BCH408 (IMMUNOCHEMISTRY)

•The branch of biomedical sciences that deals with the chemical aspects of
immunology, including response of an organism to antigenic challenge, the
recognition of self from non-self & all the biological (in vivo), serological (in
vitro) & physicochemical aspects of immunity.
Basic Definitions

•Antigens (Ag): This is any substance that can induce the formation of
antibodies & is capable of reacting specifically with the antibodies so-produced,
under appropriate conditions.

•Antibody (Ab): An antibody is an immunoglobulin molecule with a specific

amino acid sequence that enables it to interact only with the Ag that induced its
synthesis in lymphoid tissue or with Ag closely related to it.

•Immunogens (Immunizing antigen): This is an Ag that is capable of inducing a

specific immune response.

•Immunogenic substances are always antigenic, whereas Ag are not

necessarily immunogenic (e.g., autologous serum proteins).
•Complete antigen: Substances with both immunogenicity & antigenicity
(immunoreactivity).

•Haptens (Incomplete or partial antigens): Low-molecular weight molecules that

possess antigenicity (they can react with immune lymphocytes or Ab), but lack
immunogenicity (they cannot by themselves cause the production of immune
lymphocytes or Ab).

•Examples of haptens: antibiotics, analgesics, & other low-molecular weight

compounds.
•Epitopes (determinant groups or antigenic determinants): The sites either on
or within the Ag with which Ab reacts.

•Paratopes (antigen-binding site): The site on an Ab that recognizes & binds

with the Ag.

•Valence: refers to the number of paratopes of an immunoglobulin or the

number of epitopes on an Ag. It defines the theoretical number of chemical
bonds that may occur btw an Ag & an Ab.

•Immunogenicity: This is the inherent ability of an immunogen to induce a

specific immune response, resulting in the formation of Ab or immune
lymphocytes.
•Antigenicity (immunoreactivity or specific reactivity): This is the ability of an Ag
to react specifically with the Ab or lymphocyte that it caused to be produced.

•Immunocompetent cell: A cell that has the capacity to recognize Ag &/or to

synthesize Ab.

•Histocompatibility antigens (human leucocyte antigens, HLA): Ag produced by

most nucleated cells of the organism that are responsible for graft or transplant
rejection.

•Major histocompatibility complex (MHC): The aggregate of MHC Ag.

•Adjuvant: Any substance that when mixed with an Ag enhances antigenicity &
boosts immune response.

•Complement system: Comprises some heat-labile proteins that normally exist

as inactive precursors in the plasma that help in the opsonization of pathogenic
microorganisms & immune complexes.

•Opsonization: This refers to the process of making foreign bodies such as

bacteria susceptible to destruction by blood cells by coating them with opsonin
(IgG antibodies & C3b)

•Tolerogen: An Ag that is capable of inducing immunologic tolerance.

•Immunologic tolerance: This refers to unresponsiveness to an Ag that is
induced by prior exposure to that Ag.

•Allergen: This is an Ag that induces allergic response or anaphylaxis (that is, a

severe immediate hypersensitivity reaction occurring as a result of rapid
generalized mast-cell granulation).

•Vaccine: This is an attenuated Ag that induces a protective immune response

against pathogenic microbes & is used to prevent diseases.
 CONCEPTS AND TYPES OF IMMUNITY

•The term immunity, derived from the Latin “immunis” (meaning exempt), refers
to naturally acquired protection/defense against diseases e.g. measles &
smallpox.

•The immune system is an intricate collection of organs, tissues, cells, &

soluble factors that allows individuals to defend against pathogenic agents e.g.
viruses, bacteria, fungi, parasites, & tumor cells.

•The ultimate goal of this system is to prevent or limit infections or damage by

these agents.

•The immune response involves recognizing any foreign material & mounting a
reaction to eliminate it.
•There are 2 categories of immune response:

1. innate (a.k.a. non-specific; natural)

&

2. acquired (a.k.a. specific; adaptive) immunity.

•Innate immunity serves as a general protection against many different
pathogenic organisms, & as a first line of defense.

•All normal individuals are born with it, & it includes:

i. Natural mechanical barriers, e.g. the integrity of the epidermis & mucosal
membranes;

ii. Natural physicochemical barriers, e.g. the acidity of the stomach fluid;

iii. Antibacterial substances (e.g., lysozyme) present in some secretions;

iv. Normal intestinal transit & normal flow of bronchial secretions & urine, which
eliminate infectious agents from the respective systems;

v. Ingestion & elimination of bacteria & particulate matter by granulocytes,

which is independent of the immune response;
•Acquired immunity, in contrast, is directed at a particular pathogenic
organism.

•As a rule, acquired immunity is induced during the life of the individual as part
of the complex sequence of events designated as the immune response.

•In vertebrates, the innate responses trigger the acquired immune responses, &
both work together to eliminate the pathogens.
Table. Characteristics of innate versus adaptive (acquired) immunity
Specificity:

•The immune response is specific for the eliciting Ag.

•E.g., immunization with poliovirus only protects against poliomyelitis, not

against the flu.

•This specificity is due to the existence of exquisitely discriminative Ag

receptors on lymphocytes.

•Only a single or a very limited number of similar structures can be

accommodated by the receptors of any given lymphocyte.

•When those receptors are occupied, an activating signal is delivered to the

lymphocytes.

•Thus, only those lymphocytes with specific receptors for the Ag in question will
be activated.
Memory:

•This refers to the ability of the immune system to exert progressively more
intense specific responses to repeated exposures to a given Ag.

•Most immunizations involve repeated administration of the immunizing

compound, with the goal of establishing a long-lasting, protective response.

•The increase in the magnitude & duration of the immune response with
repeated exposure to the same Ag (i.e. memory of immune response) is due to
the proliferation of Ag-specific lymphocytes after each exposure.
•The numbers of responding cells will remain increased even after the immune
response subsides.

•Thus, whenever the organism is exposed again to that particular Ag, there is
an expanded population of specific lymphocytes available for activation.

•Consequently, the time needed to mount a response is shorter & the

magnitude of the response is higher.
Stages of the Acquired Immunity:

•The acquired immunity could be considered as divided into 3 separate

sequential stages.

•The 1st stage (induction) involves a small lymphocyte population with specific
receptors able to recognize an Ag or a fragment generated by specialized cells
known as Ag-presenting cells (APC).

•In the 2nd stage (amplification), the proliferation & differentiation of Ag-
responding lymphocytes is usually enhanced by amplification systems involving
APC & specialized T-cell sub-populations (T-helper cells).
•In the 3rd stage (effector), there is the production of effector molecules
(antibodies) and the differentiation of effector cells.

•Effectors cell are cells that directly or indirectly mediate the elimination of
undesirable elements.

•The final outcome, therefore, is the elimination of the microbe or compound

that triggered the reaction by means of activated immune cells or by reactions
triggered by mediators released by the immune system.
Table. Overview of the 3 main stages of the acquired immune response
Active & Passive Acquired Immunity:

•Acquired immunity can be either active or passive.

•Active acquired immunity is produced by an individual either after natural

exposure to Ag or after immunization.

•Passive acquired immunity does not involve the host’s immune response at all.
•Passive immunity occurs when preformed Ab or T-cells are transferred from a
donor to the recipient.

•It may be natural, as during pregnancy when maternal Ab cross the placenta
to the fetus, or artificially, as when Ab is injected to fight against a specific
disease.

•E.g., unvaccinated individuals who are exposed to particular infectious agents

(e.g., hepatitis A virus, rabies virus) often will be given immunoglobulins, which
are prepared from individuals who already have Ab against that particular
pathogen.

•Active acquired immunity is long lived, while passive immunity is only

temporary because the donor’s Ab or T-cells are eventually destroyed.
Types of Acquired Immune Response:

•There are 2 main types of acquired (adaptive) immunity:

1. Humoral

2. Cell-mediated
Humoral Immune Responses:

•In humoral immune responses, B-lymphocytes are activated to secrete Ab

(immunoglobulins).

•The Ab circulate in the bloodstream & permeate other body fluids, where they
bind specifically to the foreign Ag that stimulated their production.

•Binding of Ab inactivates viruses & microbial toxins (e.g. tetanus toxin or

diphtheria toxin) by blocking their ability to bind to receptors on host cells.

•Ab binding also marks invading pathogens for destruction, mainly by making it
easier for phagocytic cells of the innate immune system to ingest them.
Cell-mediated Immune Responses:

•In cell-mediated immune responses, activated T-cells react directly against a

foreign Ag that is presented to them on the surface of a host cell by antigen-
presenting cell (APC).

•T-cells can efficiently detect pathogenic microbes hiding inside host cells &
either kill the infected cells or help the infected cells or other cells to eliminate
the microbes.

•E.g., the T-cell might kill a virus-infected host cell that has viral Ag on its
surface, thereby eliminating the infected cell before the virus can replicate.

•In other cases, the T-cell produces signal molecules that either activate
macrophages to destroy the microbes that they have phagocytosed or help
activate B-cells to make Ab against the microbes.
Figure. The 2 main types of adaptive immune responses.
Notes:

•Lymphocytes carry out both types of responses.

•In the figure in the preceding slide, the lymphocytes are responding to a viral
infection.

• In the humoral response, B-cells secrete Ab that neutralize the virus.

•In the cell-mediated response, T-cells kill the virus-infected cells.

•In both cases, innate immune responses help activate the adaptive immune
responses.
 THE CELLS OF THE IMMUNE SYSTEM

•The immune system comprises 2 major components: B-lymphocytes & T-

lymphocytes.

Lymphocytes

•Lymphocytes are the white blood cells, involved in the elimination of potentially
harmful organisms or compounds.

•They make up ~ 20% of the white blood cells present in the adult circulation.

•Mature lymphoid cells (cells that secrete lymph – body fluids containing white
blood cells, majorly lymphocytes) are long-lived & may survive for many yrs as
memory cells.

•The lymphocytes interact specifically with antigenic substances & react to

non-self antigenic determinants.
•Lymphocytes differentiate from stem cells in the fetal liver, bone marrow, &
thymus into 2 main functional classes.

•They are found in the peripheral blood & in all lymphoid tissues.

Sub-populations of lymphocytes:

1.B-lymphocytes:

•Mainly derived from bone marrow cells in higher animals & from the bursa of
Fabricius in birds.

•Are responsible for the synthesis of circulating, humoral Ab (immunoglobulins).

•Are the precursors of antibody-producing cells, known as plasma cells.

2. T-lymphocytes (T-cells):

•Derived from the thymus (located at the base of the neck).

•Are involved in a variety of important cell-mediated immunologic processes

e.g. graft rejection, hypersensitivity reactions, & defense against malignant cells
& many viruses.

•T-lymphocytes also carry an antigen-recognition unit on their membranes,

known as T-cell receptor.

•T-lymphocytes have a longer life span than B-lymphocytes, & thus, are
involved in immunological memory.
Sub-populations of T-lymphocytes & their functions:

a. Helper T-lymphocytes (TH), which are involved in the induction & regulation
of immune responses.

•There are 2 functionally distinct sub-populations of T-helper lymphocytes:

i. TH1 lymphocytes, which assist the differentiation of cytotoxic cells & also
activate macrophages, which after activation play a role as effectors of the
immune response.

ii. TH2 lymphocytes, which are mainly involved in the amplification of B

lymphocyte responses.

•This Amplifying effect is mediated in part by soluble mediators (interleukins) &

in part by signals delivered as a consequence of cell-cell contact.
b. Cytotoxic T-lymphocytes, which are the main immunological effector
mechanisms involved in the elimination of non-self or infected cells.

3. Antigen-presenting cells (APC), e.g. the macrophages & macrophage-related

cells, play a very significant role in the induction stages of the immune
response by trapping & presenting both native Ag & Ag fragments in a most
favorable way for recognition by lymphocytes.

•In addition, these cells also deliver activating signals to lymphocytes engaged
in Ag recognition, both in the form of soluble mediators (interleukins such as IL-
12 & IL-1) & in the form of signals delivered by cell-cell contact.
Fig. Development & differentiation of lymphocytes from pluripotential stem cells
Table. Lymphocytes involved in immune response
Antigens (Ag) & Antibodies (Ab):

A. Antigens are non-self substances (cells, proteins, polysaccharides) that

are recognized by receptors on lymphocytes, thereby eliciting immune
response.

•The receptor molecules located on the membrane of lymphocytes interact with

small portions of those foreign cells or proteins, designated as antigenic
determinants or epitopes.

•An adult human being has the capability of recognizing millions of different
Ag, some of microbial origin, others present in the environment, & even some
artificially synthesized.
B. Antibodies are proteins that appear in circulation after immunization &
that have the ability to react specifically with the Ag used to immunize.

•Ab are also generically known as immunoglobulins as they structurally belong

to the family of proteins known as globulins (globular proteins) & from their
involvement in immunity.

•Because Ab are soluble & are present in virtually all body fluids (“humors”), the
term humoral immunity was introduced to designate the immune responses in
which Ab play the principal role as effector mechanisms.
 ANTIGEN-ANTIBODY REACTIONS

•Antigen-antibody (Ag-Ab) reaction (interaction) is a bimolecular association

between an Ag & an Ab, similar to an enzyme-substrate interaction.

•An important distinction is that Ag-Ab is reversible & does not lead to chemical
alteration in either the Ab or the Ag, in that it can be prevented or dissociated
by high ionic strength or extreme pH.

•The association between an Ag & an Ab involves various non-covalent

interactions btw the antigenic determinant (epitope), of the Ag & the variable-
region (VH/VL) domain of the Ab molecule, particularly the hypervariable
regions, or complementarity-determining regions (CDRs) (paratope).
Fig. Depiction of an Ag epitope and an Ab paratope
•The exquisite specificity of Ag-Ab interactions has led to the development of a
variety of immunologic assays, which can be used to detect the presence of
either Ab or Ag.

•These assays differ in their speed & sensitivity; some are strictly qualitative,
others are quantitative.

•Immunoassays have played vital roles in:

diagnosing diseases;

monitoring the level of the humoral immune response;

identifying molecules of biological or medical interest.

Features of Ag-Ab reactions:

•Highly specific

•Reversible

•Non-covalent.
Electrostatic (ionic or electrovalent) bonds:

•Results from the attraction btw oppositely charged ionic groups of 2 protein
side chains, e.g., an ionized amino group (NH 4+) on a lysine in the Ab, & an
ionized carboxyl group (COO-) on an aspartate residue in the Ag.

Hydrogen bonds:

•When the Ag & Ab are in very close proximity, relatively weak H bonds can be
formed btw hydrophilic groups (e.g., OH & C=O; NH & C=O; & NH & OH
groups).

•In other words, a H atom is shared btw 2 electronegative atoms.

Hydrophobic interactions:

•Hydrophobic groups, e.g. the side chains of valine, leucine, & phenylalanine,
tend to associate due to Van der Waals bonding & coalesce in an aqueous
environment, excluding H2O from their surroundings.

•The H2O molecules force hydrophobic groups together.

•Thus, the distance btw them decreases, enhancing the energies of attraction
involved.

•Hydrophobic interactions is estimated to contribute up to 50% of the total

strength of the Ag-Ab bond.
Van der Waals interactions:

•Depends upon interactions btw the “electron clouds” that surround the Ag & Ab
molecules.

•The interaction has been compared to that which might exist btw alternating
dipoles in 2 molecules, alternating in such a way that at any given moment
oppositely oriented dipoles will be present in closely apposed areas of the Ag &
Ab molecules.
•Fig. The 4 non-covalent forces that determines the strength of Ag-Ab interaction
Cross-Reactivity

•Although Ag-Ab reactions are highly specific, in some cases Ab elicited by one
Ag can cross-react with an unrelated Ag.

•Such cross-reactivity occurs if 2 different Ag share an identical or very similar

epitope.
•In the latter case, the Ab’s affinity for the cross-reacting epitope is usually less
than that for the original epitope.

•Cross-reactivity is often observed among polysaccharide Ag that contain

similar oligosaccharide residues.

•E.g., the ABO blood-group Ag are glycoproteins expressed on red blood cells.

•Subtle differences in the terminal residues of the sugars attached to these

surface proteins distinguish the A & B blood-group Ag.

•An individual lacking 1 or both of these Ag will have serum Ab to the missing
Ag.

•The Ab are induced not by exposure to red blood cell Ag but by exposure to
cross-reacting microbial Ag present on common intestinal bacteria.
•These microbial Ag induce the formation of Ab in individuals lacking the similar
blood-group antigens on their red blood cells.

•In individuals possessing these Ag, complementary Ab would be eliminated

during the developmental stage in which Ab that recognize self epitopes are
weeded out.

• The blood-group Ab, although elicited by microbial Ag, will cross-react with
similar oligosaccharides on foreign red blood cells, providing the basis for blood
typing tests & accounting for the necessity of compatible blood types during
blood transfusions.

•A type A individual has anti-B Ab; a type B individual has anti-A; and a type O
individual thus has anti-A and anti-B.
Table. Cross-reactivity in ABO blood types
Precipitation Reactions

•Ab & soluble Ag interacting in aqueous solution form a lattice that eventually
develops into a visible precipitate.

•Ab that aggregate soluble antigens are called precipitins.

•Although formation of the soluble Ag-Ab complex occurs within minutes,

formation of the visible precipitate occurs more slowly & often takes a day or 2
to reach completion.

Formation of an Ag-Ab lattice depends on the valency of both the Ab & Ag:

1.The Ab must be bivalent; a precipitate will not form with monovalent Fab
fragments.

2.The Ag must be either bivalent or polyvalent; that is, it must have at least two
copies of the same epitope, or have different epitopes that react with different
antibodies present in polyclonal antisera.
Agglutination Reactions

•The interaction btw Ab & a particulate Ag results in visible clumping called

agglutination.

•Ab that produce such reactions are called agglutinins.

•Agglutination reactions are similar in principle to precipitation reactions; they

depend on the crosslinking of polyvalent Ag.
•Just as an excess of Ab inhibits precipitation reactions, such excess can also
inhibit agglutination reactions;

•This inhibition is called the prozone effect.

•Several mechanisms can cause the prozone effect.

•First, at high Ab concentrations, the number of Ab binding sites may greatly

exceed the number of epitopes.

•As a result, most Ab bind Ag only univalently instead of multivalently.

Antibodies that bind univalently cannot crosslink one Ag to another.
Applications of Ag-Ab agglutination reaction

•Agglutination reactions are routinely performed to type red blood cells (RBCs).

•In typing for the ABO Ag, RBCs are mixed on a slide with antisera to the A or B
blood-group Ag.

•If the Ag is present on the cells, they agglutinate, forming a visible clump on
the slide.

•Determination of which Ag is present in donor & recipient RBCs is the basis for
matching blood types for transfusions.
Bacterial agglutination is used to diagnose infection

•A bacterial infection often elicits the production of serum Ab specific for

surface Ag on the bacterial cells.

•The presence of such Ab can be detected by bacterial agglutination reactions.

•Serum from a patient thought to be infected with a given bacterium is serially

diluted in an array of tubes to which the bacteria is added.

•The last tube showing visible agglutination will reflect the serum Ab titer of the
patient.

•The agglutinin titer is defined as the reciprocal of the greatest serum dilution
that elicits a positive agglutination reaction.
•E.g., if serial twofold dilutions of serum are prepared & if the dilution of 1/640
shows agglutination but the dilution of 1/1280 does not, then the agglutination
titer of the patient’s serum is 640.

•The agglutinin titer of an antiserum can be used to diagnose a bacterial

infection.

•Patients with typhoid fever, e.g., show a significant rise in the agglutination titer
to Salmonella typhi.

•Agglutination reactions also provide a way to type bacteria.

•E.g., different species of the bacterium Salmonella can be distinguished by

agglutination reactions with a panel of typing antisera.
 IMMUNOGLOBULINS

Structure

•Immunoglobulins contain a minimum of 2 identical light (L) chains (23 kDa) & 2
identical heavy (H) chains (53–75 kDa), held together as a tetramer (L2H2) by
disulfide bonds between cysteine residues in the chains.
Fig. Structure of an Immunoglobulin
Fig. Structure of an IgG
Variable (V) Region:

•The amino acid N-terminal domains of the heavy & light chains form the Ag-
binding site.

•The amino acid residues of this domain vary between different Ab molecules &
are thus known as the variable (V) regions: VL in light chains & VH in heavy.

•Most of the differences reside in the hypervariable areas of the molecule & are
usually only 6 to 10 amino acid residues in length.

•When the hypervariable regions in each chain come together along with the
counterparts on the other pair of H & L chains, they form the Ag-binding site.

•This part of the molecule is unique to the molecule and is known as the
idiotype determinant.
Constant (C) Region:

•The part of the Ab structure next to the variable region is called the constant
(C) region.

•It is made up of 1 domain in the light chain (CL) & 3 or 4 in the heavy chain
(CH).

• A CL chain may consist of either 2 kappa (κ) or 2 lambda (λ) chains but never
1 of each.

•Approximately 60% of all the human Ab molecules contain κ chains, while 40%
contain λ chains.

•Although there are no known differences in the functional properties of κ & λ

chains, there are several different types of the CH domain.

•These different types of the CH domain (γ, α, μ δ, and ε) determine the class
(isotype) of the Ab, & thereby the physiological function of a particular Ab.
Hinge Region:

•The light & heavy chains enclose an extended peptide chain between the C H1
& CH2 domains that have no homology with the other domains.

•This region is called the hinge region.

•The region is rich in proline residues & flexible, giving IgG, IgD, & IgA
segmental flexibility.

•As a result, the 2 Fab (fragment containing the Ag binding site) arms can
assume various angles to each other when Ag is bound.
Classes of Immunoglobulin

•There are 5 immunoglobulin classes in humans: IgG, IgA, IgM, IgD, & IgE.

•These 5 classes are determined by the type of H chain (γ, α, μ δ, and ε)

present in the immunoglobulin.
Immunoglobulin G (IgG)

•IgG is the most abundant in serum, constituting about 80% of the total serum
immunoglobulin.

•The IgG molecule has 2 heavy chains & 2 light chains.

•There are 4 IgG subclasses in human designated: IgG1, IgG2, IgG3, & IgG4,
according to their declining average of serum concentrations.

•The structural characteristics that differentiate the subclasses from one

another are the size of the hinge region & the number & position of the
interchain disulfide bonds between the heavy chains.

•IgG1, IgG3, & IgG4 can cross the placenta & play an important role in
protecting the developing fetus.
Immunoglobulin M (IgM)

•IgM constitutes 5%–10% of the total serum immunoglobulin; with an average

serum concentration of 1.5 mg/mL.

•It is secreted by plasma cells as a pentamer in which the 5 monomeric units

are held together by disulfide bonds, with 10 antigen-binding sites on the
periphery of the molecule.

•Each pentamer contains an additional Fc-linked polypeptide called the J

(joining) chain.

•IgM is the first immunoglobulin class produced in a primary response to an

antigen.

•It is also the first immunoglobulin to be synthesized by the neonate.

Immunoglobulin A (IgA)

•IgA consists10%–15% of the total immunoglobulin in serum.

•It is the predominant immunoglobulin class in external secretions such as

breast milk, saliva, tears, & mucus of the bronchial, genitourinary, & digestive
tracts.

•It serves an important effector function at mucous membrane surfaces, which

are the main entry sites for most pathogenic organisms.

•In serum, IgA exists as a monomer containing a J-chain polypeptide.

•IgA can cross-link large Ag with multiple epitopes.

•Binding of secretory IgA to bacterial & viral surface Ag prevents attachment of

the pathogens to the mucosal cells, thus inhibiting viral infection & bacterial
colonization.

•Breast milk contains secretory IgA & many other molecules that help protect
the newborn against infection during the first months of life, when its immune
system is not fully functional.
Immunoglobulin E (IgE)

•IgE has a very low average serum concentration of 0.3 mg/ml.

•IgE antibodies mediate the immediate (type I) hypersensitivity reactions, which

are responsible for the symptoms of hay fever, asthma, hives, & anaphylactic
shock.

•Localized mast-cell degranulation induced by IgE also may release mediators

that facilitate a buildup of various cells necessary for antiparasitic defense.
Immunoglobulin D (IgD)

•IgD constitutes about 0.2% of the total immunoglobulin in serum, & has a
serum concentration of 30 mg/mL.

•IgD, together with IgM, is the major membrane bound immunoglobulin

expressed by mature B-cells.

•IgD was first discovered when a patient developed a multiple myeloma whose
myeloma protein failed to react with anti-isotype antisera against the then
known isotypes: IgA, IgM, & IgG.
Table. Properties of human immunoglobulins
Table. Major functions of immunoglobulins
 THE COMPLEMENT SYSTEM
•The complement system comprises some heat-labile proteins that normally exist as
inactive precursors in the plasma that help in the opsonization of pathogenic
microorganisms & immune complexes.

•It is a highly conserved innate immune cascade of at least 20 serum proteins &
glycoproteins that interact to recognize & kill pathogens.

•The major actors in this system are 11 proteins designated C1 to C9, B, & D.

•It is present in the blood of all normal individuals.

•It is synthesized mainly by hepatocytes, blood monocytes, tissue macrophages &

epithelial cells of GI.
•The proteins are very powerful & act rapidly.

•Usually, the interaction of Ab molecules with Ag is followed by conformational

changes of the Ab, including changes in the spatial orientation & exposure of
biologically active domains located on the Fc region of the Ab.

•The Fc regions of Ag-bound IgG or IgM then bind & activate the complement system.

•The synthetic rates for the various complement glycoproteins increase when
complement is activated & consumed.
•Activation of the complement cascade is one of the major mechanisms for initiating
inflammation, which results in the production of powerful opsonins, chemoattractants,
& anaphylatoxins that can directly mediate cell killing through lysis.

•The key event in complement activation is the proteolytic cleavage of C3 to C3a &
C3b.

•C3 cleavage leads on to the activation of the terminal complement pathway, causing
the generation of the membrane attack complex (MAC), which assembles a lipophilic
complex capable of lyzing the plasma membranes of susceptible cells.
Pathways of the Complement System Activation

There are 3 pathways of complement activation:

•The Classic Pathway;

•The Alternative Pathway; &

•Mannan-binding lectin Pathway

The Classical Pathway:

•Initiated by an Ag-Ab reaction.

•When an Ab binds with an Ag, a specific reactive site on the “constant” portion of the
Ab becomes exposed (“activated”).

•This in turn binds directly with the C1 molecule of the complement system, thereby
initiating a cascade of sequential reactions beginning with activation of the proenzyme
C1 itself.
•The active C1 enzymes formed then activate successively increasing quantities of
other enzymes in the later stages of the system.

•Thus, the cascade is amplified.

•Multiple end products are formed, which help prevent damage to the body’s tissues
caused by the invading organism or toxin.
The Alternative pathway:

•Triggered by the yeast polysaccharide, Zymosan, in the absence of Ab.

•The alternative pathway protects body from pathogens in absence of Ab.

• The initial steps of this process are quite different from those of the classical pathway
& involve several unique serum components, namely factor P (properdin) & factors B
& D.
Fig. Activation & regulation of the complement system.
1.Opsonization & phagocytosis:

•C3b (one of the products of the complement cascade), strongly activates phagocytosis
by both neutrophils & macrophages, causing these cells to engulf the bacteria to which
the Ag-Ab complexes are attached.

•This process is called opsonization. It often enhances the number of bacteria that can
be destroyed by many hundredfold.

2.Chemotaxis :

•Fragment C5a initiates chemotaxis of neutrophils & macrophages, thus causing large
numbers of these phagocytes to migrate into the tissue area adjacent to the antigenic
agent.
3.Cytolysis:

•The complement system destroys target cells through lysis of the cell membrane.

•This is termed cytotoxicity in the case of nucleated cells, hemolysis for erythrocytes,
or bacteriolysis in the case of bacteria.

•The Lytic complex (designated C5b6789 ), which is a combination of multiple

complement factors, is one of the most important of all the products of the complement
cascade.

•It has a direct effect of rupturing the cell membranes of bacteria or other invading
organisms.
4.Anaphylotoxin activity:

•Fragments C3a, C4a, & C5a stimulate the mast cells to release histamine & other
substances, resulting in increased capillary permeability & local accumulation of fluid
in the tissue.

•These substances in turn cause increased local blood flow, increased leakage of fluid
& plasma protein into the tissue, & other local tissue reactions that help inactivate or
immobilize the antigenic agent.

5.Tissue damage:

Both the lytic complex & the inflammatory PMN's can cause considerable damage to
normal tissues, e.g. in an Arthus Reaction or in Immune Complex Disease.
6. Agglutination:

•The complement products also change the surfaces of the invading organisms, causing
them to adhere to one another, thus promoting agglutination.

7. Neutralization of viruses:

•The complement enzymes & other complement products can attack the structures of
some viruses , making them non-virulent.
 Hypersensitivity & Allergy
•Immune response, which major function is to neutralize or destroy the invading
organisms or Ag, can, in some instances cause disease resulting from its
undesirable effect directed against an exogenous Ag, or as a consequence of
an autoimmune reaction.

•Such conditions constitute hypersensitivity reactions.

•Hypersensitivity can be defined as an abnormal state of immune reactivity that

has deleterious effects on the host.

•A patient that is hypersensitive to a given compound suffers pathological

reactions as a consequence of exposure to the Ag to which he or she is
hypersensitive.

•The term “allergy” is often used to designate a pathological condition resulting

from hypersensitivity, particularly when the symptoms occur shortly after
exposure.
The different types of hypersensitivity reactions can be distinguished by:

•the time course of their development; &

•the nature of the cellular infiltrate present in the sites of a typical reaction.
Table. General characteristics of the 4 types of hypersensitivity reactions as
defined by Gell & Coombs
Type I Hypersensitivity Reactions (Allergies)

Characteristics:

•Immediate or very rapid (observed within a few minutes after the challenge);

•IgE-mediated, i.e. depends on interaction btw Ag & IgE attached to mast cells
(derived from haemopoietic stem cells in the bone marrow);

•Eosinophils & a subset of T-cells (TH2 cells) are also involved;

•Also known as anaphylactic reaction;

•Often results in the death of the animal in anaphylactic shock;

•Clinical examples: allergic rhinitis (hay fever), allergic asthma & some food
reactions e.g. peanut hypersensitivity.
•Any substance which can elicit an allergic response is referred to as an
allergen.

•An allergen can be a component of any number of microorganisms, plants or

animals, or may be a synthetic compound.

•An allergen can only be effective in eliciting an allergic reaction if the recipient
has been previously sensitized; i.e., there must be present, in the recipient’s
tissues, Ab of the IgE class directed against the allergen.

•Most Ag stimulating IgE are either inhaled or ingested. E.g. of the inhaled Ag is
ragweed pollen.

•Allergic reactions may be elicited by exposure through the air (pollen), contact
with the skin (cosmetics), ingestion (natural or artificial food products), or
injection (drugs or insect bites).
•IgE, the mediators of Type I hypersensitivity reaction, are also known as
reagins or reaginic Ab.

•They are homocytotropic; meaning that they are capable of binding to their
own mast cells & initiate allergic reaction.

•IgE is the major mediator of allergy, although in humans IgG4 can also carry
out this function with lower efficiency.

•The IgE production requires helper T-cells & T-cell-derived cytokines.

•IL-4 & IL-13 stimulate IgE production while IFN-γ inhibits it.

•Heterocytotropic Ab can initiate allergic reactions only in a species other than

the one from which the Ab was synthesized.

•E.g. rabbit IgG can cause allergic responses when transferred to guinea pigs,
but it’s unable to do so in the rabbit.
Sequence of Events in Allergic Responses

Allergy can be separated into 2 phases, a period of sensitization followed by

the allergic reaction proper.

Sensitization Phase

•Exposure of the immune system to the immunogenic allergen results in the

induction of IgE Ab, with the participation of T-cells, B-cells & macrophages.

•This induction of IgE synthesis must precede the allergic reaction itself.

•IgE Ab binds to tissue mast cells through its Fc piece.

•Mast cells have Fc receptors on their surface with an extremely high affinity for
IgE Ab.
Reaction Phase

•Re-exposure to the allergen results in the binding of the allergen to mast-cell-

bound IgE Ab.

•Cross-linking of IgE molecules on the mast cell surface by multivalent Ag is

required for the subsequent reactions.

•The large, basophilic granules present in the mast cells are released into the
tissues.

•This process of degranulation releases a variety of pharmacologically active

compounds; some directly from the granules (e.g. histamine & heparin), others
newly synthesized by the cell soon afterwards.
•These & other active compounds can lead to such conditions as asthma &
rhinitis, generally in response to allergens in pollen, dust or animal dander.

•Degranulation involves the fusing of cytoplasmic granules of the neutrophil

(lysosomes) with the phagosomes, & the emptying of its content inside the
phagosomes.
Fig. Sequence of allergic reaction as mediated by IgE
•Allergens in cosmetics can lead to rashes, & allergens in food, drugs or
injected animal poisons may result in rashes or in vascular or gastrointestinal
anaphylaxis.

•These conditions may range from very mild to severe, & are potentially fatal
(e.g., in the case of severe asthma or vascular anaphylaxis).

•The nature & severity of allergic reactions depends on the route of exposure &
the degree of sensitization, it varies considerably from one species to another,
& among humans may vary tremendously btw different individuals.
Table: Pharmacologically active compounds involved in allergy, & their effects
Early-phase versus Late-phase Allergic Reactions

•Early-phase reactions are the early or immediate consequences of the allergy,

occurring within a few minutes after exposure to the allergen, e.g. allergic skin
reaction.

•Late-phase reactions refer to long-term consequences of the allergy.

Examples:

•In the case of an allergic skin reaction (e.g. Prausnitz-Küstner), the early or
immediate phase occurs within a few minutes.

•But, over the course of subsequent hours & days, a cellular infiltrate
dominated by eosinophils will develop, which also includes neutrophils,
macrophages & TH2 cells.
•In asthmatic reactions, the late-phase response may be manifested by a
chronic inflammation & hypersensitivity of the bronchi, with important diagnostic
& therapeutic implications.

•Cromolyn sodium, can be given prophylactically to inhibit mast-cell

degranulation & prevent the initial reaction.

•In the presence of an ongoing chronic late-phase reaction, administration of

steroids may be indicated for their anti-inflammatory properties.
Evaluation of the Allergic State

•To be able to manage allergic patients, it’s important to have a simple &
accurate laboratory test to determine the specificity & severity of any allergy.

•IgE levels, & skin sensitivity to specific Ag, can be monitored in such patients.

Determination of serum IgE levels in allergic patients.

•RadioImmunoSorbent Test (RIST) can be used to measure total serum IgE.

•RadioAllergoSorbent Test (RAST), which measures the levels of circulating

IgE Ab directed against a particular allergen can also be used.

•Results of both assays correlate poorly with the degree of clinical allergy;
however, the result of RAST is somewhat better than that of RIST
•The less satisfactory results of these assays reflect the high degree of
variability among different people in the following:

i.the levels of IgE in humoral immune responses;

ii.the efficiency of its fixation to tissue mast cells;

iii.the sensitivity of mast cells to degranulation; &

iv.the sensitivity of various tissues to the effector molecules & the inflammatory

response.

•Skin testing can be very useful in determining to which allergens a particular

patient is sensitive.

•This involves injecting small amounts of various purified Ag intradermally, &

measuring the severity of the wheal & flare reaction over a period of about half
an hour.
•The wheal & flare reaction is an active cutaneous anaphylaxis reaction.

•Test diets are also very helpful in identifying food allergies.

•The patient is placed on a diet free of the most common sources of allergens
(wheat & related grains, dairy products, eggs, etc.), & various foods are added
one at a time over a period of weeks or months to determine which may be
responsible for the allergic response.
Treatment of Allergic Reaction

•Prompt treatment of a systemic anaphylactic reaction (e.g. to a food, drug or

insect bite) can be lifesaving.

Treatment Strategies:

1. Avoidance of allergens is the single most important & effective element

in
managing allergic states.

•This may include eliminating known allergens from one’s diet (e.g. nuts,
milk, eggs), removing them from one’s home (e.g. wool carpets, feather
pillows, pets & plants), & avoiding drugs to which one is sensitive (penicillin,
quinin & its derivatives).
2.Desensitization: This involves deliberate immunization with small but
increasing amounts of a particular purified allergen over a period of months or
yrs.

•The effectiveness of this procedure results from the induction of high levels
of IgG Ab, which can prevent allergic reactions by competing for the allergen &
preventing it from reaching mast-cell bound IgE.

•Desensitization has been useful for pollen & dust allergens as well as for some
animal danders (scales of hair or feathers), but its effectiveness is
unpredictable & varies btw individuals.

•It is not generally useful for food or drug allergies.

• Desensitization is associated with a certain degree of risk, since each
injection may itself induce a more or less severe allergic reaction.

• The procedure must be carried out under careful supervision.

3. Drugs can be useful in both preventing (prophylactic) & treating

(therapeutic) allergic reactions.

Examples:

• Anti-histamines inhibit binding of histamine to its receptors (but will not

effect the target tissue response once histamine is bound).

• Corticosteroids are anti-inflammatory (useful for the late-phase response),

& can also inhibit histamine synthesis.
•Cromolyn sodium is widely used as a prophylaxis for exercise & allergy-
induced asthma.

•It is known to stabilize mast cells, although it mechanism of action is not

clearly known.

•Isoproterenol, salbutamol & epinephrine can be used to counteract the effects

of the various mediators released by mast cells.

•Unlike the drugs mentioned above, they can be effective in controlling an

ongoing allergic reaction.
Type II (cytotoxic hypersensitivity)

•A cytotoxic reaction btw tissue- or cell-bound Ag & IgM or IgG Ab.

Characteristics:

•Cell-bound

•Initiated by Ab reacting with Ag on the cell membranes.

•IgM & IgG can be involved in these reactions.

•Clinical examples include organ-specific autoimmune diseases, immune

hemolytic anaemia, & transfusion reactions.

•Auto-sensitized T-cells may play a role in some diseases e.g. rheumatoid

arthritis & multiple sclerosis, although the evidence for their involvement is not
clear.
Autoimmune Hemolytic Anemia

•Patients with autoimmune hemolytic anaemia synthesize Ab directed to their

own red cells.

•Those Ab may cause hemolysis by 2 main mechanisms, namely:

intravascular hemolysis, & extravascular hemolysis

I. Intravascular hemolysis:

•If the Ab are of the IgM isotype, complement is activated up to C9, & the red
cells can be directly hemolyzed.

Rapid & massive, intravascular hemolysis is associated with release of free

hemoglobin into the circulation (hemoglobinemia), which is eventually excreted
in the urine (hemoglobinuria).

Hemoglobinuria can induce acute tubular damage & kidney failure.

IV. Extravascular hemolysis:

• If the Ab (usually IgG) fail to activate the full complement cascade, the red
cells will be opsonized with Ab (& possibly C3b,& are taken up & destroyed
by phagocytic cells expressing Fcg & C3b receptors.

• Extravascular hemolysis is usually associated with increased levels of

bilirubin, derived from cellular catabolism of hemoglobin.

• All hemolytic reactions usually lead to the mobilization of erythrocyte

precursors from the bone marrow to compensate for the acute loss.

• This is reflected by reticulocytosis &, in severe cases, by erythroblastosis.

Type III

•An immune-complex reaction btw circulating Ag & IgG Ab with subsequent

deposition in the walls of blood vessels, joints, kidneys & skin, manifesting as
serum sickness or, in the case of some inhaled Ag, extrinsic allergic alveolitis.

•Deposition of immune complexes depends on their size, charge, local

concentration of complement, & perhaps most important the nature of the Ag.

•E.g. of this type of reaction is the arthritis reaction in which Ag is injected

into the skin of an animal previously sensitized to the same Ag & has produced
Ab to that Ag.

•The preformed Ab goes to the site of the injected Ag & forms a complex,
thereby inducing complement activation & neutrophil attraction.

•The result is intense local inflammation, hemorrhage, & necrosis.

Type IV (Delayed hypersensitivity)

•A cellular immune response mediated by sensitized lymphocytes; hence, it is

cell-mediated.

•Occurs 36 - 48 hours after Ag exposure & differs pathologically from the first 3
types of hypersensitivity which are all Ab-mediated.

•Causes the local response that occurs on intradermal tuberculin testing in TB-
sensitized individuals & for the skin manifestations of contact hypersensitivity.

•T cells drive this reaction when they react with Ag & release TH1 cytokines.

•The cytokines in turn attract other cells, such as macrophages, which release
their lysosomal enzymes.
E.g. of this type of reaction is seen when PPD (purified protein derivative) is
injected into the skin of a person who has been previously infected with the
tuberculosis organism.