MODULE 2: ANTIGEN AND ANTIBODY

o Immunogens any foreign particle that elicits an

OUTLINE
immune response
I Antigen
A Parts of Antigen o All immunogens are antigens but not all antigens are
B Functions of Antigen immunogens
C Classification o Lipids and nucleic acids are only antigenic when
D Kinds of Antigen combined with proteins or polysaccharides because
E Factors Affecting Immunogenicity protein is the strongest and most common antigen
i Haptens
ii Types of Antigens on the Basis of Order of Their
Class (Origin) A. PARTS OF ANTIGEN
F Relationship of Antigens to the Host • Carrier: molecular weight over 10,000 dalton; high MW;
G Activators of Lymphocyte almost always protein in nature
H Chemical Nature of Antigens (Immunogens) o These can cause production of antibody
II Antigen Processing and Presentation • Antigenic determinant or epitope or haptene: portion of
A MHC Class I Antigen Presentation
antigen that is recognized as foreign; reacts specifically
B MHC Class II Antigen Presentation
i Antigen Presenting Cells (APCs) with an antibody receptor
C Antigen Processing and Presentation Pathway o This does not recognize the whole pathogens but only
i Cytosolic Pathway part; only those protein in nature can be recognized
ii Endocytic Pathway o Antibodies bind in epitope and this will bind a specific
D Superantigens receptor
i Conventional Antigen TCR Binding o Each epitope is recognized by different antibody
ii Superantigen Mediated Antigen-TCR Binding • Self-antigen: these are the photo antigens that can be
III Major Histocompatibility Complex/HLA
distinguished by immune system
A Biological Function
B Genetic Regions • Part of antigen that combines with specific antibody
C MHC Expression • Low molecular weight
i Class I MHC Molecules • This cannot invoke antibody production unlike carrier
ii Class II MHC Molecules
iii Class III MHC Genes
D Differential Distribution of MHC Molecule B. FUNCTIONS OF ANTIGEN
i Clinical Significance of MHC/HLA • Major: stimulates production of antibodies or immune
E MHC Molecules response
F HLA Testing • Minor: shows capability of reacting with antibody
IV Antibodies/immunoglobulin
A Immunoglobulin Structure
B Antibody structure C. CLASSIFICATION
i Two Heavy Chains • Complete antigen: with 2 parts carrier and haptene;
ii Two Light Chains perform two functions
iii Hinge Region/Mid-Region o This performs functions of antigens (major or minor)
C Properties of an Antibody • Incomplete antigen: either carrier or haptene; one
D Classification of Antibody
function is present
i According to Temperature at which they React
ii According to Occurrence o Could be only major function
iii According to Species
iv According to its Reaction with an Antigen D. KINDS OF ANTIGEN
E Immunoglobulins • Agglutinogen, Precipitinogen, Virus, Rickettsiae,
i Major Ig Classes
Toxin, Allergens
ii Immunoglobulin G
iii Immunoglobulin A
iv Immunoglobulin M
v Immunoglobulin D
vi Immunoglobulin E
F Antibody variants
i Isotype
ii Allotypes
iii Idiotype
G Enzyme Hydrolysis
V Monoclonal Antibodies E. FACTORS AFFECTING IMMUNOGENECITY
A Uses of Monoclonal Antibodies • Foreignness
i To Suppress the Immune System o Distinguishing self to non self
ii To Kill/Inhibit Malignant Cells • Molecular Size
iii Others o at least 10,000 daltons to be recognized by the
B FDA Approved Monoclonal Antibodies Used for
immune system
Immunotherapy
o Rule of thumb: The higher the molecular weight, the
more potent the antigen
I. ANTIGEN • Chemical Composition and Complexity
• Antigens: Substances recognized as foreign by body o Proteins are powerful immunogens
• This has the ability to bind to an antibody o Carbohydrates are less immunogenic than protein
o Lipids and nucleic acids are poor antigens
• It stimulates an antibody
o Polysaccharides
• Most are proteins or large polysaccharides from a foreign
o Rule of thumb: The more complex the antigen, the
organism
greater the effectiveness
o Microbes: capsules, cell walls, toxins viral capsids,
• Route, Dosage and Timing Of Parenteral
flagella, etc.
Administration
o Nonmicrobes: Pollen, egg white, RBC surface
o IV and intraperitoneal
molecules, serum proteins, and surface molecules
o Intradermal
from transplanted tissue
o Subcutaneous or intramuscular
• Proteins: strongest and most common antigens
o Antigens are foreign particles that may elicit an
immune response i. HAPTENS

BELTRAN, MACATUGGAL, MARTIN, TORRECER 10

MODULE 1: INTRODUCTION TO IMMUNOLOGY

• partial antigens ▪ The plant glycoproteins that concanavalin A (Con

• not antigenic A), Lectins and Pokeweed mitogen (PWM)
• must be coupled to a carrier molecule to be antigenic activates both T and B cells
• can react with specific antibody alone, but cannot ▪ Mitogens - substances that can induce mitosis
produce a visible reaction such as precipitation or • Immunogenicity: the ability of a substance to induce an
agglutination immune reaction
• Small foreign molecules that is not antigenic o Response is either humoral immunity or cellular
• Low molecular weight (less than 10,000) immune response
• These are called partial antigens or incomplete o Immunogenic: Substances involved in this process
antigens • III: Immunogenicity, Induced, Immunogenic (substance)
• Usually these are non-protein substances • Antigenicity: ability of a substance to bind specifically to
• Example is poison ivy which contains a chemical called the products of an immune response
catechols which is the hapten o These substances are called antigenic (naga-attach)
o AAA: Antigenecity, Attach, Antigenic
o All molecules that are immunogenic are antigenic too
ii. TYPES OF ANTIGENS ON THE BASIS OF o All antigenic molecules cannot be considered
ORDER OF THEIR CLASS (ORIGIN) immunogenic (Ex. Haptens)
1. Exogenous antigens
o Antigen processing cells such as macrophages, H. CHEMICAL NATURE OF ANTIGENS
dendritic cells (IMMUNOGENS)
o These antigens enter into the body and starts to A. Proteins
circulate and these antigens will be trapped by these o These may be pure proteins or they may be
antigen-processing cells (APC) glycoproteins or lipoproteins
o Examples: bacteria, viruses, fungi ▪ Strongest antigen
2. Endogenous antigens B. Polysaccharides
o These are body’s own cells or sub fragments or o Pure polysaccharides and lipopolysaccharides are
compounds or the antigenic products that are good immunogens
produced. ▪ Best immunogens
o These are processed by macrophages and later on will ▪ Carbohydrates are less immunogenic compared to
be accepted by cytotoxic T cells proteins because the unit of sugars are limited
o Example: Blood group antigens, HLA compared to those that are in proteins
(Histocompatibility Leukocyte antigens), etc. ▪ Carbohydrates are usually in form of glycoproteins
3. Autoantigens or glycolipids
o An autoantigen is usually a normal protein or complex C. Nucleic acids
of proteins that is recognized (can be DNA or RNA) by o Usually poor immunogenic
the immune system of patients suffering from a specific D. Lipids
autoimmune disease o Non-immunogenic
o These are antigens whose target is its own immune
system
o Examples: Nucleoproteins II. ANTIGEN PROCESSING AND PRESENTATION
• Antigen Processing
o Process by which pathogens and their products are
F. RELATIONSHIP OF ANTIGENS TO THE HOST
degraded to produce peptide antigens.
• Autoantigens o These peptide fragments combine with MHC
o antigens that belong to the host molecules present inside our cell
o do not evoke an immune response under normal o The MHC-peptide complex formed travels to the cell
conditions surface where it displays fragments to T cells for
o Example: DNA and corneal tissue recognition.
• Alloantigens • B Cells Recognize Variety of Antigens
o are from other species capable of eliciting an immune o Proteins
response o Polysaccharides
o important to consider in tissue transplantation and in o Nucleic acids
blood transfusions. o Lipids
• Heteroantigens • T Cells Recognize Only Protein Antigens
o are from other species, such as other animals, plants, o Peptides
or microorganisms. o MHC-Peptide Complex
• Heterophile antigens
o are heteroantigens that exist in unrelated plants or
animals but are either identical or closely related in A. MHC CLASS I ANTIGEN PRESENTATION
structure so that antibody to one will cross-react with • MHC Class I
antigen of the other. o present intracellular or endogenous antigens
o These are identical antigens found in the cells of o They live and replicate inside the host cells
different species
o Example: Cross-reacting microbial antigens, Human
Blood group And B antigens

G. ACTIVATORS OF LYMPHOCYTES
• There are three major categories of molecules that can
trigger the activation of lymphocytes
1. Monoclonal activators: substances that stimulate
cells expressing antigen receptors specific for an • Once the virus enters the target cell, it will release the
epitope. nucleic acid and the viral protein.
2. Oligoclonal activators: superantigens, molecules o The viral proteins are synthesized in the cytosol of
that are typically derived from bacteria. the target cell.
3. Polyclonal activators: mitogens, typically plant • These viral proteins are digested in the barrel shaped
proteins that bind to molecules present on all T cells. known as proteasome.

BELTRAN, MACATUGGAL, MARTIN, TORRECER 11

MODULE 1: INTRODUCTION TO IMMUNOLOGY

o Proteasome: barrel-shaped where viral proteins are

digested. C. ANTIGEN PROCESSING AND PRESENTATION
• Inside the proteasome, it will be further degraded in the PATHWAY
cytosol and the proteins into peptide fragments • The intracellular and extracellular antigens are eliminated
o Peptide fragments: consists of 8-15 amino acids by different pathways in immune system.
• These peptide fragments will be transported inside the • There are basically two pathways for their eliminations.
rough endoplasmic reticulum (RER) through the
Transporters Associated with Antigen Processing
i. CYTOSOLIC PATHWAY
(TAP) port
o TAP has two subunits: TAP 1 and TAP 2 • Endogenous antigens
• The alpha and beta chains of MHC I molecule are o Degraded in proteasomes
synthesized and translocated into the endoplasmic • Intracellular antigens
reticulum (ER). o Attaches with MHC Class I
• Once the peptides are transported in the RER, it binds to • MHC Class I
the newly synthesized MHC Class I molecule. • Co-receptor: CD8+
o Once they are bound, it is called MHC I Peptide o Cytotoxic T cell
Complex
• Once you have MHC I Peptide Complex, it will move to the ii. ENDOCYTIC PATHWAY
golgi apparatus then to the secretory vesicle. • Exogenous antigens
• Once it reaches the cell surface of the target cell, it will • Extracellular antigens
integrate into the membrane to be recognized by T cell. • MHC Class II
• T cell now will recognize the presented MHC I Peptide • Co-receptor: CD4+
Complex o T helper cells
• T cell has a co-receptor called CD8 • Summary of antigen presentation pathways:
o Co-receptor: important in antigen recognition 1. Endogenous cell destruction
• CD8 recognizes MHC I molecule in alpha 3 domain 2. Exogenous immune response
3. B lymphocytes antibody production
B. MHC CLASS II ANTIGEN PRESENTATION
• MHC Class II D. SUPERANTIGENS
o processing and presentation of extracellular or • Superantigens (Sags)
exogenous antigens o Special group of antigens
o The live and replicate outside the host cell o Causes non-specific activation of T cells which
o Examples: parasites and extracellular bacteria causes polyclonal activation of T cell and cytokine
release
i. ANTIGEN PRESENTING CELLS (APCs) o Induce a very strong and drastic T cell response
• This presents the peptides derived from internalized ▪ Massive activation of immune cells or massive
pathogens release of cytokines (overactivation of T cells)
o This will bind directly to the T cell receptor in MHC
molecule
o Example of superantigens: TSS and SSSS
▪ SSSS: Staphylococcal Scalded Skin Syndrome
▪ TSS: Toxic Shock Syndrome
o Escape normal antigen processing by antigen
presenting cells (APCs)
o Toxins produced by bacteria and viruses
o Well-known infection in food poisoning
• MHC Class II Presentation process:
▪ staphylococcal enterotoxin
o Bacteria may enter the host through phagocytosis or
endocytosis
o Once pathogen is engulfed, it will be followed by the i. CONVENTIONAL ANTIGEN-TCR BINDING
fusion of endosome and lysosome • Antigen Presenting cell will bind to histocompatibility
o The pathogens are digested by proteolytic molecule (MHC) then form a peptide
lysosomes and enzymes into small peptide • Displays specific antigens
fragments *TCR: T Cell Receptor
o Inside the RER is the MHC Class II molecule where it
is synthesized and assembled ii. SUPERANTIGEN MEDIATED ANTIGEN-TCR
o Once there is newly synthesized MHC molecule, it will
BINDING
go to golgi apparatus.
o From golgi apparatus, it will go to a vesicle and bind • Superantigen will bind simultaneously with T cell
this to fragment known as CLIP receptor and MHC Class II (binds directly) because
▪ CLIP: Class II – associated Invariant chain superantigen binds to variable beta region chain of
Polypeptide the T cell receptor
o Once CLIP is formed, it will go and fuse with vesicles. o The binding mimics conventional antigen-TCR binding
o This peptide fragments will bind to the peptide binding • Antigen is not specific
groove of MHC Class II molecule and transported to
cell surface
▪ Usually contains 11-15 amino acids
o Once it reaches the cell surface, T cell will recognize
through its co-receptor, CD4.
▪ The co-receptor for MHC Class II is CD4
o Once presented on the cell surface, it will be presented
to T cell and it will recognize the peptide displayed by
MHC Class II
o CD4 will bind to the beta 2 domain of the MHC • T cells get activated non-specifically
molecule

BELTRAN, MACATUGGAL, MARTIN, TORRECER 12

MODULE 1: INTRODUCTION TO IMMUNOLOGY

• Divides and differentiates into effector T cells 1. Induce the differentiation and maturation of T cell to form
o Effector T cells: Cytokines which further stimulates functional T cell repertoire
other cells 2. Present antigen to initiate immune response with a
• Activate large number of T cells with different specificity phenomenon known as MHC restriction
o Result to massive amount/quantity of cytokines which o Endogenous Ag is presented to CD8 T cell by MHC
will stimulate macrophages and other phagocytes class I molecule
• Overproduction of Cytokines o Exogenous Ag is presented to CD4 T cell by MHC
o TNF-Alpha, IL-6, IL-1 class Il molecule
▪ Results in circulatory shock, endothelial
damage and multi-organ failure B. GENETIC REGIONS
▪ Life threatening autoimmune-like response Class I gene
Classical HLA class I genes HLA-A.B.C
Non-classical LA class l genes HLA-E, F, G
region
MHC class I chain related genes
III. MAJOR HISTOCOMPATIBILITY COMPLEX/HLA Classical LA class Il genes HLA-DP, DQ, DR
Class Il gene
• Histocompatibility region
Genes associated with antigen
o “Histo”: Tissue processing
Class Ill gene Complement genes C4, C2, Bf
o “Compatibility”: Getting along region Inflammation-associated genes TNF HSP70
• Complex
o Genes localized to large genetic region containing loci
C. MHC EXPRESSION
• cluster of genes found in mammals
• Class I: On all nucleated cells (no MH on red blood cells,
• Its products play a role in discriminating self/non-self
weak expression on cells in brain)
• Participate in both humoral and cell-mediated immunity
• Class II: Found on antigen presenting cells (APC)
• Glycoprotein in nature
• Expression is increased by cytokines such as IFNa, IFNß,
• MHC act as antigen presenting structure.
IFNy and TNF
o The major histocompatibility complex is found on
• Transcription factors like CIITA (Transactivator), RFX
human chromosome 6, referred to as HLA complex.
(Transactivator) increase MHC gene expression
In mice, MHC is found on chromosome 17, referred
to as H-2 complex. • Some viruses (CMV, HBV, Ad12) decrease MH
o Terms HLA and MHC are used interchangeably. expression
o Clinically, they are relevant, because they may be • Reduction of MHC may allow for immune system evasion
involved in transfusion reactions, graft rejection, and
autoimmune diseases. D. DIFFERENTIAL DISTRIBUTION OF MHC
o Genes controlling expression of these molecules are MOLECULE
actually a system of genes known as the major Tissue MHC Class I MHC Class II
histocompatibility complex (MHC). T cells +++ +/-
o There were three kinds of molecules encoded by the B cells +++ +++
MHC; Class I, Class II, and Class III. Macrophages +++ ++
Other APC +++ +++
• Three groups of MH genes: Structurally and functionally
Thymus Epithelium + +++
distinct Neutrophils +++ -
1. Class I Hepatocytes + -
▪ recognition by CD8+ T cells Kidney + -
▪ express in all nucleated cells; highest on Brain + -
lymphocytes and low undetected on liver Erythrocytes - -
hepatocytes, neural cells, muscle cells and sperm. • Cell activation affects the level of MHC expression.
▪ Ex. HLA-A, HLA-B, HLA-C • The pattern of expression reflects the function of MHC
2. Class II molecules:
▪ recognition by CD4+ T cells o Class I is involved in the regulation of anti-viral
▪ important in antigen presentation complex (APC) immune responses
which include B lymphocytes, monocytes, o Class II involved in regulation of the cells of the
macrophages, and dendritic cells. immune system
▪ Ex. HLA-DP, HLA-DQ, HLA-DR • Anucleate erythrocytes cannot support virus replication -
3. Class III hence no MHC class I. Some pathogens exploit this (e.g.
▪ Minor histocompatibility antigen Plasmodium species.)
▪ Encode some of the complement proteins that
activate complement factor 3 by either classical
(C2, C4a, C4b) or alternative pathway (Factor B) E. MHC MOLECULES
• B cells react with antigens on its own via B cell receptors
• T cell recognize antigen in peptide form and binded to i. CLASS 1 MHC MOLECULES
MHC molecule • Present on all nucleated cells in the body
• The main function of MHC molecule is to bring the antigen • Recognition by CD8+ T cells
to cell surface for T cell recognition • highest on lymphocytes and low or undetected on liver
• HLA molecules are responsible for the compatibility of hepatocytes, neural cells, muscle cells, and sperm.
the tissues of genetically different individuals and for • involved in the recognition and rejection of grafted
the rejection of transplant kidneys and other forms of organ transplant.
• There are two main approaches to histocompatibility • For protection against viruses and parasites
testing: • These MHC class I-deficient diseased cells trigger and
o Tissue matching: determines whether the tissue from activate NK cells
one person will be rejected if transplanted to another • Chain structure: alpha 2, beta 2 macroglobulin
individual. 1. Heavy chain
o Tissue typing: determines the genotype of an ▪ a1, a2 domain: polymorphic sites
individual ▪ a3 domain: binding of CD8
• MHC genes are the most polymorphic genes present in 2. B-2 microglobulin
the genome! (Up to 250 alleles identified for some loci) 3. Peptide
• present intracellular or endogenous antigens
A. BIOLOGICAL FUNCTION

BELTRAN, MACATUGGAL, MARTIN, TORRECER 13

MODULE 1: INTRODUCTION TO IMMUNOLOGY

• If self-antigens can be infected by virus, tumors, or o To be activated before they express sufficient level of
malignancies it should be fragmented into peptide form MHC class II molecule and co-stimulatory activity
called antigen fragment • B cells
o To be able to bind it to MHC molecule which is o Constitutively express MHC class II molecule
assembled in rough ER. o To be activated for co-stimulatory activity
• Between the alpha1 and alpha2 it will form an antigen
binding pocket whereby the peptide form will attach to b. NON-PROFESSIONAL APC
the pocket. • Fibroblasts
• Once MHC molecule and peptide reaches cell surface, T • Glial cells
cell will recognize the antigen. • Pancreatic B cells
o Once recognized; T cell receptor, T cell coreceptor • Thymic epithelial cells
(CD8)
• Thyroid epithelial cells
• In MHC Class I, there are two chains:
• Vascular endothelial cells
o Alpha Chain: encoded in Chromosome 6 & MW:
45,000 Da
▪ Alpha 1 and Alpha2: makes up the antigen iii. CLASS III MHC GENES
binding pocket • minor MHC antigens
▪ Alpha3: E variant chain, and for attachment of CD8 • activate complement factor 3 by either classical (C2, C4a,
o Beta2 Macroglobulin: encoded in Chromosome 15, C4b) or alternative pathway (Factor B), TNF
MW: 12.000 Da
▪ Bound non-covalently to alpha chain F. HLA TESTING
▪ Essential for proper folding of alpha chain • There are two main approaches to histocompatibility
▪ It has no transmembrane segments or region testing:
▪ Identical in all cells o tissue matching
o tissue typing
ii. CLASS II MHC MOLECULES
• Has an antigen binding pocket between alpha1 and i. CLINICAL SIGNIFICANCE OF MHC/HLA
beta1 1. Organ transplant and bone marrow transplant.
• It presents extracellular or exogenous antigens 2. Platelet transfusion: matching between donor and
• found primarily on antigen-presenting cells, which recipient is useful to patients who are retractile to random
include B lymphocytes, monocytes, macrophages, and donor platelets
dendritic cells. 3. HLA compatibility exerts the strongest influence on long-
• Antigen Presenting Cells are divided into two: term kidney survival after a transplant.
o Professional APCs: DC, B cells, macrophages 4. HLA identical donors for bone marrow transplantation to
o Non-Professional APCs: epithelial cells, mast cells, reduce the frequency of graft vs host disease
etc. 5. Paternity testing
• It consists of two polypeptide chains: 6. Disease association: to determine an autoimmune
o Alpha: MW: 33,000 Da; alpha1 and alpha2 disease
o Beta: 28,000 Da; beta1 and beta2 (binds the CD4) MHC/HLA Related Diseases
• Alpha 1 and beta 1 domains form the antigen binding B27
Ankylosing spondylitis (C curvature of spine)
Acute anterior uveitis
pocket. B8 Celiac disease
o Binds longer peptide compare to class I molecules Multiple sclerosis
DR2
• Recognition by CD4+ T cells. Goodpasture’s syndrome
• important for antigen presentation and interactions DR3
Systemic Lupus Erythematosus (SLE)
Grave's / Myasthenia gravis
between immunocompetent cells. DR3/DR4
• For protection against bacteria, viruses and other heterozygote
IDDM / Type I Diabetes
exogenous antigens DR4 RA or Pemphigus vulgaris
• Chain structure: alpha, beta invariant chain DR5 Hashimoto's disease
1. a chain
▪ a1: polymorphic sites G. SUMMARY
▪ a2: binding of CD8 • MHC Class I
2. B chain o Receptor: CD8
▪ B1: polymorphic sites o Once recognized, it will go to cytotoxic T cell for LYSIS
▪ B2: binding of CD4 of infected cell
3. Peptide • MHC Class II
o Receptor: CD4
o Once recognized, it will be activated to T helper cells
and presented to B cells
o B cells will determine if primary or secondary response
▪ Primary response – antibody (IgM)
▪ Secondary response – memory cells (IgG)

Left to right: MHC CLASS I; MHC CLASS II

a. PROFESSIONAL APC
• Dendritic cells
o Most effective
o Constitutively express high levels of MHC class II
molecule i. HLA TESTING
o Inherent co-stimulatory activity 1. Microlymphocytotoxicity test/Complement-dependent
o Activate naive T helper cells cytolysis
• Macrophages o Dye: trypan blue or eosin
BELTRAN, MACATUGGAL, MARTIN, TORRECER 14
MODULE 1: INTRODUCTION TO IMMUNOLOGY

o Classic method using purified T lymphocytes • Monomer: A flexible Y-shaped molecule with four protein
(class I typing) and B lymphocytes chains:
2. Mixed lymphocyte culture: measurement of the degree o 2 identical light chains
of radioactivity by liquid scintillation counting o 2 identical heavy chains
3. DNA-based typing methods: nucleic acid amplification o Joined together by disulfide bonds
(PCR) and sequencing • Variable Regions
o Two sections at the end of Y’s arms
IV. ANTIBODIES/IMMUNOGLOBULINS o Contain the antigen binding sites
• Antibodies or Immunoglobulins are proteins that recognize o Identical on the same antibody, but vary from one
and bind to a particular antigen with very high specificity antibody to another.
• This group is called glycoprotein or globulins • Constant Regions
• Any antigen or microorganism may have several o Stem of monomer and lower parts of Y arms.
epitopes where different antibodies will bind. o The regions beyond the variable region of both heavy
• Made in response to exposure to the antigen. and light chain
• Each antibody has at least two identical sites that bind o Constant regions of the heavy chains are designated
antigen; called Antigen binding site. as CH1, CH2, CH3
o These forms the bases of antibody classification
• Belong to a group of serum proteins called
immunoglobulins • Valence as a determinant of Avidity
Antibody Fab IgG IgG IgM
• The basic structural unit for all immunoglobulins is a Effective antibody valence 1 1 3 Up to 10
tetrapeptide composed of two L (light) and two H (heavy) Antigen Valence 1 1 n n
Definition of Binding Affinity Affinity Avidity Avidity
chains joined together by disulfide bonds.
• Affinity: Strength of association between one antigenic
• The five classes of antibodies are IgM, IgG, IgA, IgD, and
determinant and one antibody binding site.
lgE.
• Monoclonal: Derived from as single B cell.
o IgG, IgE, IgD exists are monomer (one ‘Y’ shape
antibody) • Polyclonal: Antibody produced as different classes of
o IgA exists as Dimeric (two ‘Y’ shape antibody) immunoglobulins by many B cells clones
o IgM is the largest immunoglobulin; exist as pentamer • Avidity: overall strength of association between multiple
(five Y shape antibody molecule) antibody binding sites and multiple antigenic determinants
▪ Held together by J (joining) chain o Overall strength of binding multivalent antigens and
• Kappa and lambda (L chains) are found in all types of antibodies
immunoglobulins. o In most immune responses, the avidity decreases and
o However, H chain differ in each immunoglobulin class the affinity increases.
o More stable antigen-antibody complex
• Each immunoglobulin molecule has constant and
variable regions. • J (joining) chain: regulates the polymer formation of IgA
and IgM because IgA is dimeric and IgM is Pentameric.
• The variable region is at the amino- terminal end
o IgM: Crablike/starlike appearance
o Also known as: Fab Fragment or Fragment
Antigen Binding
• Determines the specificity of molecule for a particular i. TWO HEAVY CHAINS
antigen • Heavy Chains
o The constant region, located at the carboxy-terminal o Molecular weight varies from 50,000-90,000 dalton. It
end of the molecule has longer subunits of antibody structure.
o Also known as: Fc o Designated as u (IgM), delta (IgD), gamma (IgG),
o Responsible for binding to neutrophils, Basophils, etc. alpha (IgA), and epsilon (IgE)
• Fc Portion (constant Region) o It has more molecular weight compared to light
o This may remain the same for all antibodies. It binds to chains
the cells of the immune system o Made up of V, D, and J segments
. o Each H chains are connected to each other via 2
A. IMMUNOGLOBULIN STRUCTURE disulphide bonds in the mid-region.
o To each heavy polypeptide chain has short
Carbohydrate (CHO) chains attached in the constant
heavy chain 2.
▪ This increases the solubility of the
immunoglobulin.
o Each heavy chain contains 440 amino acids
o Each heavy chain is made of 3 constant domains:
▪ CH1(means Constant Region of Heavy chain 1)
▪ CH2
▪ CH3
• Light Blue: Heavy Chains (Constant Region) ▪ one variable (V) domain (VH)
● Dark Blue: Variable Regions of heavy chains • 5 types or classes of heavy chains in humans, all encoded
● Light Green: Light Chains (Constant Region) on the chromosome 14
o There are 2 classes; it cannot be both, only either of o G: GAMMA
the two o A: ALPHA
o Lambda (λ) or Kappa (K) o M: MU
● Dark Green: Variable region of Light Chain o D: DELTA
● Antigen Binding Sites: site where epitope or antigenic o E: EPSILON
determinants on an antigenattaches. • Variable Regions
● Disulphide Bonds/bridges: the reason why components o The amino acid sequence have great permeability.
stick together. o It is the antigen Binding Site.
*Refer to the photo above as the basis for the discussion
Note: Always remember that in antibodies, both light and heavy chain, ii. TWO LIGHT CHAINS
have both variable region and constant region.
• Light Chains are designated as:
o Kappa (κ) or Lambda (λ)
B. ANTIBODY STRUCTURE o It has less molecular weight

BELTRAN, MACATUGGAL, MARTIN, TORRECER 15

MODULE 1: INTRODUCTION TO IMMUNOLOGY

o Made up of V and J segments • Antitoxins: which neutralize specific toxins

o Each L chains is connected to the H chain via a • Complement fixing antibodies
disulfide bond • Blocking or inhibitory antibodies: combine with antigen
o W. 23,000 d but are not grossly detectable
o Each light chain is about 25,000 Dka
o Each Light chain polypeptide contains about 220
E. IMMUNOGLOBULINS
amino acids
• 5 Human Ig classes
o They are similar in structure and function but they are
o IgG: (γ) heavy chains
coded with different genes.
Found in consist of 214
o IgM: (µ) heavy chains
Kappa chain (k)
chromosome 2
60%
amino acid residues o IgA: (α) heavy chains
Lambda chain (λ)
Encoded in
40%
consist of 213 o IgD: (δ) heavy chains
chromosome 22 amino acid residues o IgE: (ε) heavy chains
• Each light chain is made of: • 4 IgG Subclasses
o one constant domain, CL o IgG1: Gamma 1 (γ1) heavy chains
o one variable domain VL. o IgG2: Gamma 2 (γ2) heavy chains
o IgG3: Gamma 3 (γ3) heavy chains
iii. HINGE REGION/MID-REGION o IgG4: Gamma 4 (γ4) heavy chains
• area that gives molecule flexibility allowing for multiple • 2 IgA subclasses
binding sites o IgA1: Alpha 1 (α1) heavy chains
• Ability to adjust for special arrangement for antigenic o IgA2: Alpha 2 (α2) heavy chains
determinants. • Human Immunoglobulin
• Mid-region has considerable flexibility o Light Chain Types (Kappa / Lambda)
• Located between the CH1 and CH2 domains o Light chain subtypes
▪ Lambda Light chain: Lambda 1 (λ1)/Lambda 2
• Stalk is known as the Stem Region (Fc region)
(λ2)/Lambda 3 (λ3)/Lambda 4 (λ4)
• Immunoglobulin Region
C. PROPERTIES OF AN ANTIBODY o Sections in an immunoglobulin molecule
• Protein in nature ▪ Most heavy chain has 1 variable domain and has 3
• High MW more than 150,000 dalton compared with constant region (CH1, CH2, CH3)
antigen ▪ Light chain has 1 variable and 1 constant
• Present in serum/plasma, CSF, saliva and seminal fluid o Each chain consists of CONSTANT and VARIABLE
• Immunogammopathies REGION encoded by the V-D or V-D-J genes.
o Overproduction of immunoglobulin or its fragment o CH: exposed to enzymes and chemicals
o Hinge region
o Disulphide bonds: the function is to stabilize the
tertiary and the quaternary structure of proteins

a. KEY EXPERIMENTS
• Proteolytic treatment of Ig with enzymes papain and
pepsin
• Chemical treatment of Ig with mercaptoethanol
• They studied the Chemical structure of immunoglobulin
D. CLASSIFICATION OF ANTIBODY • Purpose: to fragment IgG molecules.
o Upon characterization of IgG molecule, they are led to
i. ACCORDING TO TEMPERATURE AT WHICH the discovery of ‘Y’ shape structure of immunoglobulin.
THEY REACT • 1972 Nobel prize in Physiology or Medicine for
determining chemical structure of antibody
• Cold antibodies: react at 4C to room temperature o Gerald Edelman
• Warm antibodies: react at 37°C o Rodney Porter
o Albert Nisonoff
ii. ACCORDING TO OCCURRENCE
● Natural antibodies: appear without any apparent stimulus b. PAPAIN
● Immune antibodies: appear following the introduction of
an antigen
o Will appear after the introduction of antigen

iii. ACCORDING TO SPECIES

• Isoantibodies: produced after introduction of the antigen
from the same species
• Heterophile antibodies: produced after introduction of
the antigen from another species

iv. ACCORDING TO ITS REACTION WITH AN • By Rodney Porter

ANTIGEN • Proteolytic enzyme
• Agglutinins: responsible for immobilization of motile • What happens when an Ig is treated with papain?
organisms and for cell clumping o It will split the protein (CHON)
• Hemagglutinins: agglutination of RBC • Upon treatment of papain (blue circle), it will split into 3
• Precipitins: demonstrated only against soluble antigens fragments.
• Lysins: cause dissolution of antigenic cells o Papain cuts the antibody molecule just before the
• Opsonins: act upon bacterial invaders, disulphide bonds above the hinge regions.
• Neutralizing antibodies: render antigenic microorganism • Result: 3 fragments = 2 Fab + 1 Fc
(usually viruses) harmless o 2 identical fragments produce (1L and 1H)
• Allergic antibodies: react with allergens
BELTRAN, MACATUGGAL, MARTIN, TORRECER 16
MODULE 1: INTRODUCTION TO IMMUNOLOGY

o 2 Fab (fragments of antigen binding has antigen o F(c’) cleavage produces two heavy chain fragments
binding ability. that are not joined by s-s bond
• Papain splits the following fragments: • Importance of Papain and Pepsin Digestion
o 2 Fab capable of causing agglutination or precipitation o Important or useful in Immunoglobulin studies; in its
reaction structure and functions
o 1 Fc region - has no antigen binding site.It is consist o Generally, immunoglobulins are resistant to digestion
of constant chains only held together by 2 disulphide by enzymes.
bonds
▪ It crystallizes on cold storage; hence it is called i. MAJOR Ig CLASSES
Fragment Crystallizable or Crystallizable
Fragment upon purification • 5 classes/Isotypes (constant heavy chain)
▪ It carries no antibody activity. o IgG: gamma heavy chain
▪ It cannot act as antibody but it can perform o IgA: alpha heavy chain
biological activities such as: o IgM: mu heavy chain
- Placental permeability/transport o IgE: epsilon heavy chain
- attachment to phagocytic cell, o IgD: Delta heavy chain
Property IgG IgA IgM IgE IgD
- degranulation of Mast cell Physiologic
- stimulates basophil to release histamines % total Ig in Serum 75 15 9 0.004 0.2
- Opsonization Catabolic Rate (1/2
18-23 5-6.5 5-6 2.3 2.8
- Complement fixation, etc. life)
MW 150 170/400 900 190 180
Structure + +2 +4 - -
c. MERCAPTOETHANOL Biological
Agglutinating
+ +2 +4 - -
Capacity
Complement Fixing + - +4 - -
ADCC + - - - -
Mediation of Allergic
- - - +4 -
Response
Placental Transport + - - - -
Present in External
+ +4 +/- +2 -
Secretion
Receptor on B cell - - + - ?
Opsonization + - - - -
Polymeric from J
- + + - -
chain
Subclasses 4 2 - - -
• By Gerald Edelman
• It is a reducing agent ii. IMMUNOGLOBULIN G (IgG)
• Edelman found out that these antibodies containing
disulphide bonds can be broken upon addition of reducing • Four subclasses: IgG1, IgG2, IgG3 & IgG4
agent like mercaptoethanol • Monomer (one ‘Y’ shape antibody molecule)
o It splits the antibody into 2 identical chains • Highest concentration in plasma
o 2 identical Light chains have 22k Dalton each • Transported across the placenta
o 2 identical heavy chains have 53k Dalton each • Activates complement
• Opsonizes
d. PEPSIN • Main Ab in the secondary immune response
• Mediates Antibody-dependent cellular cytotoxicity (ADCC)
• React best at body temp
• Most abundant in serum
• Increases with second exposure

iii. IMMUNOGLOBULIN A (IgA)

• Two subclasses: IgA1 & IgA2
• Monomer/ =Dimer (two ‘Y’ shape antibody molecule)
• Main Ig in external secretions such as milk, tears, saliva &
respiratory & intestinal mucus
• It has secretory component
• Protects mucosal surfaces
• By Albert Nisonoff • Major protective factor in colostrum
• Antibody treated with pepsin splits the molecule below the • It is present in the secretion as dimer w/ a J (joining) chain
hinge region & secretory piece. J chain is made by B or plasma cell;
• It splits into two, however it degrades into smaller Secretory piece made by epithelial cell
fragments. (Pieces of Fragments)
• Result: 2 fragments = F(ab’)2 + 1 Fc’ iv. IMMUNOGLOBULIN M (IgM)
o Sometimes, it is written as 1F(ab’)2 + 1Fc’
o F: Fragments • Produced in the primary response
o Ab: Antigen Binding • Considered as the largest.
o ‘ (prime): variation of antigen structure that contains • Pentamer: serum (held or joined by J (joining) chains) (five
amino acids ‘Y’ shape antibody molecule)
o 2: the fragment has the binding ability like the original o With 10 antibody combining site
• One large F(ab)2 fragment • Indicates acute infection
• LMW peptides • Monomer: Ag receptor on B-cell surface
• Pepsin results in the following fragments: • 1st antibody that an infant makes
o F (ab)’2 slightly different from Fab type fragments that • Most frequently found as natural antibodies against such
not only retain the ability to bind with antigen but antigens as blood group determinants
capable of causing agglutination or precipitation • Most efficient at activating complement
reaction. • Highest agglutinating capacity

BELTRAN, MACATUGGAL, MARTIN, TORRECER 17

MODULE 1: INTRODUCTION TO IMMUNOLOGY

• Can be removed by 2 mercaptoethano: (2-ME) &

dithiothreitol (DTI)

v. IMMUNOGLOBULIN E (IGE)
• Monomer (one ‘Y’ shape antibody molecule)
• Mediates type I hypersensitivity
• Main host defense against helminth infection
• Binds to mast cells
• Triggers allergic response
• Role in response to parasites i. ISOTYPE
• Antigenic (amino acid) differences in their constant
vi. IMMUNOGLOBULIN D (IgD) • heavy regions
• Heavy chain isotypes: 9
• Monomer
• Total isotypes: 18
• Uncertain
• Present in the membrane of mature B cells
• Role in B-cell activation ii. ALLOTYPES
• Identifies mature B cells • additional antigenic features of Ig that vary among
individuals
• Results from the substitution of only one or two amino
acids in the constant regions (usually) of heavy or light
chains
• No biological significance

iii. IDIOTYPE
Left to right: IgG; IgA
• Antigenic determinants formed by the specific amino acids
in the hypervariable region
• Individual, unique differences between antibodies of
different antigen binding specificities
• Individually specific to each Ig molecule

G. ENZYME HYDROLYSIS
Left to right: IgM; IgE; IgD
• Pepsin
o One large F(ab)2 fragment
F. ANTIBODY VARIANTS o LMW peptides
• Antibody variations (shown in black). • Pepsin results in the following fragments:
o (A) Isotype: the H chain that is unique to each o F (ab)’2 slightly different from Fab type fragments that
immunoglobulin class. not only retain the ability to bind with antigen but
o (B) Allotype: genetic variations in the constant capable of causing agglutination or precipitation
regions. reaction.
o (C) Idiotype: variations in variable regions that give • F(c’) cleavage produces two heavy chain fragments that
individual antibody molecules specificity are not joined by s-s bond
Properties of Immunoglobulins
IgG IgM IgA IgD IgE
Molecular Weight 150,000 900,000 160,000 monomer 180,000 190,000
Sedimentation coefficient 7S 19 S 7S 7S 8S
Serum half-life (days) 23 6 5 1-3 2-3
Serum Concentration (mg/dL) 800-1600 120-150 70-250 1-3 0.005
Percent of Total immunoglobulin 70-75 10 10-15 <1 0.02
H chain λ (Lambda) μ (Mu) α (alpha) δ (Delta) ε (Epsilon)
H chain subclasses λ1, λ2, λ3, λ4 none α1, α2 None none
H chain molecular weight 50,000 - 60,000 70,000 55,000 - 60,000 62,000 70,000 -75,000
Constant domains (H chain) 3 4 3 3 4
Carbohydrate content (weight %) 2-3 12 7-11 9-14 12
Electrophoretic migration λ2-α1 λ1- β12 λ2- β2 λ1 λ1
Complement Fixation Yes Yes No No No
Crosses Placenta Yes No No No No
The same variable region can be present on IgM, IgD, IgG, IgE, or IgA.
Ig IgG IgM IgA IgD IgE
CD4 T-helper TH1, TH2 T independent TH2 - TH2
Molecular Mass
154 900 160 185 190
(kDa)
H chain Class Gamma mu Alpha Delta Epsilon
Subclass Gamma-1,2,3,4 - Alpha 1,2 - -
Serum half-life
23 5 6 2-3 2-3
(days)
Principal site of
Serum and tissue Serum Secretions Receptor for B Cells Mast cells
action
Principal Biological Resistance: opsonin, Resistance: precipitin, Resistance: protection of
B-cell Activation Anaphylaxis
effect secondary response primary response mucous membrane
Complement
+++ ++++ + - -
Fixation
Opsonin for
+
Macrophage PMN
Mucosal Secretion - - + - -
Crossing of Placenta + - - - -

BELTRAN, MACATUGGAL, MARTIN, TORRECER 18

MODULE 2: ANTIGEN AND ANTIBODY

V. MONOCLONAL ANTIBODIES o Inhibits VEGF

o stimulates vasculogenesis
9. Trastuzumab
o Brand: herceptin
o Breast cancer
o targets the receptor erbB2 (protein coding)
10. Cetuximab
o colorectal CA and squamous cell CA (skin CA)
o EGFR inhibitor
• Monoclonal antibody 11. Nimotuzumab
o Purified abs cloned from a single cell. o squamous cell CA
o Monoclonal antibodies are made when a cancerous o EGFR inhibitor
cell or myeloma is fused with an antibody-
producing cell to form a hybridoma.
iii. OTHERS
o Hybridomas formed by fusion of one of each cell type
(e.g., myeloma and B cell) are identified by using 1. Palivizumab
HAT, a selective medium. o RSV infections in children
▪ HAT: Hypoxanthine Aminopterin Thymidine o inhibits RSV F protein.
o Two features of the antibody-epitope relationship are 2. Abciximab
key to the use of monoclonal antibodies as a molecular o prevent coagulation in coronary angioplasty
tool. o inhibits receptor on platelets.
▪ Specificity 3. Blinatumomab
▪ Sufficiency o treat some types of acute lymphocytic leukemia
(ALL)
o attaches to CD19 protein
A. USES OF MONOCLONAL ANTIBODIES
o another part attaches to CD3
• Mabs have a single specificity rather than the ab
▪ a protein found on immune cells called T cells.
molecules present in the serum.
B. FDA-APPROVED MONOCLONAL ANTIBODIES
i. TO SUPPRESS THE IMMUNE SYSTEM
USED FOR IMMUNOTHERAPY
1. Muromonab-CD3 (OKT3) Name Type Specificity Disease indications
o Used to prevent acute rejection of organ Rheumatoid arthritis
o Binds to CD3; kidney transplants Psoriatic arthritis
Adalimumab Ankylosing spondylitis
2. Inflixmab and adalimumab (Humira)
Human TNF-a
Crohn’s disease
o inhibits TNF-α. RA, ulcerative colitis, Crohn’s Plaque psoriasis
disease Juvenile idiopathic arthritis
3. Daclizumab and Basiliximab Alemtuzumab Chronic lymphocytic
Humanized CD52
(Campath) leukemia
o Used to prevent acute rejection of transplanted Basiliximab
kidneys. Chimeric CD25 Kidney transplant rejection
(Simulect)
4. Omalizumab VEGF Colorectal cancer
o inhibits IgE. (Vascular Non-small cell lung
Bevacizumab
Humanized Endothelial carcinoma
o Moderate to severe allergic asthma. (Avastin)
Growth Renal cell carcinomas
Factor) Glioblastoma
ii. TO KILL/INHIBIT MALIGNANT CELLS EGFR
(Epidermal Colorectal cancer
1. Rituximab Cetuximab
Chimeric growth Squamous cell carcinoma
(Erbitux)
o targets CD20 on B cells factor of the head and neck
receptor)
o treat B-cell lymphomas (non-Hodgkin’s) cancer of
Rheumatoid arthritis
the lymphoid tissue, which includes the lymph nodes, Psoriatic arthritis
spleen, and other organs of the immune system. Infliximab
Chimeric TNF-a
Ankylosing spondylitis
2. Zevalin (Remicade) Crohn’s disease
Plaque psoriasis
o This is a monoclonal antibody against the CD20 Ulcerative colitis
molecule on B cells (and lymphomas) Ipilimumab
Human CTLA-4 Melanoma
o The antibody delivers radioactivity directly to (Yervoy)
cancerous B cells and can be used to treat some types Omalizumab
Humanized IgE Allergic asthma
(Xolair)
of non-Hodgkin lymphoma Non-hodgkin lymphoma
3. Tositumomab Chronic lymphocytic
o This is a conjugate of a monoclonal antibody against Rituximab
Chimeric CD20
leukemia
CD20. (Rituxan) Rheumatoid arthritis
Wegener’s granulomatosis
o Designed as a treatment for non-hodgkin lymphoma Microscopic polyangitis
4. Lymphocide Breast cancer
o Binds to CD22, a molecule found on some B-cell Trastuzumab
Humanized HER2/neu
Gastric adenocarcinoma
(Herceptin) Gastroesophageal junction
leukemias
adenocarcinoma
5. Alemtuzumab *Brand names are in parenthesis
o Targets antigen CD52 on T and B cells
o Used to treat some patients with chronic lymphocytic
leukemia (CLL); kidney transplant REFERENCES
6. Ipilimumab
o Acts to enhance the body's own immune response Notes from the discussion by Mrs. Maria Redora R. Esteban, RMT
to tumors;
o Melanoma Mrs. Maria Redora R. Esteban PowerPoint presentation: Module 2:
7. Gemtuzumab Antigens and Antibodies
o Acute myeloid leukemia
o Targets surface antigen CD 33 on leukemia cells CANVAS Notes
8. Bevacizumab
o Anti-angiogenic cancer therapy
o colorectal and breast CA
BELTRAN, MACATUGGAL, MARTIN, TORRECER 19