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Antibodies

Antibodies are glycoprotein molecules which are produced in response to an antigen, and reacts specifically with it in an observable manner

ANTIBODIES

 

       Antibodies are glycoprotein molecules which are produced in response to an antigen, and reacts specifically with it in an observable manner

 

·           Tiselius in 1937 analyzed serum by free zone electrophoresis and characterized proteins at pH 8.6

 

All proteins have negative charge and move towards anode

 


 

§           Tiselius and Kabat analyzed rabbit’s hyperimmune sera before and after absorption with immunizing agent

 

§   After absorption there was pronounced decrease in γ globulin

 

§   Hence antibody activity was traced down to γ globulin

 


 

·     γ globulin is not a homogeneous protein

 

·     In 1964 WHO international agreement selected the generic name Immunoglobulin for all antibody containing proteins

 

·     They subdivided immunoglobulin into different classes

 

Immunoglobulin classes

·     In man 5 major classes of Ig are described

 

-           Ig G    :  The major serum component

 

-           Ig M   : Macroglobulin

 

-           Ig A    : Present predominantly in secretions

 

-           Ig D    :  Important cell membrane receptor form

 

-      SgE : Antibody involved in hypersensitivity reac-209

 

Characterization of antibodies

·     Early physical – chemical studies were done with Ig G from horse, rabbit and human

 

·     Important structural features were predicted even before sophisti-cated studies were available

 

·     Molecular weight was calculated from sedimentation and diffusion studies

 

·     Asymmetrical and or non globular form by viscosity studies

 

·     Globular domain structure from unique susceptibility to proteolytic enzymes

 

·     Two antigen binding sites by hapten antibody reactions

 

·     Thus early studies predicted three functional domains and have been confirmed

 

Structure of antibodies

·     Before going into the structure of antibody, one must know the structure of proteins

 

·     Proteins are made from amino acids

 

·     Amino acids form poly peptide chains

 

·     Polypeptides form proteins

 

·     Proteins have 3 dimensional structure

 

o     If any change in the primary sequence of amino acid in polypeptide

 

o     Or in three dimensional structure there is change in the property

Digestion with enzymes

·        Rodney and Porter digested rabbit Ig with the enzyme papain

 

·              It cleaved the molecule and produced two major fractions and a small amount of short peptides

 

·     One fraction (MW 45,000) still possessed antigen binding site and was named as fragment antibody binding (Fab)

 

·     The other fragment could be crystallized, and was called Fragment crystallized (Fc)

 

·     Fab possessed antigen binding site but was monovalent

 

·              Possessed one reactive site

 

·              Could not cross link antigen molecule

 

·     When one added up the molecular weights of Fab and Fc frag-ments, plus the observation that the Fab was monovalent, it ap-peared that the original antibody contained:

 

·              Two Fab fragments and one Fc fragment

 


 

General formula for antibody

·     The general formula for antibody is (H2 L2)n

 

·     The immunoglobulins are made of 2 heavy chains and 2 light chains

 

·     These are held together by covalent bonds

 

·     These bonds are interchain disulphide bridges

 

·     Each chain is made of a number of loops

 

·     These loops are known as domains

 

·     Each domain is formed by intrachain disulphide bonds

 

·     There are 2 loop sections per L chain and 4 loop sections per H chain

 

·     There are two terminals in each chain

 

·              One is called C terminus

 

·              Other is called N terminus

 


Digestion with pepsin

 

·              When antibody molecule was treated with enzyme pepsin, it was cleaved in different fashion

 

·              A large fragment with two antigen binding sites and smaller fragments

 

·              The larger fragment was called (Fab)2 fragment

 

 

Light chain (Fig 33.6)

·     C terminus contains the constant region

 

·     N terminus contains the variable region

 

·     L chain is named as Kappa (k) and lambda (λ )

 

·              contains two domains

 

·              domain at N terminus is variable domain of light chain calledVL

 

·              domain at C terminus is constant domain called CL

 


 

Types of heavy chain

·     There are 5 different types of H chains

 

·     Based on the type of H chain the classes of antibody is determined

 

They are:

 



 

Properties and functions of immunoglobulins

Ig M

·     Ig M is the main immunoglobulin produced early in primary im-mune response

 

·     It is present on the surface of all uncommitted B lymphocytes

 

·     IgM is a pentamer and the valence is 10

 

·     Ig M is the most efficient immunoglobulin in agglutination, comple-ment fixation and other antigen antibody reactions

 

·     It plays an important role in the defense against bacterial and viral diseases

 

·     It does not cross placenta

 

Ig G

·     Ig

 

·     G has two identical antigen binding sites and is bivalent

 

·     There are four subclasses namely Ig G1,Ig G2, IgG3 and IgG4

 

·     Ig G is the predominant antibody in secondary immune response

 

·     It plays an important role in defense against bacteria, viruses

 

·     It also neutralizes toxins

 

·     It crosses placenta and is found in large quantities in newborns

 

Ig A

1.     Ig A is found mainly in secretions like milk, tears, saliva and secre-tions of respiratory, intestinal and genital tracts

 

2.     It protects the mucus membranes against microbial attack

 

3.     As many microbes enter the body through these mucus membranes, Ig A offers the first line of defense

 

4.           Each IgA molecule consists of two H2L2 units and a J chain and a secretory component.

 

5.     The secretory component is a polypeptide synthesized by epithe-lial cells and it helps IgA to pass the mucosal surface

 

IgE

·     IgE antibody is present in increased quantities in allergic individu-als\

 

·     The Fc portions of the molecule binds to mast cells and eosinophils

 

·     When this antibody combines with its antigen on the mast cell sur-face, it leads to allergic response

 

IgD

·              IgD has no antibody function

 

·        It may act as antigen receptor on cells

 

·              In serum it is present in only trace amounts


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