IMMUNOLOGY SEROLOGY

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c. subcutaneous (beneath the skin)
d. oral administration.
Chapter 3: Nature of Antigens and
the MHC Complex 5. Genetic capacity
- allows individuals to respond to
immunogens
Immunogens - linked to the MHC and to the
- Materials that trigger the immune receptors generated during T and
response of lymphocytes (macromolecules B lymphocyte development.
that is capable of triggering an adaptive
immune response by inducing the TRAITS OF IMMUNOGENS
formation of antibodies or sensitized T - ability of immunogen to stimulate host
cells in an immunocompetent host) response depends on ff. characteristics:
- Response to immunogens is caused by: a. Macromolecular size
1. The nature of the immunogen b. Chemical composition and
2. The genetic coding of MHC molecular complexity
molecules that must combine c. Foreignness.
with an immunogen before T cells d. The ability to be processed and
are able to respond to the ag presented with MHC molecules
3. Immunogen processing and
presentation to T and B cells Molecular weight:
Antigen
- refers to a substance that reacts with - 10,000 to be recognized by the immune
antibody or sensitized T cells but may not system
be able to evoke an immune response.
- Recommended: over 100,000 Daltons
- Only a few substances with < 1000 mw can
“All immunogens are antigens, but the converse is induce an immune response (exception).
not true.” - Greater MW: more potent immunogen

FACTORS INFLUENCING THE IMMUNE Immunogenicity:

RESPONSE: - determined by a substance’s chemical
composition and molecular complexity
1. Age - Best immunogens: Proteins and
- Older individuals: have a polysaccharides
decreased response to antigenic
stimulation. Proteins
- Neonates: do not fully respond to - Powerful immunogen because made of
immunogens units known as amino acids
- have epitopes.
2. Overall health - Epitopes stimulate T cells:
- malnourished, fatigued, or - essential to generate T helper
stressed are less likely to mount a cells in antibody production.
successful immune response.
3. Dose Primary structure:
- Threshold dose: allows the innate - sequential arrangement of amino
immune response to take care of acids
small amounts of pathogens and - determines the secondary structure
leave the adaptive response
- Larger dose of an immunogen = Secondary structure:
Greater immune response - the relative orientation of amino acids
- but can result in T- and within the chain
B-cell tolerance
Tertiary structure
4. Route of inoculation - embodies the spatial or three-
- actual amount of immunogen dimensional orientation of the entire
needed to generate an immune molecule
response differs depending on
inoculation route Quaternary structure
- Where immunogen entering body - is based on the association of two or
determines which cell more chains into a single polymeric
populations will be involved in the unit.
response
“Proteins may have an enormous variety of three-
These routes include: dimensional shapes”
a. intravenous (into a vein)
b. intradermal (into the skin)
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B cells: b. Differing specificities
- recognize structures that project from the c. Sequential or linear (amino acids following
external surfaces of macromolecules one another on a single chain)
- more complexity or branching = easier to d. Conformational (results from the folding
respond of one chain or multiple chains; bring
certain amino acids into close proximity
Synthetic polymers with each other so they can be recognized
- Nylon or Teflon together)
- are made up of a few simple repeating
units with no bending or folding within the Surface antibody on B cells:
molecule - Differs from those recognized by T cells
- are nonimmunogenic - react with both linear and conformational
- used in making artificial heart valves, epitopes
elbow replacements, and other medical - anything that is capable of crosslinking
appliances surface immunoglobulin molecules also
able to trigger B-cell activation
Carbohydrates - If the immunogen is a Protein, B cells may
- less immunogenic than protein (units of recognize the primary, secondary, tertiary,
sugars are more limited) or even the quaternary structure
- most often occur in the form of: - In Polysaccharides, the branch points of
a. Glycolipids (A, B, and H blood branched chains may contribute most to
group antigens) their recognition
b. Glycoproteins (Rh and Lewis
antigens) T cells
- Pure nucleic acids and lipids: - are linear and can be found anywhere in
- are not immunogenic by themselves the cell
- response generated when they are - recognize an epitope only as a part of a
attached to a suitable carrier molecule complex formed with MHC proteins on the
surface of an antigen-presenting cell.
- Systemic lupus erythematosus: when - the antigen-presenting cell (APC) must
autoantibodies are actually stimulated by a process an immunogen first and degrade it
DNA protein complex rather than by DNA into small peptides for it to be recognized.
itself.’
HAPTENS
Foreignness - are nonimmunogenic materials that, when
- Distinguishes between self and non- combined with a carrier, create new
self(immunogenic) antigenic determinants.
- Ability is acquired as lymphocytes mature - capable of reaction even without a carrier
in the primary lymphoid organs. - BUT precipitation or agglutination
- The more distant taxonomically the source reactions will not occur
of the immunogen is from the host, the - Haptens only have a single
better it is as a stimulus. determinant site and cannot form
cross-links with more than one
Antigen processing antibody molecule
- needed for the substance to elicit an may be:
immune response - complexed artificially with carrier
- involves enzymatic digestion to create molecules in a laboratory setting
small peptides (can be complexed to MHC - may occur naturally within a host and set
molecules for T and B Lymphocytes) off an immune response (Ex. Poison ivy
- The MHC molecules produced determine (Rhus radicans): contains Catechols which
responsiveness to individual antigens can couple with tissue proteins to form
the immunogens giving rise to contact
dermatitis.).
NATURE OF EPITOPES
Penicillin: drug-protein conjugate that can result
Determinant site or epitope: in life-threatening allergic response provoked by
haptens coupling with proteins
- are molecular shapes or configurations
that are recognized by B or T cells Note: antibodies recognize not only chemical
- Protein (epitopes): recognized by B features such as polarity, hydrophobicity, and
cells with few 6 to 15 amino acids. ionic charge, but the overall three-dimensional
- Large molecules may have numerous configuration.
epitopes that are capable of triggering
specific antibody production or a T-cell
response. RELATIONSHIP OF ANTIGENS TO THE HOST
Autoantigens
- antigens that belong to the host.
Epitopes may be: - do not evoke an immune response under
a. Repeating copies normal circumstances.
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Alloantigens
- are from other members of the host
species.
- are capable of eliciting an immune
response
- important in tissue transplantation and
blood transfusions.
Heteroantigens
- are from other species, such as other MAJOR HISTOCOMPATIBILITY
animals, plants, or microorganisms.
Heterophile antigens COMPLEX
● are hetero antigens that exist in unrelated
plants or animals
● are either identical to or closely related in Human leukocyte antigens (HLA)
structure so that antibody will cross-react - group of molecules that is linked to the
● Example: blood group A and B antigens are genetic capability to mount an immune
related to bacterial polysaccharides. response
- anti-A antibody is normally found - were first defined by discovering an
in individuals with blood types antibody response to circulating white
other than A and is originally blood cells
formed after exposure to - these antigens are also known as MHC
pneumococci or other bacteria molecules: determines whether
- anti-B antibody is formed after transplanted tissue is histocompatible
exposure to a similar bacterial cell
wall product MHC (Major Histocompatibility Complex)
molecules
Note: the first test for infectious mononucleosis
(IM) was based on heterophile antibody reaction
- heterophile antibody was found to react - found on all nucleated cells in the body
with sheep red blood cells, and this - play a pivotal role in the development of
formed the basis of the Paul-Bunnell both humoral and cellular immunity.
screening test for mononucleosis - Main function: bring antigen to the cell
surface for recognition by T cells
- T-cell activation will only occur
ADJUVANTS when antigen is combined with
- a substance administered with an MHC molecules
immunogen that increases the immune - may be involved in transfusion reactions,
response by: graft rejection, and autoimmune diseases.
a. prolonging the existence of
immunogen in the area Major histocompatibility complex: a system of
b. increasing the effective size of the genes controlling expression of MHC molecules
immunogen
c. increasing the number of
macrophages involved in antigen Genes Coding for MHC Molecules (HLA
processing Antigens)
● It acts by producing a local inflammatory
response that attracts a large number of MHC system:
immune system cells to the injection site. ● the most polymorphic system found in
● Aluminum salts: humans (because of many possible alleles
- the only adjuvants approved for for each loci)
clinical use in the United States. ● this polymorphism is essential to our
- used to complex with the survival
immunogen to increase its size
and to prevent a rapid escape Genes coding for MHC molecules are found on
from the tissues. the short arm of chromosome 6 and are divided
- must be injected into the muscle into three categories or classes:
to work
- Example: hepatitis B Vaccination
a. Class I molecules:
- are at three different locations, or loci,
Freund’s complete adjuvant: termed A, B, and C.
- consists of mineral oil, emulsifier, and b. Class II genes:
killed mycobacteria (0.5 mg/mL) - are situated in the D region,
- Antigen is mixed with adjuvant and then - have several different loci, known as DR,
injected. DQ, and DP.
- produces granulomas, or large areas of - have a gene that codes for the alpha chain
scar tissue, and thus is not used in and one or more genes that code for the
humans. beta chain.
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** Class I and II gene products are involved in hydrophilic amino acids, and an
antigen recognition and influence the repertoire anchor of 30 amino acids)
of antigens to which T cells can respond.
2. Lighter chain (β2-microglobulin)
c. Class III genes: - molecular weight of 12,000
- located in an area between the class I and - encoded by a single gene on
class II regions on chromosome 6 chromosome 15 that is not
- codes for complement proteins and polymorphic.
cytokines, such as tumor necrosis factor. - does not penetrate the cell
-Class III proteins are secreted proteins that have membrane, but it is essential for
an immune function, but they are not expressed on proper folding of the α chain.
cell surfaces.
X-ray crystallographic studies
Alleles ❖ indicate that the α 1 and α 2 forms an
- multiple alleles possible for each of alpha helix and that these serve as the
aforementioned loci walls of a deep groove at the top of the
- are alternate forms of a gene that code for molecule
slightly different varieties of the same - these walls functions as the
product. peptide-binding site in antigen
- An individual inherits two copies of recognition
chromosome 6, and thus there is a - This binding site is able to hold
possibility of two different alleles for each peptides that are between 8 and
gene on the chromosome (described as 10 amino acids long.
codominant), unless that person is - Is where most of the
homozygous for those alleles. polymorphism resides
❖ α 3 and ß2 regions are similar to the
Codominant: all alleles that an individual inherits constant regions found in immunoglobulin
code for products that are expressed on cells. molecules.
❖ The α 3 region reacts with CD8 on
Haplotype: cytotoxic T cells.
- MHC genes that are inherited together as
a package Nonclassical class I antigens
- Each haplotype consists of a package of
genes for A, B, C, DR, DP, and DQ. E and F antigens
- group of molecules that are not expressed
Full genotype: consist of two of each gene at a on cell surfaces
particular locus. - do not function in antigen recognition but
may play other roles in the immune
“Uniqueness of HLA antigens create a major response.
problem in matching organ donors to recipients; G antigens
antigens are highly immunogenic” - expressed on trophoblast cells during the
first trimester of pregnancy thought to
help ensure tolerance for the fetus by
Structure of Class I Molecules protecting placental tissue from the action
of NK cells.
- are expressed on all nucleated cells,
although they differ in the level of
expression. Structure of Class II Molecules
- are highest on lymphocytes and low or - are more restricted because they are
undetected on hepatocytes, neural cells, found primarily on antigen- presenting
muscle cells, and sperm. cells (B lymphocytes, monocytes,
** This explains why HLA matching is not done macrophages, and dendritic cells)
in liver transplants
- Class I antigen: a glycoprotein dimer, Major class II molecules
made up of two noncovalently linked
polypeptide chain
DP, DQ, and DR
- consist of two noncovalently bound
1. α chain polypeptide chains that are both encoded
- molecular weight of 45,000 by genes in the MHC complex.
- folded into three domains; α1 α2 - Both the α chain and the β chain are
α3 (consist of about 90 amino anchored to the cell membrane. Each has
acids each) two domains that come together to form
- is inserted into the cell membrane the peptide-binding site.
via a transmembrane segment - ends of the peptide-binding cleft are open,
that is hydrophobic (has about 25 and this allows for capture of longer
hydrophobic amino acids along peptides than in class I.
with a short stretch of about 5 DR
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- expressed at the highest level, accounting - Difference in mechanisms of
for about one-half of all class II molecules transporting of processed antigen to
on a particular cell surface.
- DR ß most highly polymorphic - Both capable of presenting an
enormous array of different
Nonclassical class II genes antigenic peptides to T cell

- DM, DN, and DO: Products of these genes

play a regulatory role in antigen
processing.

Main role of the class I and class II MHC

molecules
bind peptides within cells and transport
them to the plasma membrane, where T Role of Class I Molecules
cells can recognize them in the - Class I molecules bind peptides while still in the
phenomenon known as antigen endoplasmic reticulum.
presentation. - binding helps to stabilize the
association of the a chain of class I with
T cells: the B2–macroglobulin
- can only “see” and respond to antigens - Calnexin: an 88-kd molecule that is
when they are combined with MHC membrane bound in the endoplasmic
molecules. reticulum, and it keeps the α chain in a
- deal with two types of infectious agents: partially folded state while it awaits binding to
1. those that attack cells from the B2– macroglobulin
outside (such as bacteria) - calreticulin, tapasin, and ERp57—are
2. those that attack from the inside chaperone molecules associated with the
(viruses and other intracellular complex and help to stabilize it for
pathogens) peptide binding
- For a T-cell response to be triggered,
peptides must be available in adequate
supply for MHC molecules to bind, they Peptides
must be able to be bound effectively, and - eight to ten amino acids in length
they must be recognized by a T-cell - are derived from partial digestion of
receptor proteins synthesized in the cytoplasm.
- may include viral, tumor, or even bacterial
antigens.
Class I molecules Class II molecules - only a small fraction of them (1 percent or
less) actually induces a T-cell response.

Defective ribosomal products (DRiPs):

- present peptides that -present processed - peptides that are newly made proteins and
have been synthesized antigen to CD4 (helper) failed to fold correctly and hence are
within the cell to CD8 T cells. defective.
(cytotoxic) T cells. -mainly bind - 20% - 70% of all proteins synthesized in a
cell may fall into this category.
- the watchdogs of exogenous proteins
viral, tumor, and (those taken into the
Proteasomes:
certain parasitic cell from the outside - a large cylindrical cytoplasmic complex
antigens that are and degraded) where proteases reside:
synthesized within the - stimulate CD4 T cells
cell in the case of bacterial “Proteases digest defective or early proteins; packet
of enzymes that play a major role in antigen
infections or the presentation.”
presence of other
material that is - Peptides must be unfolded before entering
endocytosed by the the cylindrical chamber and then cleaved
cell. into the proper size.
- Once cleaved, the peptides must then be
pumped into the lumen of the
- Both class I and class II molecules endoplasmic reticulum by specialized
are synthesized in the rough transporter proteins (TAP1 andTAP2).
endoplasmic reticulum.
TAP1 and TAP2
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- are responsible for the adenosine
triphosphate– dependent transport, from
the cytoplasm to the lumen of the Binding
endoplasmic reticulum
- are most efficient at transporting peptides - Selective binding of peptides is favored by
that have 12 amino acids or less. the low pH of the endosomal
compartment.
- Tapasin: brings the TAP transporters into - HLA-DM molecules help to
close proximity to the newly formed MHC mediate the reaction by removing
molecules and mediates interaction with the CLIP fragment.
them. - peptides of approximately 13 to 18 amino
acid residues can bind (major contact
point: 7 – 10)
Binding - Hydrogen bonding takes place along the
- based on interaction of only 2 or 3 amino length of the captured peptide, in contrast
acid residues with the class I binding to class I molecules, which only bond at
groove. the amino and carboxy terminal ends.
- Different class I molecules will have - Once binding has occurred, the class II
slightly different binding affinities; protein-peptide complex is stabilized and
- Display of hundreds of class I molecules is transported to the cell surface
complexed to antigen allows CD8 positive - On the cell surface, class II molecules are
T cells to continuously check cell surfaces responsible for forming a trimolecular
for the presence of non-self antigens. (T complex that occurs between antigen, the
cells are known as surveillance cells). class II molecule, and an appropriate T-
cell receptor.
Role of Class II Molecules - If binding occurs with a T-cell receptor on
a CD4-positive T cell, the T helper cell
recruits and triggers a B-cell response,
- must be transported from the resulting in antibody formation.
endoplasmic reticulum (ER) to an
endosomal compartment before they can
bind peptides Clinical Significance of MHC
- Testing for MHC antigens has typically
been done, because both class I and class
Dendritic cells II molecules can induce a response that
leads to graft rejection.
- the most potent activators of T cells
- are excellent at capturing and digesting - MHC antigens also appear to play a role in
exogenous antigens such as bacteria. development of autoimmune diseases

Invariant chain (Ii) - While both class I and class II molecules

play a major role in antigen presentation.
- prevents interaction of the binding site
with any endogenous peptides in the - Knowing a person’s MHC type might also
endoplasmic reticulum help predict the type of allergens to which
they may be allergic.
- a 31-kd protein that is made in excess so
that enough is available to bind with all
class II molecules shortly after they are
synthesized.
- may be responsible for helping to bring a
and B chains together in the ER lumen
- serves to protect the binding site. CHAPTER 4: ANTIBODY
STRUCTURE AND FUNCTIONS
Class II invariant chain peptide (CLIP)

- a small fragment left by the protease when ANTIBODY OR IMMUNOGLOBULIN

the invariant chain was degraded
- End product when B lymphocytes are
stimulated by antigen and undergo
- is attached to the peptide-binding cleft
differentiation.
- is exchanged for exogenous peptides
Immunoglobulins
Peptides - are considered to be the humoral branch of the
immune response.
- derived from endocytosed, exogenous - They play an essential role in antigen
proteins. recognition and in biological activities related to
- reside in the endosomal compartment
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the immune response such as opsonization and
CLEAVAGE WITH PAPAIN
complement activation

Porter’s work
TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS
- was based on the use of the proteolytic enzyme
papain, which was used to cleave IgG into three
All immunoglobulin molecules are made up of a basic pieces of about equal size,
four chain polypeptide unit that consists of two large - each having a sedimentation coefficient of 3.5 S
chains called and representing a molecular weight of
➔ heavy or H chains and approximately 45,000 to 50,000 daltons.
➔ two smaller chains called light or L chains - Carboxymethyl cellulose ion exchange
chromatography separated this material into
- These chains are held together by noncovalent two types of fragments, one of which
forces and disulfide interchain bridges. spontaneously crystallized at 4ºC .This
fragment, known as the FC fragment (for
“fragment crystallizable”), had no antigen-
The basic structure of immunoglobulins was elucidated binding ability and is now known to represent
in the 1950s and 1960s by the efforts of two men: the carboxy-terminal halves of two H chains
➔ Gerald Edelman, working at the Rockefeller that are held together by S–S bonding.
Institute in the United States, and
➔ Rodney Porter at Oxford University in England. **The FC fragment is important in effector
functions of immunoglobulin molecules, which
- For their contributions, these men shared the include opsonization and complement fixation.
Nobel Prize in physiology and medicine in 1972.
- They chose to work with immunoglobulin G The remaining two identical fragments were found to
have antigen-binding capacity and were named Fab
Edelman’s work fragments (fragment antigen-binding)
- centered on using the analytic ultracentrifuge - Because precipitation would not occur if Fab
to separate out immunoglobulins on the basis of fragments were allowed to react with antigen, it
molecular weight. was guessed that each fragment represented
- He found that intact IgG molecules had a one antigen-binding site and that two such
sedimentation coefficient of 7 S (the Svedberg fragments were present in an intact antibody
unit [S] indicates the sedimentation rate in an molecule; such a molecule would be able to
analytical ultracentrifuge. form a cross-linked complex with antigen, and
the complex would precipitate.
Larger molecules will travel farther and thus have a - Each Fab fragment thus consists of one L chain
larger sedimentation coefficient. On obtaining a purified and one-half of an H chain, held together by
preparation of IgG, Edelman used 7 M urea to unfold the disulfide bonding.
molecule.

PEPSIN DIGESTION
Once unfolded, the exposed sulfhydryl bonds could be
cleaved by a reducing agent such as mercaptoethanol. Alfred Nisonoff
After such treatment, the material was subjected again to - used pepsin to obtain additional evidence for
ultracentrifugation, and two separate fractions, one at 3.5 the structure of immunoglobulins.
S and one at 2.2 S, were obtained. PEPSIN
- This proteolytic enzyme was found to cleave
The 3.5 S fraction, with a molecular weight of IgG at the carboxy-terminal side of the
approximately 50,000, was designated the H chain; the interchain disulfide bonds, yielding one single
2.2 S fraction, with a molecular weight of 22,000, was fragment
named the L chain. These two pieces occurred in equal - MW: 100,000 daltons
amounts, indicating that the formula for IgG had to be - all the antigen-binding ability, known as F(ab)2
H2L2. This is the generalized formula for all
immunoglobulins ***An additional fragment called FC’ was similar to FC
except that it disintegrated into several smaller pieces.
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- Thus, a basic picture of the four-chain unit of - CH2
the immunoglobulin molecule was obtained, - CH3
which indicated that each L chain was bonded H chain unique to each class
to an H chain by means of an S–S bond, and the ➔ IgG has an lambda H chain
H chains were joined to each other by one or ➔ IgM a Micro chain,
more S–S bonds ➔ IgA an alpha chain
➔ IgD a delta chain
➔ IgE is an epsilon chain.
THE NATURE OF LIGHT CHAINS
ISOTOPES
- a unique amino acid sequence that is common
Bence-Jones proteins to all immunoglobulin molecules of a given class
- found in the urine of patients with multiple in a given species
myeloma, were in fact L chains that were being
secreted by the malignant plasma cells. Minor variations of these sequences that are present in
- discovered in 1845 by Dr. Henry Bence-Jones, some individuals but not others are known as allotypes
who noted the peculiar behavior of these - Allotypes occur in the four IgG subclasses, in
proteins: one IgA subclass, and in the kappa light chain.
- When heated to 60ºC, they precipitate from - These genetic markers are found in the
urine, but on further heating to 80ºC, they constant region and are inherited in simple
redissolve. Mendelian fashion.
- Some of the best-known examples of allotypes
Two main types of L chains, designated are variations of the GAMMA chain known as
➔ Kappa G1m3 and G1m1
➔ lambda
- Each contained between 200 and 220 The variable portions of each chain are unique to a
amino acids, and from position specific antibody molecule, and they constitute what is
number 111 on (the amino terminus is known as the idiotype of the molecule. The amino
position number 1), it was discovered terminal ends of both L and H chains contain these
that each type had essentially the regions, which are essential to the formation of the
same sequence. antigen-binding site. Together they serve as the antigen
- constant region – same sequence recognition unit.
carboxyterminal end,
- variable region – amino-terminal Allotype = Constant region
end. Idiotype = Variable region
- DIFFERENCE: kappa and lambda lie in
the amino acid substitutions at a few
HINGE REGION
locations along the chain. (no
functional difference; both are found The segment of the H chain located between the CH1 and
in all five classes of immunoglobulin) CH2 regions is known as the hinge region.

- It has a high content of proline and

HEAVY CHAIN SEQUENCING
hydrophobic residues; the high proline content
‘ allows for flexibility.
H chain sequencing - This ability to bend lets the two antigen-binding
- demonstrated the presence of domains similar sites operate independently.
to those in the L chains—that is, variable and - The flexibility also assists in effector functions
constant regions. such as initiation of the complement cascade
- The first approximately 110 amino acids at the - Gamma, delta, and alpha chains all have a hinge
amino-terminal end constitute the variable region, but mu and epsilon chains do not.
domain. However, the CH2 domains of these latter two
chains are paired in such a way as to confer
The remaining amino acids can typically be divided up flexibility to the Fab arms.
into three or more constant regionsl with very similar
sequences, designated In addition to the four polypeptide chains, all types of
- CH1 immunoglobulins contain a carbohydrate portion, which
IMMUNOLOGY SEROLOGY
9
is localized between the CH2 domains of the two H - has the longest half-life of any immunoglobulin
chains. class, approximately 23 to 25 days, which may
help to account for its predominance in serum.
Functions of the carbohydrate include - All subclasses of IgG appear to be able to cross
➔ increasing the solubility of immunoglobulin, the placenta, although IgG2 is the least efficient.
➔ providing protection against degradation, - Fc regions of IgG have receptors with
➔ enhancing functional activity of the FC domains. macrophages, monocytes and neutrophils.
- This latter function may be the most - The subclasses mainly differ in number and
important, because recognition by FC position of disulfide bridge between the gamma
receptors correlates with the chain
presence of the carbohydrate moiety - Variability in hinge affect the ability to reach
antigen and ability to initiate biological function
like complement activation
Antibody variations. (A) Isotype—the H chain that is - IgM is much more efficient at this than IgG.
unique to each immunoglobulin class. (B) Allotype— - plays a major role in neutralizing toxins and
genetic variations in the constant regions. (C) Idiotype— viruses.
variations in variable regions that give individual - IgG is better at precipitation reactions than at
antibody molecules specificity. agglutination, because precipitation involves
small soluble particles, which are more easily
THREE-DIMENSIONAL STRUCTURE OF ANTIBODIES brought together by the relatively small IgG
molecule.
- The basic four-chain structure of all Four major subclasses:
immunoglobulin molecules does not actually 1. IgG1, 67%
exist as a straight shape y shape. 2. IgG2, 22%
- Intrachain disulfide bonds stabilize these 3. IgG3, 7% - has the largest hinge region and
globular regions number of disulfide bonds
4. IgG5, 4%
B -pleated sheet IG3 more efficient in binding complement followed by
- Folded polypeptide chain (back and forth) on IgG1, IgG2 and IgG4 have shorter hinge segments which
itself along each regions or domains. make them poor mediators of complement activation.
Major functions of IgG include the following:
Immunoglobulin fold or barrel 1. providing immunity for the newborn because
- cylindrical structure produced when the folded IgG can cross the placenta
domains of the H chains line up with those of 2. fixing complement
the L chains 3. coating antigen for enhanced phagocytosis
(opsonization)
Hypervariable regions 4. neutralizing toxins and viruses,
- strategic locations on each chain where antigen 5. participating in agglutination and precipitation
is captured within the barrel by binding to small reactions (MORE EFFICIENT IN
number of amino acids. PRECIPITATION).

complementarity-determining regions (CDRs) PROPERTIES OF IMMUNOGLOBULINS

- Three small hypervariable regions consisting of
approximately 30 amino acid residues are found
within the variable regions of both H and L
chains.

IMMUNOGLOBULIN- G

- predominant immunoglobulin in humans,

comprising approximately 75 to 80 percent of
the total serum immunoglobulins.
 IMMUNOLOGY SEROLOGY
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Property IgG 1 IgG2 IgG3 IgG4 Efficiently triggering classical complement pathway
Molecular weight 150 150 170 150 single molecule initiates reaction result multiple binding
Amino acids in hinge 15 12 62 12 sites.
region
Inter -H chain 2 4 11 2 Comparison of primary and secondary response to
disulfide bonds immunogen:
Half life (days) 14-21 14-21 7 14-21
Mean adult serum 6.98 3.8 0.51 0.56 - The primary response is characterized by a long
level lag phase, slow exponential increase in
Relative abundance 60% 32% 4% 4% antibody, and short-lived response. The
secondary or anamnestic response has a
shortened lag period, antibody rise is much
more rapid, and serum levels remain higher for
longer periods. This is caused by the large
number of antigen-specific memory T and B
cells generated during the primary response.

Immunoglobulin- M
Immunoglobulin - A
- known as a macroglobulin, Largest
● represents 10 to 15 percent of all circulating
immunoglobulin/ it is mainly found in
immunoglobulin, and it appears as a monomer
intravascular pool and not in other body fluid/
with a molecular weight of approximately
tissue
160,000
- Sedimentation rate 19s , MW 970,00
● has a sedimentation coefficient of 7 S and
- Half- life 10 days, account 5-10% in serum
migrates between the and regions on
- If treated with mercaptoethanol, it dissociates
electrophoresis,
into 5 7 s units, MW 190,000
● The H chain, called the alpha chain, has
- MW of H chain is 70,000 with 576 amino acid
molecular weight between 55,000- 60,000 and
- Pentamer form found in secretions, while
consists of about 472 amino acids.
monomer form occur in B cell surface
● Secretory IgA is synthesized in plasma cells
- 5 monomer units held together by J/ joining
found mainly in mucosal-associated lymphoid
chain=glycoprotein with cysteine residues.
tissue, and it is released in dimeric form
Linkage point of disulfide bonds between to
● IgA is synthesized at a much greater rate than
adjacent monomer (carboxy- terminal end)
that of IgG—approximately 3 grams per day in
- J chain may initiate polymerization by
the average adult—but because it is mainly in
stabilizing Fc sulfhydryl group-, J chain MW
secretory form, the serum concentration is
15,000/1 j chain per pentamer.
much lower
- Effective in agglutination and cytolytic
● Lack of complement activation actually assist in
reactions,
clearing antigen without triggering
- Maternal IgM CANNOT CROSS PLACENTA
inflammatory response thus minimizing tissue
- Earliest antibody to appear in the primary
damage
immune response but it does not persist for
● IgA receptor for neutrophils, monocytes and
long. (primary response antibody)
macrophages. Binding to these sites trigger
- Synthesized as long as antigen remains present
respiratory burst and degranulation, occur in
because there is no memory cell for IgM.
both serum and secretory IgA thus capable of
- Primary response - IgM =long log phase,
acting as opsonin.
secondary response -IgG =shortened lag phase
● Example: Success in oral Sabin vaccine induces
and much more rapid increase in antibody titer.
IgA demonstrates effectiveness as protective
- also serves as a surface receptor for antigen. In
role on mucosal surface.
the cytoplasm of the pre-B cell, chains first
appear.
Two subclasses: IgA 1 and IgA2. They differ in content by
22 amino acids, 13 of which are located in the hinge
Major Function of IgM:
region and are deleted in IgA2.The lack of this region
appears to make IgA2 more resistant to some bacterial
● Complement fixation proteases that are able to cleave IgA1.
● Agglutination
● Opsonization
● IgA2 is predominant in secretion at mucosal
● Toxin neutralization
surfaces, while IgA1 is mainly found in serum.
IMMUNOLOGY SEROLOGY
11
● IgA2, is found as a dimer along the respiratory, results in conformational changes and loss of
urogenital, and intestinal mucosa, and it also ability to bind to target cells.
appears in milk, saliva, tears, and sweat. - does not participate in typical
● IgA2 serves to keep antigens from penetrating immunoglobulin reactions such as
further into the body. The dimer consists of two complement fixation, agglutination, or
monomers held together by a J chain that has a opsonization.
molecular weight of about 15,000 - It is incapable of crossing the placenta.
● Formation of secretory IgA. IgA is secreted as a - shortly after synthesis, it attaches to basophils
dimer from plasma cells and is captured by and tissue mast cells by means of specific
specific receptors on epithelial cells. The surface proteins, termed high-affinity FC RI
receptor is actually an SC, which binds to IgA receptors, which are found exclusively on these
and exits the cell along with it. cells
- Binds in the CH3 domain on the Fc region.
Major function of secretory IgA Leaves the antigen- binding site free to interact
with specific antigen.
- Patrol mucosal surfaces - Plasma cells produce IgE located primarily in
- Acts as a first line of defense (sweat) lungs and skin
- neutralizing toxins produced by - Mast cells are also found mainly in the skin and
microorganisms in the lining of the respiratory and alimentary
- prevent bacterial and viral adherence to tracts bind specific antigen cascade event-
mucosal surfaces initiated result to degranulation of mast cells
release vasoactive amines like heparin and
histamine induce type 1 immediate
Immunoglobulin- D hypersensitivity or allergic reaction like hay
fever, asthma, vomiting, diarrhea, hives, and
life-threatening anaphylactic shock.
- was not discovered until 1965, when it was - Appearing to be a nuisance antibody, may serve
found in a patient with multiple myeloma. as protective role by triggering an acute
- It is extremely scarce in the serum, inflammation reaction that recruits neutrophil
representing less than 0.001 percent of total and eosinophil in an area to help destroy
immunoglobulins invading antigen that have penetrated IgA
- synthesized at a low level and has a half-life of defense.
only 2 to 3 days. - Play a major part in the destruction of large
- MW 180,000 migrate as fast as y protein. antigens such as parasitic worms that cannot be
- Delta H chain has a molecular weight of 62,000 easily phagocytized
and appears to have an extended hinge region - Action of IgE on mast cells. IgE binds to specific
consisting of 58 amino acids. receptors on mast cells. When antigen bridges
- found on the surface of immunocompetent but two nearby IgE molecules, the membrane is
unstimulated B lymphocytes disturbed and degranulation results. Chemical
- It is the second type of immunoglobulin to mediators are released.
appear (IgM being the first), and it may play a
role in B-cell activation and differentiation PRIMARY ANTIBODY RESPONSE
- Does not appear a protective function, not bind
complement, neutrophil, macrophages, cross
● Lag phase – no antibody is detectable or It
placenta
takes two weeks before the body produces
- Those cells bearing only IgM receptors appear
antibodies or the immune system generates a
incapable of an IgG response, while those with
primary response that is detectable within 5 to 7
both IgM and IgD receptors are capable of
days
responding to T-cell help and switching to
● Log phase – the antibody titer increases
synthesis of IgG, IgA, or IgE.
logarithmically,
● Plateau phase – the antibody titer stabilizes
Immunoglobulin- E ● Decline phase – the antibody is catabolized

SECONDARY (ANAMNESTIC) RESPONSE

- Least abundant immunoglobulin in the serum
accounting for only 0.0005 percent of total ➔ Time
serum immunoglobulins. - A secondary response has a shorter
- H chains composed 550 amino acids, lag phase, longer plateau, and more
- Disulfide bond 1 H- L chain and 2 H-H chain gradual decline
- Most heat labile of all immunoglobulins; heating ➔ Type of antibody
to 56ºC for between 30 minutes and 3 hours - IgM-type antibodies are the principal
class formed in the primary response.
IMMUNOLOGY SEROLOGY
12
- Although some IgM antibodies are - The receptors Ehrlich originally postulated are
formed in a secondary response, the the surface immunoglobulins IgM and IgD,
IgG class is the predominant type found on unstimulated B lymphocytes
formed. - The main drawback to the clonal selection
➔ Antibody titer theory was consideration of the genetic basis
- higher titer for the diversity of antibody molecules.
- The plateau levels in a secondary - In 1965, Dryer and Bennett proposed a solution
response are typically 10 fold or to this dilemma by suggesting that the constant
greater than the plate and variable portions of immunoglobulin chains
are actually coded for by separate genes

ANTIBODY DIVERSITY

- Attempts to explain the specificity of antibodies

for a particular antigen began long before the
actual structure of immunoglobulins was
discovered.
- central issue was whether an antigen selected
lymphocytes with the inherent capability of
producing specific antibody to it or whether the
presence of antigen added a new specificity to a GENES CODING FOR IMMUNOGLOBULINS
generalized type of antibody
- Tonegawa did some pioneering experiments
with DNA and discovered that chromosomes
Ehrlich’s Side-Chain Theory
contain no intact immunoglobulin genes, only
building blocks from which genes can be
● One of the first theories to be formulated was assembled.
that of Paul Ehrlich in the early 1900s, termed - confirmed the hypothesis of Dryer and Bennett
the side-chain theory. - Human immunoglobulin genes are found in
● postulated that certain cells had specific surface three unlinked clusters: H chain genes are
receptors for antigen that were present before located on chromosome 14, kappa chain genes
contact with antigen occurred. are on chromosome 2, and lambda chain genes
● New receptors would form in place of those are on chromosome 22
broken off, and this process could be repeated. - The genes cannot be transcribed and translated
Although this represented a rather simplistic into functional antibody molecules until this
explanation for antibody synthesis, two key rearrangement, assisted by special recombinase
premises emerged: enzymes, takes place.
1. the lock and-key concept of the fit of
antibody for antigen and Rearrangement of Heavy Chain Genes
2. the idea that an antigen selected cells
with the built-in capacity to respond - The selection process begins with
to it. rearrangement of the genes for the heavy
- This theory did not explain the kinetics of the chains.
immune response or the idea of immunologic - All H chains are derived from a single region on
memory. It laid the foundation for further chromosome 14
hypotheses. - The genes that code for the variable region are
divided into three groups—VH, D, and J.
- There are at least 39 VH (variable) genes,
Clonal Selection approximately
- 23 functional D (diversity) genes, and 6 J
(joining) genes.
- In the 1950s, Niels Jerne and Macfarlane Burnet - In addition, there is a set of genes (C) that codes
independently supported the idea of a clonal for the constant region.
selection process for antibody formation. - Coding for immunoglobulin H chains. Four
- key premise is that individual lymphocytes are separate regions on chromosome 14 code for H
genetically preprogrammed to produce one chains.
type of immunoglobulin and that a specific
antigen finds or selects those particular cells
- DJ regions are spliced first, and then this
capable of responding to it, causing them to
segment is joined to a variable region.
proliferate
IMMUNOLOGY SEROLOGY
13
- When RNA synthesis occurs, one constant ● Derived from a single parent antibody
region is attached to the VDJ combination. H producing cell that has reproduced many times,
chains are made first, but the cell retains its thus forming a clone. - Discovered by Georges
capacity to produce immunoglobulin of another Kohler and Cesar Milstein
class ● purified antibodies cloned from a single cell.
● Hybridoma – fusion of two different types of
Joining of these segments occurs in two steps: cells,
● These antibodies exhibit exceptional purity and
1. At the DNA level, one D and one J are randomly specificity and are able to recognize and bind to
chosen and are joined with deletion of the a specific antigen.
intervening DNA ● takes 3-6 months for the hybridoma cells to
2. a V gene is joined to the DJ complex, resulting in grow
a rearranged V(D)J gene. The VJD combination
codes for the entire variable region of the heavy PATHWAYS
chain.
1. Salvage pathway
- builds DNA from degradation of old
nucleic acids
- blocked because the myeloma cell line
employed is deficient in the required
enzymes HGPRT (hypoxanthine
Recombination signal sequence
guanine phosphoribosyl transferase)
and thymidine kinase
- The recombinase enzymes recognize specific 2. De novo pathway
target sequences that flank all immunoglobulin - makes DNA from new nucleotides
gene segments. - blocked by the presence of
aminopterin
Allelic exclusion
USES OF MONOCLONAL ANTIBODIES
- This phenomenon If a successful rearrangement
of DNA on one chromosome 14 occurs, then the - Identifying and quantifying hormones
genes on the second chromosome are not - Typing tissue and blood
rearranged. - Identifying infectious agents
- Identifying clusters of differentiation for the
Class switching classification of leukemias and lymphomas and
follow up therapy
- The variable and constant regions are joined at - Identifying tumor antigens and autoantibodies
the ribonucleic acid (RNA) level, thus conserving - Delivering immunotherapy
the DNA of the constant regions and allowing
for a later phenomenon whereby daughter Hybridoma Production
plasma cells can produce antibodies of another
type.
- Spleen cells are combined with myeloma cells in
the presence of polyethylene glycol (PEG), a
Light Chain Rearrangement surfactant
- Cells are placed in culture using a selective
- L chain rearrangement occurs only after micro medium containing hypoxanthine, aminopterin,
chains appear, micro chain synthesis represents and thymidine (HAT)
a pivotal step in the process, - Myeloma cells are normally able to grow
- exhibit a similar genetic rearrangement, except indefinitely in tissue culture, but in this case
they lack a D region.
they cannot, because both pathways for the
- Assembly and expression of the L chain locus. A
DNA rearrangement fuses one V segment to one synthesis of nucleotides are blocked.
J segment. The VJ segment is then transcribed - The remaining hybridoma cells are diluted out
along with a unique C region to form mature and placed in microtiter wells, where they are
mRNA. Unarranged J segments are removed allowed to grow. Each well, containing one
during RNA splicing. clone, is then screened for the presence of the
desired antibody by removing the supernatant
MONOCLONAL ANTIBODY