ANTIBODY STRUCTURE

Chapter 5: Cellular and Molecular Immunology

Abbas, Lichtman, Pillai 8th Ed., 2015
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3-Aug-21 PJC 3

What’s an Antibody?
• A ‘Y’ shaped protein consisting of two identical heavy chains and two identical light chains
• The end of the Y region recognises a particular epitope on an antigen
• The antigen binding sites on the variable region of a single Ig molecule are identical
• Often abbreviated to Ab or Aby
• Also known as an immunoglobulin (Ig for short) - – 5 principle types in humans (IgA, IgD, IgE, IgG, IgM)
• Expressed on naïve B cells (one that hasn’t encountered antigen yet) – both IgM and IgD on the same B cell
• Secreted by plasma cells (B cell lineage) and expressed on memory B cells
• Some antibody molecules (IgA and IgM) consists of multiples of the basic four chain ‘Y’ structure

Memory B cell

OR

Naïve B cell B cell – 1st Contact with Antigen Plasma cell

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3 Dimensional ‘Y’ Shaped Molecules

How big are they?

“An IgG would about fit in a box of about 16 nm by 12 nm by 7nm (based on
structure). So the longest dimension of the antibody would be 500 times smaller
than the diameter of a RBC.” Annemarie Honegger, University Zurich, 2016

• Image Courtesy : upload.wikimedia.org/wikipedia/commons

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Immunoglobulin Structure
H - heavy chain
L - light chain
• Two types of light chain possible
• kappa or lambda (not both)
CH1
• Either (2 x κ or 2 x λ) light chains
• Variation in CL region of light chain

C - constant regions
CL
Fold domains • Same for each class/isotype of Aby molecule
• Heavy chain constant regions (CH)
• CH1 to 3 in IgG, IgA, IgD
• CH1 to 4 in IgM, IgE
• Light chain constant regions (CL)

V - variable regions
• Antigen binding sites (vary per molecule but the two are identical)
• Variable light chain regions (VL)
• Variable heavy chain regions (VH)

SS – di-sulphide bonds
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Immunoglobulin Fold Domains

IgG, IgD, IgA IgM, IgE

• Kuby Immunology, 6th Ed. 2007

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Five human Immunoglobulin classes/isotypes

Mu
Delta
Gamma
Alpha
Epsilon

• The heavy chains can be distinguished from each other based upon the content of their constant regions (see diagram slide 8)
- Across the 5 classes, the constant region varies according to:
• Protein sequences
• Carbohydrate composition
• Size
• But, the constantC region remains the same within each class/isotype
i.e. all IgA molecules within an individual have the same HeavyC and LightC composition
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Immunoglobulin Fragments
• Enzymatic digestion with papain
• Produces two Fab fragments plus
• Fc portion
• Enzymatic digestion with pepsin
• Produces one F(ab’)2 fragment
• Reduction of di-sulphide bonds with mercaptoethanol
• Produces two separate light chains and
• Produces two separate heavy chains
• Monoclonal Abs can be made against fragments
• Research applications

Fab – fragment antigen-binding

Fc – fragment crystalisation
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Y shaped molecules

• IgM exists as a pentamer

• IgA (including the secretory form at mucosal surfaces) exists primarily as a dimer
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• IgA is most prevalent overall (serum + tissue)

in Serum

IgA and IgE

Parasites
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Immunoglobulin Subclasses
• Only IgG and IgA have subclasses in humans
• IgG
• IgG1, IgG2, IgG3, IgG4
• Occur due to variations in the number of S=S bonds in the heavy chains
• Subclasses vary in prevalence in the body (concentration)
• Vary in function

• IgA
• IgA1, IgA2
• Subclass A1 predominates
• Relative proportions vary according to tissue
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• Binding to Fc receptors – includes

opsonisation and antibody dependent
cell mediated cytotoxicity
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Immunoglobulins as immunogens
• Immunoglobulins contain epitopes that can induce an immune response in other individuals or species
• Three types of antigenic determinants (epitopes) on immunoglobulin (Aby) molecules
1. Isotypic – epitopes on the constant regions of both the heavy and light chains of an antibody class
(g, µ, a, d, e) that are the same within a species.
- i.e. all IgG1 molecules in all mice have the same set of epitopes
- The epitopes may be different between mouse IgG, IgA, IgM, IgE, IgD.

YY
2. Allotypic- epitopes
3. Idiotypic – epitopes
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Allotypic and Idiotypic Determinants

• Allotypic epitopes - on the constant region of both heavy and
light chains of an antibody class that vary between individuals
of the same species i.e. Mouse strain A and Mouse strain B
Isotypic epitopes (same within a species)

• Idiotypic epitopes - on the variable region of both heavy and light chains
- Epitope variations between individual antibody molecules of the same class (g, µ, a, d, e) or subclass within an individual
3-Aug-21 PJC 17

Valency and Avidity of Antibodies (Abbas Ch 5)

Avidity is the overall strength of binding between an antigen &
an antibody molecule due to the number of contact points

• The epitopes are too far apart for both ends of the variable regions to bind
• IgE doesn’t have a hinge region – no flexing

• The antibody molecule is able to bind to the epitopes on two identical antigens
• IgG, IgA, IgD have a hinge region which allows flexing

• IgM has 10 identical binding sites but cannot flex (no hinge regions)
• Even if the affinity is low, with 10 binding sites, the avidity can still be very high

•
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• Affinity is the strength of

binding between one antibody
binding site and one epitope

• The better the fit, the better the

strength of the bond
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Antigen-Antibody Complexes (Abbas Ch 5)

• At an optimal ratio of Aby to Ag, large immune complexes are formed

• In vitro, these are visible as precipitates
• In vivo, large immune complexes can result in disease e.g. rheumatic fever, glomerular nephritis
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Detection of diphtheria toxin: Antigen-Antibody Complexes

Corynebacterium
diphtheriae strains

Filter paper strip soaked with anti C.diphtheriae toxin

antibodies (antibodies that can bind to the toxin) NB: Only toxigenic strains are pathogenic – encoded by a toxin pos phage
 3-Aug-21 PJC 21

B cells and Antibody Production

1° Antigen

Inside the Bone Marrow

Production
requires
T cell help

2° Antigen

• Upon primary exposure to an antigen, a naïve mature B cell either differentiates into a plasma cell or a memory B cell
• Plasma cells produce/secrete antibodies
- Production of IgG, IgA or IgE requires T cell help (steps not shown above)
- IgM can be produced by B cells independently of T cell help
• Memory cells express antibody on their surface but do not produce antibody (become plasma cells) until reactivated by their
specific antigen upon reinfection/re-exposure
- Long lived memory B cells produce either IgG, IgA or IgE (all require T cell help)
- Short lived memory cells only produce IgM (no T cell help)
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Primary vs. Secondary Immune Response - BASIC

• Immune memory allows a more rapid and larger immune response after 2° contact
• Immunisation utilises this knowledge
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Antibody Production following Immunogenic Stimulus

• NB: The actual time will vary according to type of antigen, route of administration/point of contact
• For vaccination, the presence or absence of adjuvant, species, strain (genetics) of animal will also influence the IR
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Study Questions
1. Excluding disulphide bonds, what are the four primary components of an immunoglobulin molecule?
2. Which part of the immunoglobulin molecule is responsible for antigen recognition?
3. Name two cell types that express immunoglobulin on their surface?
4. Name the five classes of human immunoglobulin? Draw a basic schematic diagram of each.
5. Which cell type is responsible for actively secreting immunoglobulin? Which of the human immunoglobulin isotypes in not
produced by this cell?
6. What must happen for a memory B cell to transform into a ________ cell and begin producing antibody? Which isotype of
antibody will be produced if there is no T cell help?
7. Which two human immunoglobulins have an extra fold domain in their heavy chains?
8. Which part of the immunoglobulin molecule is responsible for biological activity (once antigen has been bound to the Aby
molecule)?
9. Using diagrams, explain the fragments that are produced when an immunoglobulin molecule is treated with a) pepsin, b)
papain and c) mercaptoethanol
10. Which human immunoglobulin is the most prevalent in serum and which is able to cross the placenta?
11. Which human immunoglobulin can best activate complement?
12. Which human immunoglobulin (only one) can bind to the Fc receptors on PMN, Mϕ, NK cells?
13. What is the main structural difference between the human subclasses of IgG?
14. During a humoral immune response, which immunoglobulin isotype is produced first? Which is produced second?
25-Jul-23 PJC 1

ANTIBODY FUNCTION

Chapter 13: Cellular and Molecular Immunology

Abbas, Lichtman, Pillai 8th Ed., 2015
25-Jul-23 PJC 2

ELECTRONIC WARNING NOTICE FOR COPYRIGHT STATUTORY LICENCES

WARNING

This material has been reproduced and communicated to you by or on behalf of Curtin
University in accordance with section 113P of the Copyright Act 1968 (the Act)

The material in this communication may be subject to copyright under the Act. Any
further reproduction or communication of this material by you may be the subject of
copyright protection under the Act.

Do not remove this notice.

25-Jul-23 PJC 3

Humoral Immunity è Production of Antibodies

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Acquired IR to extracellular bacteria Dendritic Cell

Importance of T cells
≈ 8 hours in contact

≈ 2-3 days

Th1

Th2

IgG2

IgG4 IgD
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Immunoglobulin “Y shaped” globular molecules

• Primary function:
- Antibodies stick to target molecules
- Epitopes on antigens

Antibody molecules on a virus

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Antibodies bind to specific epitopes on antigens

• In this example, the bacteria is the antigen/immunogen and the surface molecules are the epitopes (antigenic determinants)
- Two different immunoglobulin molecules have bound to two different epitopes
• The antigen binding sites on the variable region of a single Ig molecule are identical
- The variable regions (both L, H) are encoded by the same respective gene segments
25-Jul-23 PJC 7

IgG attached to Streptococcus pyogenes

Localization and orientation of polyclonal IgG at the bacterial

surface. (A and B) Negative staining EM was used to visualize the
localization and orientation of IgG bound to the bacterial surface. Gold-
labeled IgG is found either widely scattered (A, 10,000 µg/ml IgG) or
found along the midsection of the surface proteins (B, 1 µg/ml IgG) of
wild-type S. pyogenes. Bar, 100 nm. Images are representative of two
independent experiments. (C and D) High magnification shows single
IgG molecules bound either via Fab (C, 10,000 µg/ml IgG) or via Fc (D,
1 µg/ml IgG). Two representative images with pseudocolor variants are
shown of each experiment. Bar, 25 nm.

DOI: 10.1084/jem.20120325 | Published December 10, 2012

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Biological role of Antibody/Antigen interactions

• Activation of complement
• Antibody Dependent cell-mediated cytotoxicity (ADCC)
• Passive Immunity
• Neutralisation
• Toxins
• Viruses

• Opsonisation
• Agglutination
• Immobilisation of bacteria/RBC/particles/other molecules
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• Binding to Fc receptors – includes

opsonisation and antibody dependent
cell mediated cytotoxicity
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Serum

IgA and IgE

Parasites
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Activation of Complement – What is complement?

• The complement system consists of a series of plasma proteins that are part of the innate immune system
- Innate immune system = not adaptable and does not change over a lifetime
• Functions of the complement system
- Induce cell lysis (formation of the membrane attack complex - MAC)
- To promote an inflammatory response (includes many processes and functions) including;
• degranulation of eosinophils, basophils/mast cells
• chemotaxis of leucocytes
• aggregation of platelets
- Promote phagocytosis (opsonisation of antigen/particles with complement C3b – recognised by phagocytes)
- Remove immune complexes (also via C3b)

• 3 activation pathways
Y
- Classical Pathway – Antibody mediated
- Alternative Pathway
- Lectin Pathway Y
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Antibody Activation of Complement

• Cell Lysis via the Classical Pathway
• Cascade of biological reactions leading to formation of the Membrane Attack Complex
• Many, many holes per cell
• Initiated by Antibody/Antigen binding
- (IgG and IgM) IgG3++, IgM++ , IgG1+, IgG2±
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Antibody Dependent cell-mediated cytotoxicity (ADCC)

• Natural Killer cells (NK cells) detect antibody coated cells via the exposed Fc receptor(s)
- A mechanism for the removal of tumour cells and virally infected cells
- Release toxic granules to kill the target cell
• Antibody isotype: only IgG (?ADCC with IgE, parasites and eosinophils – see next)
• NK cells can also target a cell by recognising the absence of surface MHC class I molecules
- See Missing self hypothesis (Brian discussed concept in L3)
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Immunoglobulin E (? Also an example of ADCC)

• Least abundant isotype
• Main function is the immune response to parasites
- Once manufactured, IgE binds to the surface of the parasite
- Fc receptors on eosinophils and mast cells bind to the Fc portion of IgE
- Degranulation of eosinophils (release of peroxidase, lysosomal enzyme, ‘major basic protein’)
25-Jul-23 PJC 16

IgE and Allergy

• Allergic response – usually an inappropriate IR
- Can trigger very powerful inflammatory reactions
- 1° IR to allergen = IgE bound to basophils/mast cells
- 2° IR to allergen = degranulation/release histamine
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Passive Immunity – Placental Transfer

• Maternal transfer of immunoglobulins from mother to foetus

• Antibody isotype transferred during gestation: only IgG

• Subclasses transferred: IgG1, IgG3, IgG4 , (not IgG2)

• Foetal levels of IgG increase rapidly around 3-4 months gestation

• Confers immunity to infection upon the foetus
• Newborns only manufactures their own IgG around 3-4 months/4-8 months after birth
• 1-3 months prior to birth a foetus begins to manufacture their own IgM

• One negative consequence of placental transfer

• Haemolytic disease of the newborn – antibodies from the mother’s immune system react with foetus’ RBC
• Usually occurs if mother has anti-Rhesus D antibodies and the child’s RBC are Rhesus D antigen positive

• Not covered in haematology until 3rd year. Basic blood grouping done late 2nd year.
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Passive Immunity – Placental Transfer

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Passive Immunity – Anti-venom

• Antibodies against a particular venom (antigen) are administered to an affected patient
• Snake anti-venoms (IV)
• Box Jellyfish (IV preferable but IM possible)
• Funnel Web spider (IV)
• Stonefish, Redback spider – (IM, but diluted and IV if life threatening ????)
• Antibody isotype: purified IgG

• Direct quote from Wikipedia.

“Antivenom (or antivenin or antivenene) is a biological product used in the treatment of venomous bites or stings. Antivenom is
created by milking venom from the desired snake, spider or insect. The venom is then diluted and injected into a horse, sheep, rabbit, or
goat. The subject animal will undergo an immune response to the venom, producing antibodies against the venom's active molecule
which can then be harvested from the animal's blood and used to treat envenomation.”

• Describing antibody specificity – an example

• CSL’s Stonefish anti-venom is manufactured in horses
• The immunoglobulins contained in this preparation can be described as being: “horse anti-stonefish venom IgG antibodies”
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Neutralisation of Toxins
• Neutralisation of bacterial exotoxins (secreted toxins)
• Prevent the binding and entry of the toxin into the target cell(s)
• Neutralisation of venom following passive administration
• Antibody isotype: IgG
• Removal of the antigen antibody complexes formed by phagocytosis or complement
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Nagler reaction with Clostridium perfringens

C. perfringens No antitoxin
antitoxin on this half on this half

Lecithinase breaks down phospholipids

producing opaque soapy deposits.
Neutralisation of lecithinase by
antitoxin, no opaque deposits.
NB: Growth of organism not inhibited

Test organism: Likely ID?

Positive control
Clostridium perfringens

How would you describe the specificity of the antibody used if this solution was made up of IgG molecules made in a goat?
25-Jul-23 PJC 23

Neutralisation of Virus Particles

• Antibodies bind to the surface antigens of a virus particle
• Blocks adhesion of the virus to the mucosal epithelium/cell membrane
- Prevents viral entry into the cell
• Antibody isotypes: IgG, IgA, IgM
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Phagocytosis without antibodies

Foreign agents/particles engulfed and destroyed
• Neutrophils (PMN)
• Monocytes (in blood) èMacrophages (in tissue)
• Dendritic cells
- Antigen Processing Cells derived from monocytes
• Eosinophils
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Antibody Opsonisation and Phagocytosis

• Opsonisation is the process of an antibody binding to an epitope on an antigen molecule located on a target micro-organism/particle,
effectively coating the particle and marking it for destruction by phagocytosis
• The coating molecule is referred to as the ‘opsonin’ (in this case, antibody molecules)
• Phagocytes involved:
• Macrophages
NB: one of the biological roles of
• Polymorphonuclear cells (PMN) the Fc portion of immunoglobulin
• Antibody isotype: Only IgG ?IgM
25-Jul-23 PJC 26

Immobilisation of bacteria/agglutination of particles

• Agglutination (clumping) enhances phagocytosis

- Reduces the number of infectious units/particles to be dealt with by the immune system

• Antibody isotypes:

• IgM +++

• IgA ++

• IgG+

IgG
 25-Jul-23 PJC 27

Immunoglobulin D
• IgD is expressed on mature B cells together with IgM
• IgD functions as an antigen specific B-cell receptor (BCR) for B cell activation
i.e., the binding of antigen stimulates the B-cell to either become a memory B cell or a plasma cell
- When stimulated, a memory B cell converting to a plasma cell undergoes isotype switching to IgM
(after interaction with native antigen) OR IgG, IgA, IgE (requires T cell help)

1°Antigen

Some memory B cells can turn into IgD plasma

cells but most turn into IgG, IgA, IgE plasma cells

2°Antigen

IgD

• Small quantities of IgD secreted/released by mature memory B cells that have been activated by antigen upon secondary exposure
• IgD seems to be important in binding to microbes and their products in the upper respiratory tract
- Antigen bound IgD is recognised by and can activate basophils and mast cells
- These cells then produce antimicrobial factors
25-Jul-23 PJC 28

Summary Slide #1 (from Abbas)

Both include
agglutination
and
immobilisation

IgE

after complement
activation

Intermediate steps for

IgG, IgA, IgE
NOT shown
25-Jul-23 PJC 29

Summary Slide #2 (using different images)

ADCC

NK cells (IgG)

Eosinophils (IgE)
 25-Jul-23 PJC 30

Study Questions
1. Which pathway of complement may be activated by antibody binding to antigen?
2. What is the end result of the activation of this pathway?
3. In ADCC, which immune cell is activated, which isotype of immunoglobulin is involved, and which cells are targeted by this
immune response?
4. Only one class of immunoglobulin is transferred placentally. Which one, and what biological purpose does this passive
transfer have?
5. Use a schematic diagram to explain how antibody molecules can neutralise bacterial toxins/venom? Label ALL interacting
particles and components
6. IgG, IgA and IgM can all bind to viral particles. How does this attachment inhibit viral infection?
7. Describe the process of antibody opsonisation. Which isotype of immunoglobulin is involved?
8. Why is IgM the isotype of immunoglobulin that has the best agglutinating ability? Why is IgA next best?
9. Briefly describe the events that occur during the primary and secondary exposure of the immune system to an allergen?
10. Why can the release of histamine cause asthma in susceptible people? i.e. what biological effect does it have?
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Enough Aby for different Ag? - Antibody Gene Segments

λ Light chains (3 segments) – Variable (1/30), Joining (1/3), Constant (1/3) = 270
κ Light chains (3 segments) – Variable (1/40), Joining (1/5), Constant (1) = 200

Heavy chains (4 segments) – Variable (1/40), Diversity (1/25), Joining (1/6), Constant (1) = 6000
• Only 1 constant region calculated because IgA, IgE, IgM, IgG can all be produced as part of an antibody
molecule against the same epitope of the same antigen

• 270 x 6000 = 1,620,000 different Lambda light chain antibody molecules possible to different Antigens