IMMUNOLOGY AND SEROLOGY

LECTURE / WEEK NO.2/ MACARUBBO L.

ANTIBODY STRUCTURE AND FUNCTION  Single, unique variable region (amino-terminal end)
I. Tetrapeptide structure of immunoglobulins  One or more constant regions (carboxy-terminal end)
II. Light and heavy chains  Fc fragment
III. Three-dimensional structure of antibodies o Has no antigen-binding ability
IV. Characteristics of specific immunoglobulins o For fragment crystallizable
V. Antibody diversity theories o Represents the carboxy-terminal halves of two H
VI. Genes coding for immunoglobulins chains
VII. Monoclonal antibody o Held together by S–S bonding
o Important in effector functions of
ANTIBODIES immunoglobulin molecules
 Opsonization
 Soldiers of the body wherein they interact with the  Complement fixation
pathogenic substances.
 Fab fragment
 Are immunoglobulins
 Two identical fragments were found to have antigen-
o Glycoproteins found in the serum protein of the
binding capacity (Fragment antigen binding)
blood approx. 20% of plasma proteins.
o One L chain
o 82% to 96% polypeptide and 2% to 14%
o One-half of an H chain
carbohydrate.
o Held together by disulfide bonding
o Five major classes: IgG, IgM, IgA, IgD, IgE
o Obtained by papain digestion of an
 Are the key element of the humoral immune response
immunoglobulin
TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS
BASIC STRUCTURE OF IMMUNOGLOBULINS
 Basic four-chain polypeptide
o Two large heavy (H) chains  𝐹(𝑎𝑏’)2
o Two small light (L) chains o Obtained by pepsin digestion
 Held together by noncovalent forces and disulphide o Two antigen binding sites together
interchain bridges o Fc’ portion in pieces
 The basic structure of immunoglobulins was discovered  An Additional fragment called Fc’ was
by Gerald Edelman and Rodney Porter in the 1950’s and similar to Fc except that it disintegrated
1960’s. into several smaller pieces.
 IgG most abundant of all antibodies.
H-chain

L- chain

IMMUNOLOGY AND SEROLOGY: ANTIBODY STRUCTURE AND FUNCTION 1

 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.2/ MACARUBBO L.
LIGHT CHAINS (BENCE JONES PROTEINS)
 Found in the urine of patients with multiple myeloma
 Discovered in 1845 by Dr. Henry Bence Jones
 L chains secreted by malignant plasma cells
 Two types - identical carboxyl terminal end
o st
Kappa (κ) – 60%; coded 1 in DNA transcription
o Lambda (λ)
 Contain between 200 and 220 amino acids
 Constant region
 Variable region
 60°C – precipitate in the urine. 80°C – redissolve
HEAVY CHAINS
 Variable region
o First approximately 110 amino acids
 Constant regions
o Remaining amino acids
o Three or more regions with very similar
sequences designated CH1, CH2, and CH3
 Are unique to each class and give each immunoglobulin
type its name
o IgG: γ chain
o IgM: μ chain
o IgA: α chain
o IgD: δ chain
o IgE: ε chain
 Isotype: A unique amino acid sequence that is common
to all immunoglobulin molecules of a given class in a
given species
 Allotypes: Minor variations of isotype sequences that are
present in some individuals but not others
o Occur in 4 IgG subclasses, in one IgA subclass
and in kappa L chain.
 Idiotype: Variations in variable regions that give
individual antibody molecules specificity
o Variable portion of each chain are unique to
specific antibody molecule.
IMMUNOLOGY AND SEROLOGY: ANTIBODY STRUCTURE AND FUNCTION
HINGE REGION
 The segment of H chain located between the CH1 and
CH2 regions.
 Has a high content of proline, allowing flexibility, and
hydrophobic residues
 Flexibility
o Allows two antigen-binding sites to operate
independently
o Assists initiation of complement cascade
CARBOHYDRATE PORTION
 Found in all types of immunoglobulins
 Localized between the CH2 domains of the two H chains
 Increases the solubility of immunoglobulin
 Provides protection against degradation
 Enhances functional activity of the Fc domains
THREE-DIMENSIONAL STRUCTURE OF ANTIBODIES
 Folded into compact globular subunits stabilized by
intrachain disulphide bonds
 Immunoglobulin fold
 Does not exist as Y shape but in fact folded into compact
globular subunits based on the formation of balloon
shaped loops at each domain.
 Antigen is captured within the fold by binding to a small
number of amino acids at strategic locations on each
chain known as hypervariable regions.
o 3 small hypervariable regions consisting of
~30 amino acids found within H and L
chains. Each of these regions are called
Complementary determining regions
(CDR’s)
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 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.2/ MACARUBBO L.

IgG IgG: LABORATORY TESTING

 Predominant immunoglobulin in humans (75% to 80% of  Agglutination and precipitation reactions take place in
the total serum immunoglobulins) vitro
 Has the longest half-life of any immunoglobulin class (23  IgG is better at precipitation reactions than at
days, predominance in serum) agglutination
 Four subclasses: o Precipitation involves small soluble
o IgG1: 66% particles, which are more easily brought
o IgG2: 23% together by the relatively small IgG
o IgG3: 7% molecule.
o IgG4: 4%
IgM
 Differ in number and position of the disulphide bridges
between the γ chains  Known as a macroglobulin
 All subclasses can cross the placenta o Has a molecular weight of about 900,000 d
 Macrophages, monocytes, and neutrophils have Fc o Accounts for 5% to 10% of all serum
receptors specific to the Fc region of IgG, increasing the immunoglobulins
efficiency of phagocytosis  Has half-life of 6 days (much shorter than that of IgG)
 A high diffusion coefficient allows IgG to enter  Can exist as:
extravascular spaces more readily than other o Monomer (on surface of B cells)
immunoglobulin types o Pentamer (found in secretions tears, urine)
 IgG plays a major role in neutralizing toxins and viruses
Pentamer form (5)
IgG3  Held together by J chains (joining chains), which are
 Has the largest hinge region linkage points for disulfide bonds between two adjacent
 Has the largest number of interchain disulphide bonds monomers.
 Is the most efficient at binding complement  J chain is present per pentamer; ~MW – 15,000
 Is induced in response to protein antigens  Has a star-like shape with 10 antigen-binding sites

IgG2 and IgG4

 Have shorter hinge segments

 Are poor mediators of complement activation
 Are involved in responses to polysaccharide antigens
 All subclasses have the ability to cross the placenta
except IgG2.

MAJOR FUNCTIONS OF IgG  Has a high valency

 Found mainly in the intravascular pool
 Providing immunity for the newborn (because IgG can
 Cannot cross the placenta
cross the placenta)
 Known as the primary response antibody
 Fixing complement
o Appears first after antigenic stimulation and
 Coating antigen for enhanced phagocytosis
in the maturing infant
(opsonization)
o Synthesized only as long as antigen remains
 Neutralizing toxins and viruses
present
 Participating in agglutination and precipitation reactions
 No memory cells

IMMUNOLOGY AND SEROLOGY: ANTIBODY STRUCTURE AND FUNCTION 3

 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.2/ MACARUBBO L.

 Primary versus secondary response SUBCLASSES OF IgA

 Primary response
 IgA1
o Increased IgM, followed by IgG in the
o Mainly found in serum
presence of antigen
o Acts as anti-inflammatory agent
o Both enters lag period
o Downregulates IgG-mediated phagocytosis,
 Secondary response
chemotaxis, bactericidal activity, and
o IgG will increase since it has subsequent
cytokine release.
contact with the immunogen (antigen-
 IgA2
specific memory T and B cells)
o Predominantly found in secretions at
mucosal surfaces
o Is a dimer along the respiratory, urogenital,
and intestinal mucosa
o Keeps antigen from penetrating farther into
the body
o Is more resistant to some bacterial
proteinases that are able to cleave IgA1

SECRETORY IgA

FUNCTIONS OF IgM  Is synthesized in plasma cells found mainly in

mucosalassociated lymphoid tissue
 Complement fixation
 Is released in dimeric form
 Agglutination
 Is captured by secretory component (SC) on epithelial
 Opsonization
cells.
 Toxin neutralization
 Secretory Component (SC) has a molecular weight of
o IgM most efficient of all immunoglobulins at
70,000 is later attached to the Fc region around the hinge
triggering the classical complement
portion of alpha chains.
pathway
 IgM also serves as a surface receptor for antigen. Mu
chains 1st appear in the cytoplasm of pre B-cell.

IgA

 Accounts for 10% to 15% of all circulating

immunoglobulins in serum
 One variable and three constant regions
 Serum IgA: Appears as a monomer with a molecular
weight of approximately 160,000 d has a coefficient
sedimentation of 7S
 Migrates between beta and gamma region on
electrophoresis.

IMMUNOLOGY AND SEROLOGY: ANTIBODY STRUCTURE AND FUNCTION 4

 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.2/ MACARUBBO L.
SECRETORY IgA FUNCTIONS
 Patrols mucosal surfaces and acts as a first line of
defense
o Neutralizes toxins produced
microorganisms
o Prevents bacterial and viral adherence to
mucosal surfaces
 Passively transfers immunity to newborn during
breastfeeding
 Complexes of IgA and antigen are easily trapped in
mucus and eliminated by the ciliated epithelial cells of
the respiratory and intestinal tracts
 Aggregation of IgA immune complexes may trigger the
alternate complement pathway
 Neutrophils, monocytes, and macrophages possess
specific receptors for serum and secretory IgA
 Binding to these sites triggers a respiratory burst and
degranulation of the cells involved
 Both forms of IgA can thus act as opsonins, or promoters
of phagocytosis
o Oral immunizations such as Sabin vaccine.
IgD
 Extremely scarce in the serum
 Less than 0.001% of total immunoglobulins
 Has a molecular weight of approximately 180,000 d
 The δ H chain
o Has a molecular weight of 62,000
o Appears to have an extended hinge region
consisting of 58 amino acids.
 Is more susceptible to proteolysis than
immunoglobulins
 Has a short half-life (1 to 3 days)
 Found on the surface of immunocompetent but
unstimulated B lymphocytes
 Appears second (after IgM)
 May play a role in B-cell activation
 Plays a role in regulating B-cell maturation and
differentiation
IMMUNOLOGY AND SEROLOGY: ANTIBODY STRUCTURE AND FUNCTION
 Secreted form in serum does not appear to serve a
protective function
o Does not bind complement
o Does not bind to neutrophils or
by
macrophages
o Does not cross the placenta
IgE
 0.0005 % of total serum immunoglobulins. MW –
190,000 making it an 8S molecule.
 Has a carbohydrate content of 12%
 Ability to activate mast and basophils
 Has an ε H chain composed of around 550 amino acids
that are distributed over one variable and four constant
domains
 Produced by plasma cells that are located primarily in the
lungs and skin.
 Does not participate in complement fixation,
agglutination, or opsonization
 Is incapable of crossing the placenta
 Attaches to basophils, eosinophils and tissue mast cells
through high-affinity Fc ε RI receptors
 Most heat labile of all immunoglobulins heating to 56°C,
loss of ability to bind to target cells.
IgE FUNCTION: ALLERGIC REACTIONS
 Type I immediate hypersensitivity results
o Hay fever, asthma, vomiting and diarrhea,
other hives, life-threatening anaphylactic shock.
IgE FUNCTION: PARASITIC INFECTIONS
 Eosinophils play a major part in the destruction of large
antigens, such as parasitic worms, that cannot be easily
phagocytized.
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 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.2/ MACARUBBO L.

ANTIBODY DIVERSITY THEORIES  More than one gene controls synthesis of a particular
immunoglobulin
SIDE-CHAIN THEORY o H chains
 Variable-region genes: VH, D, and J
 Established by Paul Ehrlich in the early 1900s
 Constant-region genes: set of C
 Postulated that certain cells had specific surface
genes
receptors for antigen that were present before contact
o L chains: lack a D region
with antigen occurred
 Through a random selection process, these individual
 If antigen introduced, combines with the proper
segments are joined to commit that lymphocyte to
receptors to break off and enter the circulation as
making antibody of a single specificity
antibody molecules
 Postulated that process could be repeated with further MONOCLONAL ANTIBODIES
contact with antigens
 Mainly used for diagnostic testing and therapeutic
 TWO KEY PREMISES EMERGED:
purposes
o Lock and Key concept of the fit of antibody
o In vitro diagnostic testing
for antigen.
o Delivery of therapeutic agents in diseases
o Idea that an antigen selected cells with the
 Developed based on knowledge that B cells are
built-in capacity to respond to it.
genetically pre-programmed to synthesize very specific
1950S: JERNE AND BURNET’S CLONAL SELECTION FOR antibody
ANTIBODY FORMATION  Are derived from a single parent antibody producing cell
 Lymphocytes are genetically pre-programmed to that has reproduced many times
produce one type of immunoglobulin  Hybridoma
 A specific antigen finds the particular cells capable of o Fusion of an activated B cell with a
responding to it, causing them to proliferate laboratory grown myeloma cell that cannot
make its own DNA because of deficiency of
 Would require a large number of genes
HGPRT.
1965: DRYER AND BENNETT  Production involves:
o Immunizing a mouse with a certain antigen
 Constant and variable portions of immunoglobulin chains
o Harvesting spleen cells
are coded for by separate genes.
o Combining spleen cells with myeloma cells
in the presence of PEG
GENES CODING FOR IMMUNOGLOBULINS
o Selecting fused cells and screening for
 Chromosomes contain building blocks from which genes presence of desired antibody
can be assembled (Tonegawa 1987)  In 1975 Georges Kohler and Cesar Milstein discovered a
 Human immunoglobulin genes are found in three technique to produce antibody arising from single B-cell.
unlinked clusters:
o H chain genes on chromosome 14
o κ chain genes on chromosome 2
o λ chain genes on chromosome 22
 Rearrangement is needed for genes to become
functional antibody molecules

IMMUNOLOGY AND SEROLOGY: ANTIBODY STRUCTURE AND FUNCTION 6

 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.2/ MACARUBBO L.

SUMMARY  Several genes code for a particular immunoglobulin;

through a random selection process, these individual
 The basic structural unit for all immunoglobulins is a
segments are joined to make antibody of a single
tetrapeptide composed of two L and two H chains joined
specificity.
together by disulfide bonds.
 Monoclonal antibodies are made when a cancerous cell
 The five classes of antibodies are IgM, IgG, IgA, IgD, and
or myeloma is fused with an antibody-producing cell to
IgE.
form a hybridoma.
 Kappa and lambda (L chains) are found in all types of
immunoglobulins, but the H chains differ for each
immunoglobulin class.
 Each immunoglobulin molecule has constant and
variable regions.
o Variable region (Fab fragment) determines
the specificity of that molecule for a
particular antigen.
o Constant region (Fc fragment) is responsible
for binding to effector cells.
 The five different types of heavy chains are called
isotypes.
 Minor variations in a particular type of heavy chain are
called allotypes.
 The variable portion of L and H chains unique to a
particular immunoglobulin molecule is known as the
idiotype.
 IgG is small and easily penetrates into tissues.
 IgM is large and excels at complement fixation.
 IgA has an SC that protects it from enzymatic digestion
while it patrols mucosal surfaces.
 An extended hinge region gives IgD an advantage as a
surface receptor for antigen.
 IgE binds to mast cells to initiate a local inflammatory
reaction.
 The primary response to an antigen takes 5 to 7 days
before antibody can be detected.
 The secondary response to antigen occurs more rapidly
and persists for a longer time.
 Ehrlich’s side-chain theory is based on the antigen
selecting the correctly programmed B lymphocyte.
 The clonal selection hypothesis postulated that
lymphocytes are generally pre-endowed to respond to
one antigen or a group of antigens.

IMMUNOLOGY AND SEROLOGY: ANTIBODY STRUCTURE AND FUNCTION 7