Part II

Immunology

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 ANTIBODY

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 ANTIBODY

• Humoral basis of immunity

• Secreted by plasma cell
• Present on B cell membrane
• React with antigen - specifically observable
• Antibody - phagocytosis, phagocytosis,
ADCC
• Sera - high antibody levels - ‘immune sera’

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 ANTIBODY

• Fractionation of immune sera - soluble

albumin, insoluble globulin
• Globulins - water soluble pseudoglobulin,
insoluble globulin
• Most antibodies - globulins
• Tiselius (1937) - serum proteins - albumin,
alpha, beta, gamma globulin
• Antibody activity – gamma globulin fraction

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 ANTIBODY

Serum electrophoresis showing γ globulin nature of antibodies

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 ANTIBODY

• 1964 – WHO - immunoglobulin

• Protein - known antibody activity and other
proteins related to them by chemical structure
• Includes - abnormal proteins – myeloma
• Macroglobulinemia
• Cryoglobulinemia

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 ANTIBODY

• Immunoglobulins - synthesised by plasma

cells and by lymphocytes
• Immunoglobulins - structural and chemical
concept
• Antibody - functional and biological concept
• All antibodies - immunoglobulins
• All immunoglobulins - may not be antibodies

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 ANTIBODY - STRUCTURE

ENZYME DIGESTION
Rabbit lgG antibody - to egg albumin
digested by papain
split - two fractions
Insoluble fraction Soluble fraction
Crystallised in cold Bound with egg albumin
Fc (crystallisable) Fab (antigen binding)

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 ANTIBODY STRUCTURE

Each molecule of immunoglobulin

Split by papain - three parts
One Fc - Two Fab
When treated with pepsin
Two Fab fragments - held together
Bivalent - F(ab)2
Fc portion – digested - small fragments

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 ANTIBODY STRUCTURE

Basic structure of an immunoglobulin molecule and the fragments obtained

by cleavage by papain and pepsin
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 IMMUNOGLOBULIN CHAINS

• Each molecule - 4 polypeptide chains

• Smaller chain - Light (L) chain
• Longer chain - Heavy (H) chain
• ‘L’ chain attached to ‘H’ chain by disulphide
bond
• Two ‘H’ chains - joined together by 1-5 S-S
bond

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 IMMUNOGLOBULIN CHAINS

H chain-structurally, antigenically distinct

Designated by Greek letter
Immunoglobulin class H chain
lg G  (gamma)
lg A α (alpha)
lg M  (mu)
lg D  (delta)
lg E  (epsilon)

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 IMMUNOGLOBULIN CHAINS

• L chains - similar in all classes of

immunoglobulins
• Two varieties - kappa (K), lambda ()
• A molecule of immunoglobulin may have
either kappa or lambda, never both
• Kappa andlambda named after Korngold
and Lapari
• Each light chain is bound to a heavy chain
by interchain and intrachain disulphide
bonds
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 IMMUNOGLOBULIN CLASSES

• Antigen combining site - at amino terminus

- composed of both ‘H’ and ‘L’ chains
• Amino terminal - amino acid sequence-
variable – variable region - antigen
recognition
• Carboxy terminal - constant region-
effector functions
• Infinite range - antibody specificity

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 IMMUNOGLOBULIN

Structure of immunoglobulin molecule

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 HYPERVARIABLE REGION

• Amino acid sequence of variable region of L

and H chains
• Consist of relatively invariable and highly
variable zones
• Highly variable - three in L four in H
• Hypervariable - ‘hot spots’ sites on
hypervariable regions that make contact with
epitope ‘complementary determining region’
or CDR

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 IMMUNOGLOBULIN CLASSES

• IgG
• IgA
• IgM
• IgD
• IgE

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 IgG

• Major serum immunoglobulin 80% of total

• Mol wt - 150,000(7S)
• Half-life - 23 days
• Level raised - chronic malaria, kala azar,
myeloma
• lgG - transported across placenta
• Natural passive immunity - newborn

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 IgG

• lgG - binds microorganism - enhances their

phagocytosis
• Extracellular killing - target cell - coated
with lgG antibody
• Mediated through recognition of surface of
Fc fragment
• lgG complex - platelet Fc receptor
• Aggregation of vasoactive amines

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 IgG

Structure of immunoglobulin molecule

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 IgG

• Participates in - complement fixation,

precipitation, neutralisation of toxin and
viruses
• General purpose antibody
• Protective against infections agents
• Passively administered - suppress homologous
antibody
• IgG late antibody, initial immune response IgM
• Subclasses - lgG1, lgG2, lgG3, lgG4

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 IgA

• Seondmost abundant class

• 10-13% of serum immunoglobulin
• Half-life of 6-8 days
• Major immunoglobulin – colostrum,
saliva, tears

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 IgA

• Two forms - Serum lgA, Secretory lgA

• Serum lgA - monomer
• IgA on mucosa, in secretions - dimer
• Two monomeric units - together at the
carboxy terminal by glycopeptide - J chain
(joining)
• Dimeric secretory lgA - synthesised by
plasma cells
• J chain also synthesised by plasma cells
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 IgA

Secretory IgA molecule

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 SIgA

• Secretory lgA - contains glycine-rich

polypeptide, called the secretory component
or secretory piece
• Produced by mucosal/glandular epithelial
cells
• Dimeric lgA - binds receptor on epithelial
cell, endocytosed transported to luminal
surface

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 SIgA

• During the process - part of receptor

remains attached to lgA dimer
• Secretory component
• Secretory piece - protects lgA from
denaturation - by bacterial proteases in the
intestinal mucosa
• SlgA much larger than serum lgA

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 SIgA

• Selectively concentrated in secretions and

on mucus surfaces
• Role in local immunity against respiratory
and intestinal pathogens
• Resistant to digestive enzymes
• Inhibit adherence of organism to mucosa
activates - alternate complement pathway
• Promotes phagocytosis and intracellular
killing of microorganisms

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 IgM

• 5-8% of serum immunoglobulin

• Half-life of 5 days
• Heavy molecule - millionaire molecule
• lgM – polymer - five peptide subunits
• Polymerisation of subunit depends on J chain
• Most lgM - intravascular
• Earliest immunoglobulin to be synthesised

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 IgM

IgM molecule
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 IgM

• Not transported across placenta

• lgM in fetus/newborn - indicates
intrauterine infections
• Short lived, disappear earlier than lgG
• Presence in serum - recent infection

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 IgM

• Unique structural features - suited to

biological role
• Protect - microorganism and other large
antigens
• Function lgM lgG
• Immune hemolysis single molecule 1000
molecule

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 IgM

• Opsonisation 500-1000 times more

effective than lgG
• Bactericidal action 100 times more
• Bacterial agglutination 20 times more
• Neutralisation of toxin, virus-less active
than lgG

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 IgD

• Resembles lgG structurally

• Concentration-3 mg/100 ml serum
intravascular
• Surface of unstimulated B lymphocyte
• Recognition receptor for antigen

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 IgE

• 1966 Ishizaka - atopic reagin molecule

• Structurally resemble lgG
• Affinity for surface of tissue cells (mast
cells)
• Mediates- Prausnitz-Kustner reaction
• Does not cross placenta
• Does not fix complement
• Elevated levels – asthma, hay fever, eczema

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 IgE

• Produced in lining of respiratory and

intestinal tract
• Responsible for anaphylactic type of
hypersensitivity
• Protection by mast cell degranulation and
release of inflammatory mediators

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 ABNORMAL IMMUNOGLOBULIN

• Structurally similar proteins - pathological

processes
• Multiple myeloma – 1847, Bence-Jones-
multiple myeloma light chain
immunoglobulin - kappa or lambda
• In single patient - kappa/lambda, never
both

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 ABNORMAL IMMUNOGLOBULIN

• Myeloma - plasma cell dyscrasia

• Unchecked proliferation of one clone of
plasma cells - monoclonal
• Multiple myeloma may affect plasma cell
synthesis of lgG, lgA, lgD, lgE

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 ABNORMAL IMMUNOGLOBULIN

• Waldenstrom’s microgloblinemia -
lgM-producing plasma cells
• Heavy chain disease - lymphoid
neoplasia over production of Fc part of
heavy chain
• Cryoglobulinemia – gel or precipitate
forms on cooling serum

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 IMMUNOGLOBULIN SPECIFICITIES

ISOTYPE
• Variations in the constant region of the
heavy chain of immunoglobulin

Isotypes, allotypes and idiotypes of antibodies

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 IMMUNOGLOBULIN SPECIFICITIES

ALLOTYPE
• Multiple alleles for genes leading to subtle
amino acid differences in some members of
a species

Isotypes, allotypes and idiotypes of antibodies

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 IMMUNOGLOBULIN SPECIFICITIES

IDIOTYPE
• Determinants that arise from heavy and
light chain variable region

Isotypes, allotypes and idiotypes of antibodies

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 ANTIBODY DIVERSITY

• Presence of a large number of antibodies

that bind different antigens/pathogens
• Significance – to develop engineered
antibodies for therapeutic purpose

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 CLASS SWITCHING

• On antigenic stimulation, the H chain VDJ

unit can join any constant heavy gene
segment and express the particular
antibody class
• This process is class switching
• Significance – class switch recombination
deficiencies
• Hyper IgM syndromes

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