the antigen binding sites of adjacent IgE are cross linked by allergens, several mediators are released by the cells and immediate [anaphylactic] hypersensitivity reaction occurs. Although IgE is in trace amounts in normal seen [approx 0.004%], persons with allergic reactivity have greatly increased amounts and IgE may appear is external secretions. • IgE does not fix complement. It does not cross placenta. • IgE is important host defense against certain important worm infection – stronglyloides trichenella, ascaris and hook worms. • IgE levels increased in these infections. • Because the worms are too large to be phagocytosed, they are killed by Eosinophils that release worm destroying enzymes. • IgE specific for worm proteins binds to receptor on Eosinophils triggering ADCC response. ISOTYPES, ALLOTYPES AND IDIOTYPES. • As Ig are proteins they themselves can be antigenic. • So this property allows us to subdivide them into Isotypes, allotypes and Idiotypes. • Isotype – are defined by antigenic i.e., a acid differences in their constant region. Though they are antigenically different all isotypes are found in all normal humans. Eg. IgG and IgM are different isotypes – the constant region of heavy chains √ and µ is different antigenically. Similarly H chains of IgG, IgM, IgA, IgD and IgE are also antigenically different so different isotypes. IgG further divided into four subclasses IgG1-IgG4 based on ‘H’ differences. • Similarly IgA - IgA1 & IgA2. • k and λ chains are different isotypes as then constant region differ antigenically. • Allotype – These are additional antigenic features of Ig that vary among individuals. • This is due to genes that code for L & H chains are polymorphic and individuals can have different alleles. • Eg. √ H contains an allotype called Gm because one or two a.acid difference in this will lead to different antigenicity to the molecule. • Each individual will inherit different allelic genes that code for one or another a.acid at the Gm sites. • Allotypes refered to as Gm in case of √ H chains – abbreviation of gamma. Allotypes related to KL is referred as inv an abbreviation of patients name. • Idiotypes – These are antigenic determinants formed by specific amino acid in hyper variable regions. Any of these is antigenic determinant is idiotype. Each idiotype is unique for the Ig molecule produced by a specific clone of plasma cells [i.e. Ab producing cells]. • Anti idiotype antibody will react only with hyper variable region of the specific Ig molecule that induced it. IMPORTANT FUNCTION OF IMMUNOGLOBULINS Immunoglobulins Major functions IgG - Main antibody is secondary response - Opsonize bacteria [ easy to phagocytose] - Fixes complement – enhance bacterial killing - Neutralizes bacterial toxins and viruses - Crosses the placenta IgA - Secretory IgA prevents attachment of bacteria and viruses to mucous membranes. - Does not fix complement. IgM - Produced in the primary response to an antigen Fixes complement Des not cross placenta Antigen receptor on the surface of B cells IgD - Uncertain - Found on surface of many B cells, lymphatic leukemia cells. - Also in serum IgE - Mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to antigen [allergen]. - Defends against worm infection by causing release of enzymes from Eosinophils. - Does not fix complement - Main host defense against helminth infections. PROPERTIES OF HUMAN IMMUNOGLOBULINS Property IgG IgA IgM IgD IgE 75 15 9 0.2 0.004 1000 200 120 3 0.05 3. Sedimentation coefficient 7S 7S 19 S 7S 8S 4. Mol. Wt [x 1000] 150 170 or 400 900 180 190 5. Serum half life [days] 23 6 5 2-8 1-5 Monomer Monomer 1. % of total IgM serum [approx] 2. Serum concn. [mg/dl][approx] 6. Structure Monomer Monomer Monomer Or Or Dimer Pentamer Property IgG IgA IgM IgD IgE 8. Complement fixation + - + - - 9. Transplacental passage + - - - - 10. Mediation of allergic responses - - - - + 7. H-chain symbol Property IgG IgA IgM IgD IgE 11. Found in secretions - + - - -/+ 12. Opsonization + - Indirectly - - 13. Antigen receptor on B cell - - + + - 14. Polymeric form has J chain - + + - - • IgA - 11s form in secretions [ eg. Saliva, milk, tears] and fluids of resp. tract, G.I tract, Genital tract. • IgM – Indirectly opsonises by activating complement. Producing C3b which is an opsonin.
