Antibodies

 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 calledVL

 

·              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