2 Fab region - Fab or Fragment for

Chapter 5 Antibody Structure and o

antigen binding
Function o Fc or Fragment crystalline – is the that
crystallizes upon purification
Antibodies
 One or more constant regions (carboxy-terminal
 are immunoglobulins end)
o Glycoproteins found in the serum o CH1
o 82% to 96% polypeptide and 2% to 14% o CH2
carbohydrate o CH3
o Five major classes: IgG, IgM, IgA, IgD,  Single unique variable region (amino-terminal
IgE end)
 are the key element of the humoral immune o VH (variable heavy chain)
response o VL (variable light chain)
 Immunoglobulins are the slowest moving  Fc fragment
proteins o Has no antigen-binding ability
 appear primarily in the gamma (γ) band with o represents the carboxy-terminal halves
serum electrophoresis at pH 8.6 of two H chains
o serum proteins are albumin and globulin o Held together by S-S bonding
o There 3 fragments of globulin: o Important in effector functions of
 alpha globulin immunoglobulin molecules
 beta globulin  Opsonization – process in which
 gamma globulin – antibodies antibodies will help to attract
primarily appear phagocytic cells.
 Immunoglobulins are considered to be the main  Complement fixation
humoral element of the adaptive immune  Fab fragment
response. o “fragment for antigen binding”
 They play an essential role in antigen o precipitation would not occur if Fab
recognition and in biological activities related to fragments were allowed to react with
the immune response such as opsonization and antigen, it was guessed that each
complement activation. fragment represented one antigen-
 Immunoglobulins are divided into five major binding site and that two such fragments
classes on the basis of a part of the molecule were present in an intact antibody
called the heavy chain. molecule.
o These classes are designated as IgG, o a single monomer has two Fab – Fab1
IgM, IgA, IgD, and IgE (with Ig being and Fab2
the abbreviation for immunoglobulin). o Papain is an enzyme that can digest
o The heavy chains are γ, μ, α, δ, and ε, immunoglobulin and upon digestion,
respectively. immunoglobulin monomer is divided
 each type of immunoglobulin is made up of a into three fragments
number of regions called domains, which  2 Fab
consist of approximately 110 amino acids each.  1 Fc
o consist of One L chain and
TETRAPEPTIDE STRUCTURE OF o one-half of an H chain
IMMUNOGLOBULINS o Held together by disulfide bonding
 basic four-chain polypeptide - tetrapeptide o Obtained by papain digestion of an
o two large heavy (H) chains immunoglobulin
o two small light (L) chains  F(ab’)2
 Held together by noncovalent forces and o obtained by pepsin digestion
disulfide interchain bridges o two antigen-binding sites together
 2 main domains of the immunoglobulins o Fc’ portion in pieces
 o Pepsin is an enzyme that can further hinge region
digest the Fc portion into several
 is the segment of H chain located between the
light chains (bence jonce proteins) CH1 and CH2 regions
 It has a high content of proline and hydrophobic
 Discovered in 1845 by Dr. Henry Bence jones
residues;
 L chains secreted by malignant plasma cells
 the high proline content allows for flexibility
 Two types
o the ability to bend lets the two antigen-
o Kappa (κ)
binding sites operate independently and
o Lambda (λ)
engage in an angular motion relative to
 Each contained between 200 and 220 amino each other and to the FC stem
acids; from position number 111 onward (the o assist initiation of complement cascade
amino terminus is position number 1), it was
and binding to cells with specific
discovered that each type had essentially the
receptors for the Fc portion of the
same sequence
molecule
o Constant region
 Gamma, delta, and alpha chains all have a hinge
 Variable region - the amino-terminal end region, but mu and epsilon chains do not.
 all κ L chains have an almost identical carboxy-
terminal end; the same is true of λ chai carbohydrate portion
 found in all types of immunoglobulin
Heavy chains  localized between the CH2 domains of the two
 Heavy-chain sequencing demonstrates the H chains
presence of domains similar to those in the L  it increase the solubility of immunoglobulin
chains—that is, variable and constant regions  it provides protection against degradation,
 Variable region  it enhances functional activity of the FC
o first approximately 110 amino acids domains
 Constant regions
o remaining amino acids
three-dimensional structure of antibodies
 immunoglobulin molecules does not actually
o three or more regions with very similar
exist as a straight Y shape but is in fact folded
sequences designated CH1, CH2, and
into compact globular subunits based on the
CH3
formation of balloon-shaped loops at each of the
 Are unique to each class and give each
domains
immunoglobulin type its name
o Intrachain disulfide bonds stabilize these
o IgG : γ H chain
globular regions
o IgM : μ chain
o immunoglobulin fold
o IgA : chain,
 Within each of these regions or domains, the
o IgD : δ chain polypeptide chain is folded back and forth on
o IgE : ε chain. itself to form what is called a β-pleated sheet.
 Isotype: a unique amino acid sequence that is  The folded domains of the H chains line up with
common to all immunoglobulin molecules of a those of the L chains to produce a cylindrical
given class in a given species. structure called an immunoglobulin fold
 Allotypes:  Antigen is captured within the fold by binding to
o Minor variations of these sequences that a small number of amino acids at strategic
are present in some individuals but not locations on each chain known as hypervariable
others regions.
o Allotypes occur in the four IgG  Three small hypervariable regions consisting of
subclasses, in one IgA subclass, and in approximately 30 amino acid residues are found
the κ L chain. within the variable regions of both H and L
 Idiotype: the variable portions of each chain are chains
unique to a specific antibody molecule o Each of these regions, called
complementarity-determining regions
 (CDRs), is between 9 and 12 residues o Fixing complement
long o Coating antigen for enhanced
o They occur as loops in the folds of the phagocytosis (opsonization)
variable regions of both L and H chains o Neutralizing toxins and viruses
 The antigen-binding site is actually determined o Participating in agglutination and
by the apposition of the six hypervariable loops, precipitation reactions
three from each chain.  All subclasses are able to participate in the
 Antigen binds in the middle of the CDRs, with secondary immune response, an enhanced and
at least four of the CDRs involved in the binding quicker response to antigen, although their
 a small number of amino acids can create an appearance depends on the triggering antigen.
immense diversity of antigen-binding sites  IgG1and IgG3 are induced in response to protein
antigens, whereas IgG2 and IgG4 are associated
DIFFERENT TYPES OF with polysaccharide antigens
IMMUNOGLOBULIN  Macrophages, monocytes, and neutrophils have
receptors on their surfaces that are specific for
IgG
the FC region of IgG.
 predominant immunoglobulin in humans, o enhances contact between antigen and
comprising approximately 70% to 75% of the phagocytic cells and generally increases
total serum immunoglobulins the efficiency of phagocytosis. IgG1 and
 has the longest half-life of any immunoglobulin IgG3 are particularly good at initiating
class, approximately 23 days, which may help to phagocytosis, because they bind most
account for its predominance in serum strongly to FC receptors
 Four subclasses:  IgG has a high diffusion coefficient that allows
o IgG1, 66% it to enter extravascular spaces more readily than
o IgG2, 23% other immunoglobulin types
o IgG3, 7% o it is distributed almost equally between
o IgG4, 4% the intravascular and extravascular
spaces.
 All IgG have the ability to cross the placenta
except for IgG2  it plays a major role in neutralizing toxins and
viruses.
 Differ mainly in the number and position of the
disulfide bridges between the γ chains  IgG Laboratory Testing
o Agglutination and precipitation
 Variability in the hinge region affects the ability
to reach for antigen and the ability to initiate reactions take place in vitro
important biological functions such as o IgG is better at precipitation reactions
complement activation than at agglutination
 IgG3  precipitation involves small
o has the largest hinge region soluble particles, which are
more easily brought together by
o has the largest number of interchain
the relatively small IgG
disulfide bonds;
molecule
o it is the most efficient at binding
complement, IgM
 IgG1, IgG2 and IgG4 have shorter hinge
 known as a macroglobulin
segments, which tend to make them poor
o it has a sedimentation rate of 19 S,
mediators of complement activation.
 IgG1and IgG3 are induced in response to protein which represents a molecular weight of
approximately 900,000.d
antigens, whereas IgG2 and IgG4 are associated
o It accounts for between 5% and 10% of
with polysaccharide antigens
 Major functions of IgG all serum immunoglobulins
o Providing immunity for the newborn  The half-life of IgM is about 6 days, much
shorter than that of IgG
because IgG is the only antibody that
can cross the placenta
 If IgM is treated with mercaptoethanol, it  Primary response vs Secondary response
dissociates into five 7 S units, each having a o primary response is characterized by a
molecular weight of 190,000 and a four-chain long lag phase, a slow increase in
structure that resembles IgG. antibody, and a short-lived response.
 Can exist as o The second or anamnestic response is
o Monomer (on surface of B cells) distinguished by a shortened lag period,
o Pentamer (found in serum secretions) a much more rapid rise in antibody, and
 Pentamer form higher serum levels for a longer period
o held together by a J or joining chain, of time.
serve as linkage points for disulfide o secondary response is the result of the
bonds between two adjacent monomers larger number of antigen-specific
 J or joining chain is a memory T and B cells generated during
glycoprotein made in plasma the primary response.
cells that contains several  Functions of IgM
cysteine residues o complement fixation,
 Linkage occurs at the carboxy- o agglutination
terminal end of two of the μ o opsonization
chains, and it appears that the J o toxin neutralization
chain may initiate  IgM is the most efficient of all immunoglobulins
polymerization by stabilizing at triggering the classical complement pathway
FC sulfhydryl groups so that because a single molecule can initiate the
cross-linking can occur. reaction as a result of its multiple binding sites.
 The J chain also facilitates  The larger number of binding sites also makes
secretion at mucosal surfaces IgM more efficient at agglutination reactions,
 One especially with multivalent antigens
 J chain is present per pentamer.  IgM forms a potent defense against many
o Has a star-like shape with 10 antigen- bacterial diseases.
binding sites  IgM also serves as a surface receptor for antigen
o The Fab arms can bend out of the plane o Mu chains first appear in the cytoplasm
to bind two or more separate antigens or of the pre-B cell.
multivalent antigens. o When they associate with the early
o Has a high valency
surrogate L chains, rearrangement of the
 helps to overcome the fact that genes controlling L chain synthesis is
they tend to have a low affinity begun.
for antigen. o Later, as L chains are synthesized, IgM
o Because of its large size, IgM is found
monomers are formed and become
mainly in the intravascular pool and not inserted into the plasma membrane.
in other body fluids or tissues. o The presence of membrane IgM
o It cannot cross the placenta.
classifies lymphocytes as immature B
o IgM is known as the primary response cells.
antibody
 it is the first to appear after
antigenic stimulation and the
IgA
first to appear in the maturing
infant  IgA represents 10% to 15% of all circulating
 synthesized only as long as immunoglobulin.
antigen remains present  Serum IgA: appears as a monomer with a
o No memory cells molecular weight of approximately 160,000, has
o IgM can be used to diagnose an acute a sedimentation coefficient of 7 S, and migrates
infection, as its presence indicates a between the β and γ regions on electrophoresis.
primary exposure to antigen
  The H chain, called the α chain, has a molecular health of newborns by passively
weight between 55,000 and 60,000 and consists transferring antibodies and greatly
of about 472 amino acids. decreasing infant death from both
o These amino acids comprise ne variable respiratory and gastrointestinal
and three constant regions infections.
o IgA is not capable of fixing complement
Subclasses of IgA by the classical pathway, although
 IgA1 aggregation of immune complexes may
o mainly found in serum. trigger the alternate complement
o act as an anti-inflammatory agent. pathway
o Lack of complement activation may
o downregulates IgG-mediated
actually assist in clearing antigen
phagocytosis, chemotaxis, bactericidal
without triggering an inflammatory
activity, and cytokine release.
response, thus minimizing tissue
damage
 IgA2
 neutrophils, monocytes, and macrophages
o predominantly found in secretions at
possess specific receptors for IgA
mucosal surfaces
 Binding to these sites triggers a respiratory burst
o found as a dimer along the respiratory,
and degranulation.
urogenital, and intestinal mucosa;
o occurs for both serum and secretory
o it also appears in breast milk, colostrum,
IgA, indicating that they are capable of
saliva, tears, and sweat.
acting as opsonins, or promoters of
o Because mucosal surfaces are a major
phagocytosis
point of entry for pathogens, IgA2
serves to keep antigens from penetrating
farther into the body.
Immunoglobulin D (IgD)
o Is more resistant to some bacterial
proteinases that are able to cleave IgA1  It is extremely scarce in the serum, representing
 Secretory IgA less than 0.001% of total immunoglobulins.
o synthesized in plasma cells found  It is synthesized at a low level and has a half-life
mainly in mucosal-associated lymphoid of only 1 to 3 days.
tissue  The molecule has a molecular weight of
o is released in dimeric form approximately 180,000 and migrates as a fast γ
o is captured by secretory component (SC) protein.
on epithelial cells  The delta (δ) H chain
 Secretory IgA functions o has a molecular weight of 62,000
o patrol mucosal surfaces and act as a first o appears to have an extended hinge
line of defense. region consisting of 58 amino acids
 It plays an important role in  Most of the IgD is found on the surface of
neutralizing toxins produced by immunocompetent but unstimulated B
microorganisms lymphocytes.
 helps to prevent bacterial and  It is the second type of immunoglobulin to
viral adherence to mucosal appear (IgM being the first)
surfaces.  may play a role in B-cell activation,
o Complexes of IgA and antigen are easily  Plays a role in regulating B-cell maturation and
trapped in mucus and then eliminated by differentiation
the ciliated epithelial cells of the  Because of its unusually long hinge region, IgD
respiratory or intestinal tract. is more susceptible to proteolysis than other
o This prevents pathogens from colonizing immunoglobulins.
the mucosal epithelium.  In the secreted form in the serum, IgD does not
o Because IgA is found in breast milk, appear to serve a protective function because
breastfeeding helps to maintain the
 o it does not bind complement, o Typical reactions include hay fever,
o it does not bind to neutrophils or asthma, vomiting and diarrhea, hives,
macrophages and life-threatening anaphylactic shock
o it does not cross the placenta o Recently developed anti-IgE antibody
that targets free IgE has been used as
therapy for allergies and asthma.
 IgE appears to be a nuisance antibody; however,
it may serve a protective role by triggering an
Immunoglobulin E (IgE) acute inflammatory reaction that recruits
neutrophils and eosinophils to the area to help
 is best known for its very low concentration in
destroy invading antigens that have penetrated
serum
IgA defenses
 it has the ability to activate mast cells
 Eosinophils, play a major part in the destruction
andbasophils.
of large antigens such as parasitic worms that
 It is the least abundant immunoglobulin in the
cannot be easily phagocytized
serum,
 accounting for only 0.0005% of total serum
immunoglobulins
 The epsilon (ε) or H chain is composed of Antibody Diversity Theories
around 550 amino acids that are distributed over
Side-chain Theory
one variable and four constant domains.
 Paul Ehrlich in the early 1900s, termed the side-
 A single disulfide bond joins each ε chain to an
chain theory.
L chain and two disulfide bonds link the H
 Ehrlich postulated that certain cells had specific
chains to one another.
surface receptors for antigen that were present
 IgE is the most heat-labile of all
before contact with antigen occurred
immunoglobulins; heating to 56°C for between
 Once antigen was introduced, it would select the
30 minutes and 3 hours results in conformational
cell with the proper receptors, combination
changes and loss of ability to bind to target cells
would take place, and then receptors would
 IgE does not participate in typical
break off and enter the circulation as antibody
immunoglobulin reactions such as complement
molecules
fixation, agglutination, or opsonization.
 New receptors would form in place of those
 It is incapable of crossing the placenta
broken off, after which this process could be
 Shortly after synthesis it attaches to basophils, repeated.
Langerhans cells, eosinophils, and tissue mast
 Although this represented a rather simplistic
cells by means of specific surface proteins,
explanation for antibody synthesis, two key
termed high-affinity FC ε RI receptors, which
premises emerged:
are found exclusively on these cells o first, the lock-and-key concept of the fit
 Produced by plasma cells that are located
of antibody for antigen
primarily in the lungs and skin o second, the idea that an antigen selected
 Mast cells are also found mainly in the skin and
cells with the built-in capacity to
in the lining of the respiratory and alimentary
respond to it.
tracts.
 When two adjacent IgE molecules on a mast cell 1950s: Jerne and Burnet’s clonal selectin or antibody
bind specific antigen, a cascade of cellular formation
events is initiated that results in degranulation of
 lymphocytes are genetically preprogrammed to
the mast cells with release of vasoactive amines
produce one type of immunoglobulin and that a
such as histamine and heparin.
specific antigen finds or selects those particular
o these mediators induces what is known
cells capable of responding to it, causing them to
as a type I immediate hypersensitivity or
proliferate.
allergic reaction
 The receptors Ehrlich originally postulated are
the surface immunoglobulins IgM and IgD,
 which are found on unstimulated B o The selection process begins with
lymphocytes. rearrangement of the genes for the H
 Repeated contact with antigen would continually chains
increase a specific lymphocyte pool. o All H chains are derived from a single
 Would require a large number of genes region on the long arm of chromosome
 The main drawback to the clonal selection 14.
hypothesis was consideration of the genetic  Variable-region genes: VH, D,
basis for the diversity of antibody molecules. and J
 If separate genes were present to code for  Constant-region genes: set of C
antibody to every possible antigen, an genes
overwhelming amount of DNA would be o L chains: lack a D region
needed.  During the process of B-cell maturation, the
pieces are spliced together to commit that B
1965: Dyer and Bennett
lymphocyte to making antibody of a single
 suggesting that the constant and variable specificity.
portions of immunoglobulin chains are actually Monoclonal Antibodies
coded for by separate genes.
 There could be a small number coding for the  Mainly used for diagnostic testing and
constant region and a larger number coding for therapeutic purposes
the variable region. o In vitro diagnostic testing
 This would considerably simplify the task of o Delivery of therapeutic agents in
coding for such variability. diseases
 although all lymphocytes start out with identical  Developed based on knowledge that B cell are
genetic germline DNA, diversity is created by a genetically preprogrammed to synthesize very
series of recombination events that occur as the specific antibody
B cell matures  Monoclonal antibodies are so called because
they are derived from a single parent antibody-
Genes Coding for Immunoglobulins producing cell that has reproduced many times,
thus forming a clone.
 chromosome contain no intact immunoglobulin
genes, only building blocks from which genes  Every cell in the clone is just like every other
can be assembled cell; the antibody produced by each cell is
exactly the same as that of every other cell.
 Human immunoglobulin genes are found in
three unlinked clusters: Hybridomas
o H chain genes are located on
chromosome 14,  fusion of an activated B cell with a myeloma cell
o κ chain genes are on chromosome 2, that can be grown indefinitely in the laboratory
o λ chain genes are on chromosome 22 o Myeloma cells are cancerous plasma
cells.
 The genes cannot be transcribed and translated
o Normally, plasma cells produce
into functional antibody molecules until this
rearrangement, assisted by special recombinase antibody, so a particular cell line that is
enzymes, takes place not capable of producing antibody is
chosen.
 rearrangement involves a cutting and splicing
o this cell line has a deficiency of the
process that gets rid of much of the intervening
DNA, resulting in a functional gene that codes enzyme hypoxanthine-guanine
for a specific antibody. phosphoribosyltransferase (HGPRT)
that makes it incapable of synthesizing
Rearrangement of Heavy-Chain Genes nucleotides from hypoxanthine and
thymidine, which are needed for DNA
 More than one gene controls synthesis of a
synthesis.
particular immunoglobulin
 Production involves:
o immunizing a mouse with a certain
antigen
o Harvesting spleen cells
o Combing spleen cells with myeloma
cells in the presence of PEG
o Selecting fused cells and screening for
presence of desired antibody