Antigen & Antibody

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- Family of circulating, structurally-related glycoproteins produced in response to exposure to antigens
- First to be discovered
- Specificity: Each individual antibody protein is capable of binding specifically with one unique epitope
- Mediators of humoral Immune Responses against all classes of microbes & many foreign substances
- Greatest discrimination between different antigens
- Greatest antigens -binding strength
- Blood = cells (RBCs, WBCs & Platelets) + fluid (plasma)
- Serum= Plasma - coagulation (clotting) factors
- Physical separation according to solubility characteristics into albumins & globulins
- Separation by migration in electric field (electrophoresis) → most antibodies are found in γ (gamma) globulins
- All Antibody molecules share same basic structural characteristics:
1. Diversity
• Only small Ag-binding portions with high variations
• Different Antibodies bind high number of structurally diverse Antigens
2. Effector functions & common physicochemical properties ➔ Non-Ag-binding portions with few variations
- :
• Include all protein molecules which contain domains with Ig fold structure
• Example of molecules in Immune System:
1. Membrane-bound IgG
2. T-Cell Receptor
3. Class I MHC
4. CD4 coreceptor of T-Lymphocytes
5. CD28 costimulatory receptor
6. ICAM-1 adhesion molecule
- Antibody structure (Y-shaped):
• Monomeric symmetric structural basic CORE unit of 4 chains:
✓ 2 identical Heavy Chains, each one composed of:
❖ 1 Variable region domain
❖ 3 or 4 Constant region domains:
 Anchors membrane-bound Antibodies in
plasma membranes of B-Lymphocytes
 Found only in secreted Abs
 Determine tissue distribution of Antibodies
❖ Amino-terminal Variable (V) regions: their
Amino Acid sequences vary among Antibodies
produced by different B-Lymphocyte clones &
participate in Ag recognition)
❖ Carboxy-terminal Constant (C) regions
✓ 2 identical Light Chains, each one composed of:
❖ 1 Variable region domain
❖ 1 Constant region domains:

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  Do NOT participate in effector functions
 NOT directly attached to cell membranes
❖ Amino-terminal Variable (V) regions: their Amino Acid sequences vary among Antibodies produced
by different B-Lymphocyte clones & participate in Ag recognition)
❖ Carboxy-terminal Constant (C) regions
❖ 2 classes (isotypes):
 Κ (kappa) & λ (lambda), distinguished by their carboxyl-terminal C regions
 Each Ab molecule has either 2 identical κ or 2 identical λ Light Chains
 NO known differences in function between κ & λ.
 In humans: κ≈60%, & λ≈40%
 Skewed (Markedly changed ratio) is often used clinically for Diagnosis of B cell lymphomas
because neoplastic cells derived from one B cell clone produce single species of Antibod
molecules, all with same Light Chain.
• Fab region (Antigen-binding):
✓ 2 identical
✓ Each consists of complete Light Chain (VL & CL) associated with VH & CH1 fragment of Heavy Chain
✓ Contain Antigen-binding sites.
❖ Paired 1 Variable region of Heavy chain + 1 Variable region of Light chain
❖ Each Ab molecule has ≥2 Antigen-binding sites.
❖ Can accommodate linear epitope of ≈6 AAs.
❖ Constant regions are distant from Ag-binding site & do NOT participate in Ag recognition
• Fc region:
✓ Composed of 2 identical disulfide-linked peptides (each containing CH2 & CH3 domains of heavy cahin)
✓ Bind to complement, & Fc receptors (FcRs) → effector functions
✓ Their constant domain ➔ Crystallizable (self-associate) into lattice
✓ Effector functions of Antibodies are mediated by binding of CH of Fc to other cells & plasma proteins:
❖ Effector functions of Antibodies are initiated ONLY by Abs with bound Ags (NOT by free Ab):
 ≥2 adjacent Ab Fc are required to trigger various effector functions
 To ensure specific target to eliminate Antigens which are recognized by Ab, & that circulating
free Abs do not trigger effector functions wastefully, & inappropriately.
❖ FcR & complement-binding sites of Abs are found within CH of different isotypes
❖ Microbial Ag-complexed IgG binds, through its Fc, to γ heavy chain-specific FcRs expressed on
phagocytes (PMNLs & macrophages) → phagocytosis
❖ IgE binds to IgE-specific FcRs expressed on mast cells → degranulation
❖ Ag-complexed IgG or IgM bind, through its Fc, to complement protein (C1q) → activation of classical
complement pathway → inflammatory mediators, & microbial phagocytosis & lysis
• Flexibility of Antibody molecules:
1. Mainly due to Hinge region:
✓ Between CH1 & CH2
✓ 10 to 60 Amino Acid residues
✓ Unfolded & flexible conformation which permit independent movement of Antigen-binding site relative
to rest of molecule.
2. Some due to ability of each VH domain to rotate with respect to adjacent CH1 domain

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 • Complementarity Determining Regions (CDRs)
✓ Hypervariable regions
✓ Found in variable regions of antibody
✓ Form surface complementary to 3D-shape of bound Antigen
✓ Antigen binding is primarily function of CDRs which form multiple contacts with bound Antigen
✓ Spread out of antigen-binding site to form broad planar surface to accommodate conformational
epitopes of large macromolecules
✓ In number of Antibodies, CDRs close up to form Ag-binding cleft to accommodate small molecules (as
monosaccharides & drugs)
✓ Composed of 6 segments (3 in VH & 3 in VL brought together to form Ag-binding site)
✓ Short (each ≈10 AA sequence residues)
✓ Greatest variability ( distinct interaction surfaces & specificities among different Antibodies)
✓ Hold in place by more conserved sequences of framework regions which mostly determine folding/
making up Ig domain of V region.
✓ Proceeding from amino terminus of either VH or VL, they are called:
❖ CDR1
❖ CDR2
❖ CDR3 → is the most variable & Most extensive contact is with CDR3
• Linkage in Antibody )‫(الجدول هاظ مش مهم كثير‬:
Linkages/ Associations Between (pairing of) 2 Heavy chains Between Heavy chain & Light chain
Interactions of chains of
each Antibody molecule
Covalent

Covalent - By disulfide bonds: - By disulfide bonds Between cysteine residues

• In IgG, between cysteine residues in CH2 in carboxy terminus of LC & CH1 domain of
domains, close to Hinge region. Heavy Chain
• In other isotypes, in different locations

Non-Covalent Between third CH3 domains Between VL & VH domains & Between CL & CH1
domains.
• Antibodies forms:
1. Membrane-bound:
• Anchored in plasma membrane
• Found on surface of B-Lymphocytes
• Function as naïve B-Lymphocyte membrane Ig receptors for Ag ( Ag recognition → B-Lymphocyte
activation → differentiation into plasma cells → Antibodies of same specificity as Ag receptor)
• Carboxy terminus include 2 segments:
A. Transmembrane: hydrophobic AA residues.
B. Cytoplasmic (intracellular juxtamembrane):
❖ With positively charged AAs which bind to negatively charged phospholipid head
groups on inner leaflet of plasma membrane & anchor Antibody in membrane.
❖ IgM & IgD: short; 3 AA residues
❖ IgG & IgE: long; 20-30 residues
2. Secreted:
• Transported out of cell
• Found in plasma, & other extracellular fluids as mucosal secretions & interstitial fluid of tissues

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 • Antigen binding → Antigen elimination (requires Antibody interaction with other Immune System
components, as complement, & cells as phagocytes & eosinophils)
• Carboxy terminus tail piece of hydrophilic Amino Acid residues.
✓ IgM: long (21 residues) which is involved in pentamer formation
✓ IgG: short (3 residues)
• Antibody-mediated effector functions include:
✓ Neutralization of toxins
✓ Activation of complement
✓ Opsonization of pathogens for enhanced phagocytosis
✓ Ab-dependent cell-mediated cytotoxicity by which Antibodies target infected cells for lysis
by cells of innate Immune System
✓ Ab-mediated mast cell activation
3. Monomers: secreted IgG & IgE, & all membrane-bound
4. Multimers (≥2 covalently-joined monomers):
• Secreted IgM (pentamer) & IgA (dimer)
• Bind to Antigens more avidly than monomers, even if both contain Fab regions w individually bind
Antigen equally well
• Formed by interactions between Tail pieces:
✓ Regions located at carboxyl-terminal ends of μ & α HCs
✓ Contain:
❖ Joining (J) chain: 15-kD polypeptide
❖ Disulfide-bonded to tail pieces →serves to stabilize & to transport multimers across
epithelial cells from basolateral to luminal surface

• Cleavage of Antibody:
1. Pepsin:
✓ Site of cleavage: distal to hinge region
✓ Results:
❖ 1 bivalent Fab
❖ Fc fragment ➔ peptide fragments
2. Papain:
✓ Site of cleavage: on hinge region
✓ Results:
❖ 2 identical Fab
❖ 1 Fc

• Antibody molecules are divided into classes (isotypes) which are further subdivided into subclasses
(subtypes) according to differences in structure of their Constant domain of Heavy chain
• Amino Acid sequence of CH is the same in all antibodies of one isotype or subtype, but different in
antibodies of other isotypes or subtypes
• Antibodies of different species differ from one another in C regions & in framework parts of V regions

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 Isotype of Subtypes ( H Chain) Serum Serum half- Secreted Functions
antibody concentration life (days) form
(mg/mL)
IgA IgA1 & IgA2 3.5 6 Mainly Dimer; Mucosal immunity
(α1 or α 2) also Produced in walls of GI & RT
monomer,
trimer
IgD None Trace 3 Monomer Naïve B cell antigen receptor
(δ)
IgE None 0.05 2 (shortest) Monomer Defense against helminthic
(ε) parasites, immediate
hypersensitivity
IgG IgG1-4 13.5 ( largest ) 23 (Longest) Monomer Opsonization, complement
(γ1, γ2, γ3 or γ4) activation, Antibody-dependent cell
mediated cytotoxicity, neonatal
immunity, feedback inhibition of B
cells
IgM None 1.5 4 Pentamer Naïve B cell antigen receptor
(µ) (monomeric form), complement
activation
- IgG:
• Has the longest half-life
• Due to Binding to neonatal Fc receptor (FcRn):
✓ Specific FcR
✓ Structurally resemble class I MHC
✓ Transport IgG across cells without targeting them to lysosomes preventing rapid degradation
✓ Transport of maternal IgG across Blood Placental Barrier During gestation
✓ Transfer of maternal IgG across neonatal intestine
✓ In adult vertebrates, FcRn bind, & direct micropinocytosed IgG molecules away from lysosomal
degradation in acidic endosomes, release them when vesicles fuse with cell surface to neutral pH &
return them to circulation
• IgG1 & IgG2: most long-lived & most efficient in terms of effector functions
• IgG3: relatively short-lived (binds poorly to FcRn)
• Applications in treatment:
✓ Injected fusion proteins, biologically active protein segment + human IgG Fc segment which binds to FcRn
→ ↑ in vivo half-life:
1- CTLA4-Ig:
❖ Extracellular segment of CTLA-4 receptor w binds to B7 co-stimulators, + human IgG Fc segment
❖ immunosuppressive agent & Used in treatment of Rheumatoid Arthritis
2- TNFR-Ig:
❖ Extracellular segment of type II TNF + Human IgG Fc segment)
❖ Blocks inflammatory actions of TNF
❖ Used in treatment of certain autoimmune diseases as Rheumatoid Arthritis & psoriasis
- Antibody isotype (class) switching:
• Naive B-Lymphocytes initially express only membrane-bound IgM & IgD
• Mechanism:
✓ Single clone of activated B-Lymphocytes produces secreted Antibodies with:
❖ Different CH (different effector functions)

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 ❖ Identical VH (same Ag-binding specificity as the original membrane-bound Ig receptor for
Antigens)
✓ Rationale:
❖ Different B-Lymphocyte clones produce more useful isotypes to eliminate Antigen
❖ To prolong effectiveness of humoral Immune Response (IgG with long half-life).
❖ IgG (phagocytosis), & IgE (destruction of helminths)
- Features Related to Antigen Recognition:
1. Antibody Fine Specificity for Antigens:
• Highly specific recognition distinguishing small differences in chemical structures
• e.g. between 2 linear protein epitopes differing by only single conservative AA substitution
2. Cross-reaction:
• Binding of ≥2 Antigens by the same Antibodies
• Antibodies produced against Antigens of one microbe may bind to different but structurally
similar Antigens of other microbes or self molecules (immunologic diseases)
3. Antibody Diversity:
• any individual makes high number of Abs with distinct specificity
• Mechanism:
▪ Random recombination (occur exclusively in Lymphocytes) of limited set of inherited
germline DNA sequences to form functional genes which encode VH & VL as well as by
addition of nucleotide sequences D recombination process
▪ Rationale: binding to different Antigens ➔ ↑Antibody repertoire = total collection of
Antibodies with different specificities.
4. Affinity Maturation:
• ↑average binding affinity of Abs for specific Ag as T cell-dependent humoral Immune
Responses to protein Antigens evolves
• In primary Immune Response, affinity has Kd 10−7 to 10−9 M
• In secondary Immune Response, affinity Antibodies increases with Kd of ≤10−11 M
• Mechanism:
▪ Somatic point mutation in V regions of Antibodies → activated B-Lymphocytes generate
different V domains, some of whcih bind Ag with greater affinity than did the original V
domains → high affinity Antibodies preferentially bind to Antigen & as a result of
selection, these B-Lymphocytes become dominant with each subsequent exposure to
Antigen
▪ Rationale: ↑ability of Antibodies to neutralize toxins & infectious microbes w is dependent
on tight binding of Antibodies achieved by high-affinity & high-avidity interactions.
- Antigen Recognition by Antibody:
• Reversible non-covalent binding interactions include electrostatic forces, hydrogen bonds, van der Waals
forces, & hydrophobic interactions.
- The valency of the antibody:
• Refers to how many binding sites an antibody may have
• Monovalent ➔ 1 Antigen-binding site of Ig bind to one epitope
• Bivalent ➔ 2 Antigen-binding site of Ig bind to 2 epitope
• Secreted IgM → 10 binding sites
• Secreted IgA → 4 binding sites

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- Affinity of Antibody:
• Strength of specific binding between single Antigen-binding site of Antibody & single epitope of Antigen
• Same for each epitope of polyvalent Antigen.
• Represented by dissociation constant (Kd):
✓ indicates how easy to separate Antigen-Antibody complex into its constituents
✓ Smaller Kd indicates higher or stronger affinity as lower concentration of Antigen & of Antibody is
required for complex formation.
✓ Kd of Antibodies produced in typical humoral Immune Response usually varies ≈10-7 M-10-11 M
- Avidity of Antibody:
• Sum of total binding strengths between Antibody & Antigen
• Much greater than affinity of any 1 Antigen-binding site
• Depends on affinity & valency of interactions
• Repeated identical epitopes widely spaced on cell surface (or monovalent Ags):
❖ Monovalent binding ➔↓ avidity vs ↑affinity of each
❖ Bivalent binding ➔ ↑avidity
❖ Polyvalent binding (10 identical Ag-binding sites of IgM simultaneously) ➔ very ↑avidity vs affinity of
each

- Injection of Antibody molecules from one species into another:

• Cause serum sickness
• Recipient recognizes them as foreign, mounts Immune Response & makes antibodies largely against C
regions of introduced Antibody
- Sequence differences in antibodies:
• Allotypes of Antibodies (polymorphic variants):
✓ Smaller differences in different individuals of same species reflect inherited polymorphisms in genes
encoding Antibody CH & CL
✓ Anti-allotypic Antibody which recognizes allotypic determinant.

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 • Idiotypes of Antibody:
✓ In the same individual , Differences between Antibody V regions are concentrated in CDRs
✓ Anti-idiotypic Antibody which recognizes some aspect of CDRs of another Ab.
- Theories:
• Potential mechanism of immune regulation that individuals produce anti-idiotypic Antibodies against their
own Antibodies which control Immune Response
- Antibody synthesis:
• Synthesized only by cells of B lymphocyte lineage
• Healthy 70-kg adult human produces ≈2-3g Abs every day
• IgA is the largest antibody synthesis (2/3)
• Ig expression:
✓ Stem cell: NONE
✓ Pre-B cell (synthesizes Ig polypeptides):
❖ Cytoplasmic μ Heavy Chains + surrogate Light Chains = Pre B cell receptor
❖ Pre B cell receptor , Non-covalently associated with Igα & Igβ ( 2 other integral membrane
proteins which serve signaling functions & are essential for surface expression of IgM & IgD)
✓ Immature B-Lymphocytes (+μ) → membrane IgM + synthesize κ or λ Light Chains which associated
with Heavy Chain proteins
✓ Mature B-Lymphocytes (+μ & δ) → membrane IgM & IgD + synthesize κ or λ Light Chains which
associated with Heavy Chain proteins
✓ Activated B cell: low rate secreted Ig; isotype switching; affinity maturation
✓ Antibody-secreting plasma cell: high rate secreted Ig; reduced rate membrane Ig
- Monoclonal Antibodies:
• Normally, millions of different B-Lymphocytes clones, each B-cell has Antibodies with same Ag-binding site
& different from Antibodies produced by other clones
• Polyclonal Antibodies: different Antibodies with different specificity which bind to different epitopes on
antigen
• Production of Monoclonal antibodies:
✓ Fusion of B-Lymphocyte from immunized mice, with myeloma cell (immortal monoclonal tumor of
Ab-secreting plasma cells) produce Hybridoma
✓ Hybridoma secrete Monoclonal antibodies
✓ Only one clone of antibodies with single specificity which bind to single epitope on antigen
✓ Uses:
1. Identification of unique phenotypic markers (clusters of differentiation; CD) to various cell types
2. Functional analysis of cell surface & secreted molecules
3. Immunology diagnosis of many systemic & infectious Disease by detecting particular antigens or
antibodies in blood, urine, or tissues in immunoassays
4. Tumor Identification by determining tissue source of tumors by HP/S of tumor sections using
labeled monoclonal antibodies specific for various cell proteins
5. Treatment by specific targeting of cells & molecules involved in pathogenesis of many diseases:
❖ Antibodies against cytokine TNF ➔ treatment for Rheumatoid Arthritis & other inflammatory
diseases
❖ Antibodies against CD20 ➔ treatment for B cell leukemias & for depleting B-Lymphocytes in
certain autoimmune disorders
❖ Antibodies against epidermal growth factor receptors to target cancer cells

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 ❖ Antibodies against vascular endothelial growth factor (cytokine which promotes
angiogenesis) in patients with colon cancer
• Limitations of Monoclonal antibodies:
✓ Human anti-mouse Antibody (HAMA): Antibodies against mouse Ig block function or enhance
clearance of injected monoclonal Ab & can cause serum sickness
✓ Solutions:
1. Genetic engineering → genes encoding Ag-binding sites of mouse monoclonal Antibody are
inserted into complementary DNA (cDNA) encoding human myeloma protein → hybrid gene →
hybrid protein expressed which retains Antigen specificity of original mouse monoclonal but
has core structure of human Ig (= Humanized Antibodies)
2. Fully human monoclonal Antibodies derived using phage display methods or in mice with B-
Lymphocytes expressing human Ig transgenes.

- All substances recognized by & bind specifically to Antibodies or TCRs, whether or NOT they stimulate Immune
Responses
- Any biologic molecule including:
1. Simple intermediary metabolites as autacoids (physiologically active substances as serotonin, bradykinin,
or angiotensin, produced by & acting within body), & hormones
2. Macromolecules as Carbohydrates (polysaccharides), proteins, Nucleic acids, & lipids
- Immunogens:
• Molecules able to stimulate Immune Responses, activate Lymphocytes, & generate specific antibodies
• All immunogens are antigens but NOT all antigens are immunogens
- Haptens:
• Free small chemicals as dinitrophenol, antigenic (bind to Abs), but NOT immunogenic
• Hapten-carrier complex:
✓ Multiple copies of hapten are conjugatedto large Carrier molecule as protein or polysaccharide
✓ Able to generate Antibodies specific for hapten
- Epitope (antigenic determinant):
• Any shape or surface on molecule
• May be delineated on any type of compound, but NOT restricted to macromolecules)
• Any Antibody recognizes & binds only specifically to particular epitope
• Nature of epitopes depends on conformation:
1. Conformational epitopes:
✓ Amino Acids residues NOT in sequence
✓ Spatially juxtaposed in folded polypeptide chain)
✓ Accessible in native protein & Lost by denaturation (unfolding)
2. Linear epitopes:
✓ Adjacent Amino Acids residues in sequence
✓ May be:
❖ Accessible to Antibodies if they appear on external surface or in region of extended
conformation in native protein, as well as in denatured protein
❖ Inaccessible to Antibodies in native conformation & appear ONLY in denatured protein.

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 3. Neoantigenic determinants:
✓ New epitopes, result from post-synthetic structural modifications (as by glycosylation,
phosphorylation, ubiquitination, acetylation, & proteolysis= peptide bond cleavage).
- Types of Antigens )‫(مهمم مع الجدول‬:
• Polyvalent (multivalent): Presence of multiple identical epitopes
1. Macromolecules (usually much bigger than Ag-binding region of Antibody):
✓ Binding & bridging, cross-linking, clustering, bringing close together of multiple Antigen-BCRs:
❖ This is essential step to initiate B-Lymphocyte activation & humoral Immune Response
✓ of ≥2 Antibodies (optimal triggering of many effector functions of Abs)
2. Cell surfaces: microbes: protein or polysaccharide
3. Polysaccharides & Nucleic Acids: many identical epitopes may be regularly spaced
• NOT polyvalent: most globular proteins (unless they are in aggregates).
Protein Conformation Denaturation
Native folding unfolding Diagram
Antigen epitopes Accessibility to antibodies
1-Conformational (=AAs residues Accessible Inaccessible
NOT in sequence but spatially (lost)
juxtaposed in folded polypeptide
chain).

2-Linear a. Accessible Accessible Accessible

(=adjacent a- b. Inaccessible Inaccessible Accessible
Accessible AAs
residues in b-
Inaccessible
sequence)

3-New created by proteolysis & by Inaccessible Accessible

post-synthetic structural (absent)
modifications as by glycosylation,
phosphorylation, ubiquitination,
acetylation).

- Sizes of Antigen-Antibody (immune) complexes:

• Large complexes:
✓ Formed in zone of equivalence
✓ [correct]s of polyvalent Antigens + specific Antibodes
✓ Extensive cross-linking of attached molecules
✓ Most or all of Antigens & Antibodies are complexed

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 • Smaller complexes:
✓ Formed by:
❖ ↑[Ag] (zone of Ag excess) ➔ Free Antigens displace Ag bound to Ab
❖ ↑[Ab] (zone of Ab excess) ➔ free Antibodes displace bound Antibody from Ag
• In vivo
✓ Antigen/Antbody complexes
✓ Deposited either in
▪ Peripheral Blood Circulation
▪ In tissues ➔ deposits inflammatory reaction ➔ immune complex diseases

- In adaptive Immune System, 3 molecules bind antigens )‫(الجدول مهم‬:

Feature Antigen-binding molecules
Immunoglobulin T-Cell Receptor (TCR) MHC molecules
Antigen-binding site Made up of three CDRs in Made up of three CDRs in Peptide-binding cleft
VH & three CDRs in VL Vα & three CDRs in Vβ, made of α1
domains domains
and α2 domains (class I
MHC) and α 1 and β1
domains (class II MHC) &
α

Nature of antigen that may Macromolecules Peptide-MHC complexes Peptides

be (proteins, lipids,
bound polysaccharides) & small
chemicals

Nature of antigenic Linear & conformational Linear determinants of Linear determinants of

determinants recognized determinants of various peptides only 2 or 3 peptides; only some
Macromolecules & amino acid residues of a amino acid residues of a
chemicals peptide bound to an MHC peptide
of a peptide

Affinity of antigen binding Kd10-7-10-11 M ; average Kd10-5-10-7 M Kd10-6-10-9 M; extremely

affinity of Igs increase stable binding
during immune response
On-rate & off-rate Rapid on-rate, variable Slow on-rate, slow off- Slow on-rate, very slow
off-rate rate off-rate

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