BASIC IMMUNOLOGY

I. DEFINITIONS
• Acquired (specific / adaptive) Immunity:
A type of host defences that is stimulated by microbes that invade host tissues. In other
words, acquired immunity adapts to the presence of microbial invaders.
• Adjuvants:
Substances which enhance the immune response against the immunogens.
• Affinity:
The binding strength of an antigen (having a single antigenic determinant) with a single
combining site of an individual antibody. Affinity is the sum of attractive and repulsive forces
operating between the antigenic determinant and the antibody combining site. Affinity also refers
to the equilibrium constant (Keq) that describes the Ag-Ab reaction.
Keq = [Ag-Ab] / [Ag] X [Ab]
• Antibody (Ab):
A specific protein that is produced in response to an immunogen and reacts with antigens.
• Antigen (Ag):
A substance that reacts with the products of a specific immune response (i.e. antibodies).
• Agglutination test:
A test in which antibodies react with particulate antigens resulting in their agglutination
(or clumbing). Note that the agglutination test works only with the particulate antigens.
• Agglutinin:
Antibodies used to agglutinate particulate antigens in the agglutination test. Theoretically,
all antibodies can agglutinate antigens, but IgM due to its high valence is good agglutinin.
• Avidity:
A measure of the overall binding strength of an antigen (having many antigenic determi-
nants) with a multivalent antibody. Avidity is influenced by both the valence of the antibody and
the valence of the antigen.
• Complements:
20 different serum proteins that are produced by a variety of cells, including hepatocytes,
macrophages and gut epithelial cells. Some complements bind to the immunoglobulins or to cell
membranes. Others are proenzymes that when activated cleave one or more complement proteins
to yield fragments that activate cells, increase vascular permeability or opsonize bacteria.
• Complement activation:
Alteration of complement proteins, enabling them to interact with another complements.
• Complement fixation:
Utilization of a complement by an antigen-antibody complex.

• Complement inactivation:
Denaturation (usually by heat) of an early complement component resulting in a loss of its
hemolytic activity.
• Conventional T-dependent antigens:
Antigens which monoclonally (or oligoclonally) activate a small fraction of T-cells. Only
one in 104 - 105 of the T-cell population is able to recognize that type of antigens.
• Convertase:
An altered complement protein, which acts as a proteolytic enzyme for another comple-
ment component.
• Countercurrent electrophoresis test:
A test in which the antigen and the antibody are placed in wells punched out of an agar gel.
The antigen and antibody are then electrophoresed into each other where they form a precipitation
line. The test works only if the Ag and Ab have opposite charges. This test is primarily qualitative,
although from the thickness of the band you can get some measure of quantity.
• Cross reactivity (Multispecificity):
The ability of an individual antibody combining site to react with more than one antigenic
determinant. Cross reactions arise because the cross reacting antigen shares an epitope in common
with the immunizing antigen.
• Domains:
Globular folded regions of the immunoglobulin molecule that contain intra-chain disulfide
bonds. Domains may be ligh chain domains (VL and CL) or heavy chain domains (VH, CH1-CH3
(or CH4).
• Direct coombs test:
A test used to detect if antibodies are bound to the RBCs surface antigens or not. In this
test, the patients RBCs are washed and then incubated with a second antibody (or an antihuman
globulin - also called coombs reagent). If antibodies have been fixed on the RBCs surface, the
RBCs in this case will agglutinate when incubated with the antihuman globulin.
• Epitope (Antigenic determinant):
The portion of an antigen that combines with the products of a specific immune response.
• Fab region:
A region that contains the antigen binding sites of the antibody. The Fab region consists of
two identical fragments that contain the light chains and the VH and CH1 domains of the heavy
chains. Each Fab region is monovalent, whereas the original molecule was divalent. An antibody is
able to bind a particular antigenic detrminant, because it has a particular combinations of VH and
VL. Thus different combinations of VH and VL result in antibodies that can bind different
antigenic determinants.
• Fc region:
A region whose principle function is to mediate the effector functions of immunoglobulins.
Normally, the ability of an antibody to carry out an effector function requires the prior binding of
an antigen. However, there are exceptions for this rule. The Fc region contains the remainder of the
two heavy chains (each containing a CH2 and CH3 domains).
• Haptens:
Small molecules which could never induce an immune response when administered by
themselves (i.e. non-immunogenic). Free haptens, however, can react with the products of the
immune response. This means that haptens have the property of antigenicity, but not immunogen-
icity. It was found that when a hapten is conjugated (or coupled) with an immunogenic carrier
molecule, it can induce an immune response.
• Hapten-carrier conjugates:
Immunogenic molecules, in which haptens have been covalently attached to an immuno-
genic carrier molecule. Hapten-carrier conjugates are characterized by having native antigenic
determinants of the carrier as well as new determinants created by the hapten.
• Hemagglutination test:
A test in which antibodies react with the erythrocyte antigens resulting in their agglutina-
tion (or clumping).
• Hemagglutinin:
Antibodies used to agglutinate the erythrocyte antigens in the hemagglutination test.
• Hinge region:
 A region of the immunoglobulin molecule, at which the arms of the antibody forms a Y-
shape, because there is some flexibility in the molecule at this point.
• Immunoglobulins:
Glycoprotein molecules that are produced by the plasma cells in response to an immuno-
gen and function as antibodies.
• Immunogen:
Substance that induces a specific immune response.
• Incomplete antibodies:
Antibodies which bind to the red blood cells, but do not cause their agglutination.
• Indirect coombs test:
A test used to detect circulating antibodies (in the blood serum) against someone else’s
RBCs. In this test, the washed RBCs are incubated with a test serum. If the serum contains anti-
bodies to antigens on RBCs surface, the antibodies will bind to the surface of the RBCs. In the
second step, the RBCs are incubated with an antihuman globulin. If antibodies have been bound
to the the RBCs surface in the first step, the RBCs will agglutinate when incubated with the anti-
human globulin (also called coombs reagent).
• Innate immunity:
A type of host defences that is usually present in healthy individuals, prepared to block the
entry of microbes and to rapidly eliminate microbes that do succeed in entering the host tissues.
• Passive hemagglutin test:
A test in which the antibodies react with the soluble antigens coating the surface of the
erythrocyte RBCs resulting in their agglutination. This test is performed just like the agglutina-
tion test and used to detect antibodies to soluble antigens (such as viral antigens, polysaccharides,
or haptens).
• Pathogen Associated Molecular Patterns (PAMPS):
Broad molecular patterns that are present in many different pathogens and can be reco-
gnized by the innate immune system. In other words, PAMPs are the antigenic determinants reco-
gnized by the innate immune system. An example of PAMPS is the microbial cell wall compo-
nents which are recognized by complements.
• Pattern Recognition Receptors (PRRs):
Receptors that have the ability to recognize the PAMPS (or the pathogen associated
molecular patterns). A particular PRR can recognize a molecular pattern that may be present on a
number of different pathogens and thus enabling the receptor to recognize a wide variety of
organisms. PRRs (such as complement) are therefore non-specific in their action.
• Qualitative agglutination test:
Agglutination tests used in an qualitative manner to detect the presence of an antigen or an
antibody. For example to determine a person’s blood group, the patient RBCs are mixed with an
antibody to a blood group antigen, and a positive test is indicated by the agglutination of these par-
ticulate antigen.
• Radial Immuno-diffusion (Mancini) quantitative precipitation test:
A test in which an antibody is incoporated into the agar gel, and different dilutions of the
antigens are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts
with the antibody and when the equivalence point is reached a ring of precipitation is formed. The
diameter of this ring is proportional to the log of the antigen concenteration (since the amount of
antibody is constant). Thus, by running different concenterations of a standard antigen, one can
generate a standard curve from which we can quantitate the amount of an antigen in an unknown
sample. The test is used in the determination of immunoglobulin levels in patient samples.
• Specificity of antibody:
Ability of an individual antibody combining site to react with only1 antigenic determinant.
• Superantigens:
Antigens which polyclonally activate a large fraction of T-cells (up to 25%). Examples of
these antigens include: Staphylococcal enterotoxins (food poisoning), Staphylococcal shock tox-
ins, and Streptococcal pyrogenic exotoxins. The diseases associated with the exposure to these
superantigens are in part due to hyper-activation of the immune system and subsequent release
of biologically active cytokines by the activated T-cells.
• TH-1 cells:
A type of T-helper cells that is produced in the thymus and help the CD8+ pre-cytotoxic
cells to differentiate into cytotoxic cells.
• TH-2 cells:
Another type of T-helper cells that is also produced in the thymus, but help the the B cells
to differentiate into the plasma cells, which in turn secrete antibodies.
• Valency of antibody:
Number of antigenic determinants that an individual ntibody molecule can bind. The valency
of all antibodies is at least two and in some instances more.

II. QUESTIONS AND ANSWERS

Q1: State the factors affecting Immuogenicity.
A1: (I) Contribution of the immunogen:
• Foreignness - Only foreign molecules are immunogenic.
• Size - The larger the molecule is, the more immunogenic it is likely to be.
• Composition - The more complex, the more immunogenic the substance will be.
• Physical form - Particulate antigens are more immunogenic than the soluble ones.
(II) Contibution of the biological system:
• Genetic factors - Substances may be immunogenic in one species, but not in another.
• Age - Usually the very young and very old individuals have a diminished ability to in-
duce an ammune response against the immunogen.
(II) Method of administration:
• Antigen dose - There is a dose of antigen above or below which the immune response is
not optimal.
• Route - Generally the subcutaneous route is better than the intravenous or intra gastric.
• Adjuvants - Adjuvants are substances that can enhance the immune response.
Q2: State the factors affecting Antigen-Antibody reactions.
A2: Factors affeccting the antigen-antibody reactions include:
• Affinity: The higher the affinity of the antibody to the antigen, the more stable will be
the interaction. Thus, the ease with which one can detect the interaction.
• Avidity: Reactions between multivalent antigens with multivalent antibodies are also
more stable, and thus easily detected.
• Ag-Ab ratio: The ratio of the antigen and the antibody influences the detection of the
AgAb complex. This is because the size of the complex formed is related to both the
concenteration of the antigen and the antibody.
• Antigen physical form: Particulate antigens are easily agglutinated by antibodies.
Q3: What are the functions of complements?
A3: Complements: (1) Can opsonize bacteria for enhanced phagocytosis, (2) Can activate
various cells including PMNs and macrophages, (3) Can participate in regulating antibody respon-
ses, (4) Can increase vascular permeability, and (4) Can aid in the clearance of immune com-
plexes and apoptotic cells. Complements also contribute to inflammation and tissue damage.
Q4: Write about the classical complement pathway?
A4: It is dependent upon antibodies to be operational. In this pathway:
• C1 (which is a multi-subunit containing 3 different proteins; C1q, C1r, and C1s) binds to
the Fc region of the IgG and IgM antibody molecules that have interacted with the antigen.
The binding of C1q to the antibody must cross link at least two antibody molecules and re-
quires calcium and magnesium ions.
• The binding of C1q to the antibody activaties C1r, which in turn activaties C1s. The result
is the formation of an activated “C1qrs”. C1qrs is an enzyme which cleaves:
o C4 into two fragments; C4a and C4b.
o C2 into two fragments; C2a and C2b.
The C4b and C2a fragments bind to the membrane to form the C4bC2a complex, whereas
the C4a and C2b fragments are released into the surrounding microenvironment.
• The resulting C4bC2a complex is a C3 convertase, which cleaves C3 into two fragments;
C3a (which is released into the microenvironement) and C3b (which binds to the membr-
ane in asscociation with C4b and C2a).
• The resulting C4b C2a C3b complex is a C5 convertase. The generation of C5 convertase
is the end of the classical pathway.
Q5: Write about the lectin pathway?
A5: The lectin pathway is very similar to the classical pathway, except that it is antibody
independent. In this pathway:
• The mannose binding lectin (MBL) – which is similar to C1q – binds to the bacterial (or
pathogen) surface, resulting in the association of two serine proteases; MASP-1 (which is
similar to C1r) and MASP-2 (which is similar to C1s).
• The formed tri-molecular complex MBL/MASP-1/MASP-2 results in the formation of an
activated “MASPs”. MASPs is an enzyme which cleaves:
o C4 into two fragments; C4a and C4b.
o C2 into two fragments; C2a and C2b.
The C4b and C2a fragments bind to the membrane to form the C4bC2a complex, whereas
the C4a and C2b fragments are released into the surrounding microenvironment.
• The resulting C4bC2a complex is a C3 convertase, which cleaves C3 into two fragments;
C3a (which is released into the microenvironement) and C3b (which binds to the membr-
ane in asscociation with C4b and C2a).
• The resulting C4b C2a C3b complex is a C5 convertase. The generation of C5 convertase
is the end of the lectin pathway.
Q6: Write about the Membrane Attack Pathway?
A6: In this pathway, the C5 convertase from the classical (C4bC2aC3b), lecctin (C4bC2a
C3b), or alternative (C3bBbC3b) pathway cleaves C5 into two fragments; C5a (which remains in
the fluid phase) and C5b (which rapidly associates with C6 and C7).
At the same time, C8 binds followed by several molecules of C9. The C9 molecules form a
pore in the pathogen membrane, through which the cellular contents leak resulting in lysis (which
occur due to the membrane physical damage). The complex consisting C5bC6C7C8C9 is known as
“Membrane Attack Complex (MAC)”.
Q7: What is the function of the Protein S (vitronectin)?
A7: This protein binds to the soluble C5bC6 complex (which have been dissociated from
the membrane), and thus preventing the binding of the C5bC6 complex to other bystander cells,
which may in turn result in their damage.
Q8: Write about the amplification loop of C3b formation in the alternative pathway.
 A8: In this pathway, Factor B binds to the C3i (resulted from the spontaneous hydrolysis of
C3) and become susceptible to Factor D. Factor D cleaves Factor B into Bb. The C3iBb complex
acts a C3 convertase and cleaves C3 into C3a and C3b. Once C3b is formed, Factor B will bind to
it and becomes susceptible to cleavage by Factor D. the resulting C3bBb complex is a C3
convertase that will continue to generate more C3b, and thus amplifying C3b production.
Q9: Write about the chemical nature of immunogens.
A9: The vast majority of immunogens are proteins (which may be pure proteins or may be
glycoproteins or lipoproteins). In general, proteins are usually good immunogens. Immunogens
may be also pure polysaccharides or lipopolysaccharides, which are also good immunogens.
Nucleic acid, on the other hand are usually poor immunogens. However, they may become
immunogenic when they are complexed with proteins. Lipids were found to be non-immunogenic
at all. Some glycolipids and phospholipids, however, can stimulate T-cells and produce a cell-
mediated immune response.
Q10: Describe the nature of the antigen-antibody reactions.
A10: In Ag-Ab reactions, the antigenic determinant of the antigen fits into a cleft formed by
the combining site of the antibody. This site is present in the Fab portion of the molecule and is
constructed from the hypervariable regions of the heavy and light chains. The Ag-Ab reactions are
thus can be viewed as a lock and its key, in which a key (i.e. Ag) fits into a lock (i.e. Ab).
Q11: Write about the nature of the bonds involved in the Ag-Ab reactions.
A11: The bonds that hold the antigen (Ag) into the antibody combining site are all non co-
valent in nature. These include: electrostatic bonds, Van der Wals forces, and hydrophobic bonds.
Since Ag-Ab reactions occur via non-covalent bonds, they are reversible by their nature.
Q12: Write about the general functions of immunoglobulins.
A12: The primary function of immunoglobulins is to act as antibodies and bind specifica-
lly to one or more few closely related antigens. In fact, each immunoglobulin binds to a specific
antigenic determinant. Often the binding of an antibody to an antigen has no direct biological eff-
ect. Rather, the significant biological effects are a consequence of secondary “effector functions”
of antibodies, such as:
(1) Complement fixation that leads to cell lysis as well as the release of other
biologically active molecules.
(2) Binding of various cell types (such as phagocytic cells, lymphocytes, platelets,
mast cells, and basophils) to the immunoglobulins which activate these cells to
perform some functions.
Q13: Write about the structure of immunoglobulins.
A13: Although different immunogens can differ structurally, they all are built from the
same basic units, these are:
A. Heavy and Light chains: All immunoglobulins have a four chain structure (two identical
light chains and two identical heavy chains).
B. Disulfide bonds: The heavy and light chains and the two heavy chains are held together by
inter-chain disulfide bonds and by non-covalent interactions. There are also intra-chain di-
sulfide bonds within each of the polypeptide chains.
C. Variable (V) and constant (C) regions.
D. Hinge region: At which the molecule forms a Y-shape.
E. Domains, which are globular folded regions
F. Carbohydrates: These are usually attached to the CH2 domain
in most immunoglobulins.
Q14: Write about the cellular barriers of the innate (non-specific) immunity.
A14: The following cells are main line of defense in the non-specific immune system.
Polymorphonuclear cells (PMNs) and Neutrophiles arrive to the site of infection, where
they “phagocytose” invading organisms & kill them. Macrophages also function in phagocytosis
and intracellular killing of microorganisms. Moreover, they act as antigen presenting cells, which
are required for the induction of specific immune rsponses. Natural killer (NK) and lymphokine
activated killer (LAK) cells, which can non-specifically kill virus infected and tumor cells. Eo-
sinophils have proteins in their granules that are so effective in killing parasites.
3. COMPARISONS
1. Compare between innate immunity and acquired immunity.
Innate immunity Acquired immunity
Innate immunity shows no specificity, Acquired immunity demonstrate a high
since its components can recognize degree of specificity. This is because
broad molecular patterns that can be components of the acquired immune
Specificity
found in many pathogens, and thus they system (such as antibodies, and the B
can recognize and react with a wide and T cells receptors) recognize and
variety of different microorganisms react with only particular pathogens
Always present and ready to be Requires some time to react to an
Time
mobilized upon infection invading organism

Does not have an immuno- Demonstrates immunological memory

Memory
logical memory and reacts more rapidly on subsequent
exposure to the same organism
2. Compare between the classical, lectin, and alternative complement pathways.
Classical Lectin Alternative
MBL, MASP-1,
C3, Factor B, Factor D,
Components C1, C2, C3 and C4 MASP-2, C2,
and properdin
C3 and C4
Antibody dependent? Yes No No
Require
Yes Yes
divalent cations?
Generate prokinin? Yes No
Generate Anaphylotoxin? Yes Yes
C3 Convertase C4bC2b C3bBb
C5 convertase C4bC2bC3b C3bBbC3b
FactorH, Factor I,
Requlatory components C1-INH, C4-BP, Factor I
DAF, CR1
3. Compare between the activities and controlling factor(s) of the different comple-
ment fragments.

Activity Effect Controlling factor(s)

Prokinin (Fluid
C2a / C2b ??
accumulation) Edema C1 – INH *

Basophil and Mast cells

degranulation, Enhanced
C3a and C4a Anaphylaxis C3a – INA
vascular permeability,
Smooth muscle contraction
C3b Fcators I and H
Opsonin
Phagocytosis C4 binding protein and
C4b (phagocyte activation)
Factor I
 Vascular permeability,
Smooth muscle con-
traction, chemotaxis, Anaphylaxis (most
C5a Phagocyte activation, potent) and C3a – INA
Stimulation of respiratory Inflammation
burst and inflamma-
tory cytokines
Inflammation and ]

C5b, C6 and C7 Chemotaxis Protein S (vitronectin)

Tissue damage
* The deficiency of the C1-INH inhibitor is usually associated with the development of hereditary
angioedema.
4. Compare between the antigenic determinants recognized by the B cells and those
recognized by the T cells.

Determinants recognized by B cells Determinants recognized by T cells*

The antigenic determinants recognized
by the B cells are created by the 1o seq- The antigenic determinants recognized
uence of residues in the polymer (linear by the T cells are created by the 1o seq-
Composition
or sequence determinants) and/or by the uence of amino acids in proteins.
2o, 3o, or 4o molecule structure
(conformational determinants)
The antigenic determinants recognized
by the B cells are small and are limited The antigenic determinants recognized
Size
to approximately 4-8 residues by the T cells are small and are limited
(amino acids and/or sugars) to approximately 8-15 amino acids

In theory, each 4-8 residues can const- In theory, each 8-15 residues can cons-
itute a separate antigenic determinant. titute a separate antigenic determinant.
Number In practice, the no. of antigenic deter- In practice, the no. of antigenic deter-
minants / antigen is much lower than minants per antigen is much lower than
that would theoretically be possible that would theoretically be possible
* Note that: T - cells CAN NOT recognize polysaccharide or nucleic acid antigens. That’s
why polysaccharides are T-independent antigens, whereas proteins are T-dependent antigens. Note
also that the antigenic determinants recognized by the T-cells are limited to those portions of the
antigen that can bind to the MHC molecules.
5. Compare between the clinical implications of the different human immunoglobulins.

Increases in: Decreases in:

All infections, Hyperimmunization, Liver Selective IgG and IgA deficiency,
IgG
diseases, and Rheumatoid arthritis and Chronic lymphoblastic leukemia
Actinomycosis, Malaria, Infectious
IgM Chronic lymphoblastic leukemia
mononucleosis, and Trpanosomiasis
IgA Liver cirrhosis, and Rheumatoid arthritis Acute and chronic lymphoblastic leukemia
IgD Chronic infections and IgD myelomas ???
IgE Hay fever, Asthma, and IgE myelomas Congenital agammaglobulinemia