On centrifugation blood gives rise to fluid and a cellular fraction. The fluid fraction is known as plasma and cellular fraction includes Red Blood Cells, leukocytes and platelets. The plasma consists of all proteins responsible for blood clotting and small soluble molecules and macro molecules of blood. If this plasma is allowed to clot, the fluid fraction that remains after clotting is known as serum. A classical experiment by A. Tiselius and E. A. Kabat in 1939 suggested that antibodies reside in serum. In their experiment they immunized Rabbit with protein Ovalbumin and divided the serum into two aliquots. Electrophoresis of one serum aliquot produced 4 peaks. These four peaks were denoted as α, β and γ globulin. The other serum aliquot was reacted with Ovalbumin and precipitate formed was discarded. The remaining serum proteins were electrophoresed which revealed significant drop in γ globulin. This suggested that γ globulin fraction of serum reacts with antigen so this γ globulin fraction was identified as immunoglobulin or antibody.
Structure of Antibody:
Antibody molecule consists of four peptide chains. Out of four two are identical light peptide chains of molecular weight 25000 and two are identical heavy chains of molecular weight 50,000.s Each light chain is bound to heavy chain by disulphide bond and by non covalent interactions such as salt linkage, hydrogen Bonds and hydrophobic bonds to form heterodimer. Similarly, two heavy chains are bounded by non covalent interaction and by disulphide bond. The highly variable region of about 110 Amino acid is present on both heavy chain and light chain which defined the specificity of antibody. These segments of highly variable sequence are called V region; VL in light chain and VH in heavy chain. These variable regions are termed as complimentarity determining region (CDR) on which antigens come to bind. The remaining region of antibody beyond variable region are known as constant or C region; CL on light chain and CH on heavy chain.
The constant region of light chain has two basic amino acid sequences so there are two types of light chains, one is known as Kappa (κ) & other is Lambda (λ). Similarly the constant region of heavy chain show 5 basic amino acid sequence pattern namely μ ,δ, γ, ε and α which gives rise to five types of immunoglobulin known as IgM, IgD, IgG, IgE and IgA respectively. These five different heavy chains are called isotypes.
Enzymatic cleavage of antibody:
When antibody is treated with enzyme papin, it give rise to three fragments. Two of which are identical and third one is different. The two identical fragment are known as Fab fragments ("fragment, antigen binding"). These fragments have antigen binding activity with molecular weight 45, 0000. The third one is termed as Fc fragment ("fragment, crystallizable") without antigen binding activity. It is names so because it is found to be crystallized when kept under cold condition. When antibodies are digested with pepsin, it generates a single 100,000 MW fragment known as F (ab') fragment which has antigen binding activity. Fc is not found to be present because it is digested into multiple fragments.
Type of Immunoglobulin
IgG is the most abundant class of immunoglobulin which counts about 80% of total serum immunoglobulin. They are the effective complement activator. IgG class of immunoglobulin also mediate phagocytes.
IgM constitute about 5%-10% and its concentration is in serum is 1.5 mg/ ml. It is the first antibody which is produced in primary response to the antigen. It is also firstly synthesized by the neonates. This class of immunoglobulin is present on the membrane of the B cell as monomeric form and also secreted by B cells in pentameric form. The five monomers are bind together by J chain so J chain is required for the polymerization of IgM. Because of its pentameric form it is strong complement activator.
IgA constitute about 10-15% of total serum immunoglobulin. It is present in external secretions such as breast milk, saliva, tears and mucus of genitourinary track, digestive track. It is normally secreted in dimeric form but in serum it is present in monomeric form. It provides immunity against certain bacteria like Salmonella, vibro and virus like Polio.
IgE concentration is too low in serum (0.3mg/ml). These classes of antibodies are involved in hypersensitivity reaction.
IgD constitute about 0.2% of total serum immunoglobulin. It is expressed on the membrane of B-cell along with IgM. It was first discovered in patient of multiple myeloma.
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