This presentation explains the general structure of immunoglobulins, action of papain, pepsin and mercaptoethanol on the structure of Igs and its functions.

1. DR. R. RENUKA, M.SC.,M.PHIL.,PH.D.
ASSOCIATE PROFESSOR OF BIOCHEMISTRY
V.V.VANNIAPERUMAL COLLEGE FOR WOMEN
VIRUDHUNAGAR, TAMILNAD, INDIA.

2. ANTIBODY
• An antibody, also known as an immunoglobulin (Ig), is a specialized
glycoprotein that is produced by plasma cells (activated B cells) after
stimulation by an antigen. Antibodies are the functional basis of humoral
immunity. Antibodies occur in the blood, in gastric and mucus secretions,
and in breast milk. Antibodies in these bodily fluids can bind pathogens
and mark them for destruction by phagocytes before they can infect cells.

3. GAMMAGLOBULIN
Serum treated with OVA
Ag
Immunized serum
The immunoglobulins derive their name from the finding
that they migrate with globular proteins when antibody-
containing serum is placed in an electrical field.

4. FORMS OF Ig
• Igs exist in two forms;
• Soluble Igs or which are found in dissolved condition in the blood serum and
body fluids.
• Serum Igs are heterogenous and polyclonal in nature as they are generated
against multiple epitopes of the antigens.
• Membrane bound Igs which are found on the surface of B cells. They are also
called as surface bound Ig or B cell receptors (BCRs).
• They play vital role in the initial stages of B cell activation.

5. HISTORY

6. GENERAL STRUCTURE OF ANTIBODIES
• Y- shaped heterodimer, composed of four polypeptide chains.
• Two identical short light (L) chains of molecular weight 25,000
Da each and
• Two identical long heavy (H) chains of molecular weight
50,000 Da or more.
• All the 4 PP chains are linked with each other by disulphide
bonds and non-covalent interactions such as hydrogen bonds,
hydrophobic bonds, ionic bonds and Vander walls forces.
• All chains have 2 ends – an amino terminal end (NH3) and a
carboxy terminal end (COOH).

7. TYPES OF H AND L CHAIN

9. VARIABLE AND CONSTANT REGIONS
• Each heavy and light chain in an immunoglobulin
molecule contains an amino-terminal variable
(V) region that consists of 100 to 110 amino acids
and differ from one antibody to another. The
remainder of each chain in the molecule – the
constant (C) region exhibits limited variation
that defines the two light chain subtypes and the
five heavy chains subclasses.
• The amino terminal portions, corresponding to
the variable regions (Fab portion), bind to
antigen; effector functions are mediated by the
carboxy-terminal constant regions (Fc region).

10. VARIABLE REGION
• The first 110 amino acid residues near amino terminal end of both L and H
chains constitute variable region and are mentioned as VL and VH
respectively.
• These regions represent Ag binding site or paratope of an antibody.
• Some zones (hot spots) within variable region show relatively higher
variability in amino acid sequences. These are called as hyper variability
region or complementarity determining regions(CDRs).
• There are 3 hot spots in L chain and 4 hotspots in H chain. These sites make
actual contact with epitope.

11. CONSTANT REGION
• Remaining part of Ig molecule at the base of the stalk of Y or in the C-
terminal end is called constant regions.
• Length; 104 amino acids for L chain; 330 AA for 𝜸, 𝜶 𝒂𝒏𝒅 𝜹 heavy
chains and 440 AA for 𝝁 𝒂𝒏𝒅 𝜺 heavy chains.
• AA sequence of constant region shows uniform pattern.
• A single Ab has 2 identical light chains and 2 identical heavy chains.

12. DOMAINS
• H and L chains further folded into domains
due to the presence of intrachain disulphide
bonds.
• Within a domain, a loop like structure of 60
AA is present.
• Each light chain has 2 domains; one VL
(variable) domain and the other is CL
(constant) domain.
• Each heavy chain has four domains; one
variable (VH) domain and 3 constant ( CH )
domains which are named as CH1,CH2 and
CH3.

13. • Ig M and Ig E have an additional constant
heavy chain domain called CH4.

14. HINGE REGION

15. FLEXIBILITY OF
HINGE REGION

16. ENZYMATIC DIGESTION OF ANTIBODY
REVEALS DIFFERENT FRAGMENTS

17. FUNCTIONS OF
ANTIBODIES

18. FUNCTIONS OF ANTIBODIES

20. FUNCTIONS OF ANTIBODIES

22. GENERAL FUNCTIONS OF ANTIBODIES
• Neutralization of infectivity (Fab fragment),
• Phagocytosis,
• Antibody-dependent cellular cytotoxicity (ADCC),
• Complement-mediated lysis of pathogens or of infected cells: Antibodies
activate the complement system to destroy bacterial cells by lysis
• Transcytosis, mucosal immunity & neonatal immunity
• Another function is unique to Immunoglobulin E (IgE), which is ‘activation
of mast cells, eosinophils and basophils’.

23. Antibodies are secreted into the blood and mucosa, where they can block the infectivity of
pathogens (bacteria, viruses, parasites and fungi), inactivate or neutralize foreign substances such
toxins. Neutralization generally occurs as a result of interfering with an organism’s attachment to
host tissues.

24. Phagocytosis
Antibodies facilitate phagocytosis of foreign substances by a process called opsonization.
The internalization and degradation of antibody-coated pathogens by macrophages and
neutrophils via FcRs (Fc receptors are protein molecules present on the surfaces of
macrophages and neutrophils which can bind the constant region of immunoglobulin
molecules) is a critical antibody function for clearance of pathogens in vivo.

25. Complement-mediated lysis of pathogens or of infected cells
• Antibodies (IgM and most IgG subclasses) activate the complement
system which can result in the lysis of organisms or of infected cells. An
important byproduct of the complement cascade is C3b, which is a
protein fragment that can bind non-specifically to cell and Ag-Ab
complexes.
• Organisms or Ag-Ab complexes bound by complement can be
internalized by phagocytic cells, with the resultant clearance.
Internalization through complement receptors on antigen-presenting cells
(APCs) can also result in the processing of antigen for presentation to T
lymphocytes.

26. Antibody-Dependent Cellular Cytotoxicity (ADCC)
• Antibodies have shown anti-microbial activity either directly or through interactions with
FcRs or complement. ADCC occurs when antibody forms a bridge between an infected
target cell (virus infected cells of the host) and an FcR-bearing effector cell, particularly
natural killer (NK) cells. The result of this three-way interaction is the death of the target cell,
either by lysis or apoptosis.

27. Transcytosis, Mucosal Immunity and
Neonatal Immunity
• Some antibodies can move across epithelial layers (depends on the
property of the constant region of that antibody molecule) via a process
called transcytosis. IgA is the major immunoglobulin that undergoes
transcytosis and is available in secretory form (sIgA) in the mucosal
surfaces of respiratory, gastrointestinal and urogenital tracts.
• In mammalian species including humans, most subclasses of IgG can
cross the placental barrier which confers protection of developing foetus
against pathogens. This passive immunization of developing foetus
occurs during the third trimester of gestation.

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