INTRODUCTION TO IMMUNOLOGY Types of Immunity:

It is the study of the molecules, cells, organs, and systems

responsible for the recognition and disposal of foreign
(nonself) material; how body components respond and interact;
the desirable and undesirable consequences of immune
interactions; and the ways in which the immune system can be
advantageously manipulated to protect against or treat
disease.

HISTORY:
Virtually the entire history of immunology has been recorded How Immune System Activates:
within the last 100 years, and it is only in the recent past that
the most significant part of this history has been written. It was
not until the 1960s that the cells responsible for the immune
response were identified and characterized.

First written records of immunological experimentation dates

back to 1500s when the Chinese developed variolation which is Innate/Natural Acquired/Adaptive
a process of deliberately exposing an individual to material It is the ability of the It is a type of resistance that is
from small pox lesions. individual to resist characterized by specificity for
infection by means of each individual pathogen, or
normally present body microbial agent, and the ability
functions. to remember a prior exposure,
Scientist Contribution which results in an increased
Edward • Introduced smallpox vaccination in the response upon repeated
Jenner 1700s exposure.
• Vaccination came from latin word • Non-specific • Reinforcement
“vacca” which means cow • Exists even before • Inducibility
• Cross-immunity is a phenomenon in invasion of foreign • Specificity
which exposure to one agent agents • Specificity
produces protection against another • Components are • Diversity
agent preformed • Memory
Louis Pasteur • Introduced live attenuated vaccine • Non-adaptive • Specialization
• During 1880-1881 he introduced • Lacks immunologic • Self-limitation
chicken cholera and anthrax vaccine memory • Discrimination
• During 1885 he student the spinal
cords of rabid animal and developed
anti-rabies vaccine Innate Immunity:
Elie • He observed that foreignobjects EXTERNAL
Metchnikoff introduced into transparent starfish A. Structural / Physical Barriers
larvae became surrounded by motile • Unbroken skin
cells that attempted to destroy these • Mucuous membranes in Respiratory and GI tract
invaders. • Ciliated epithelium
• He called this process phagocytosis, • Lacrimal apparatus
meaning cells eat cells. • Sweat, sebaceous glands
• Introduced cellular immunity.
Almoth • He linked cellular immunity and B. Mechanical Barriers
Wright humoral immunity by showing that the • Peristaltic movement of intestines
immune response involved both • Shedding of cells
cellular and humoral elements. • Coughing, sneezing, mucous secretion
• Flushing action of urine
Role of Immune System:
• Defending the body against infections C. Chemical Barriers
• Recognizing and responding to foreign antigens • Acidity in urine
• Lactic acid and 5.0 pH of female genital tract
• Defending the body against the development of tumors
• 1.0pH of stomach
• Lysozyme in tears and saliva (attacks gram + )
• Normal flora that exhibits competitive exclusion
(e.g.,Candida albicans)
• Lactic acid in sweat
• Lactoferrin in breastmilk
 INTERNAL DEFENSE INTERNAL DEFENSE
It is designed to recognize molecules that are unique to B. CELLULAR
infectious organisms (e.g., mannose which is a carbohydrate 1. Neutrophil
found in microorganism but not evident on human cells.) • 50-70% of circulating WBCs
A. HUMORAL • Primary granules( Primary granules( azurophilic
1. Physiologic Factors azurophilic granules) : granules) :
• Body temperature myeloperoxidasemyeloperoxidase; elastase;
• Oxygen tension proteinase 3; lysozyme; cathepsin G; and defensins,
• Hormonal Balance small proteins that have antibacterial activity.
• Secondary granules: collagenase, lactoferrin,
2. Basic Polypeptides lysozyme, reduced nicotinamide adenine
• Spermin – pH dependent polyamine found in semen; dinucleotide phosphate (NADPH)
inhibits growth of gram + bacteria oxidase
• Defensin – present it human neutrophil, are cationic • Tertiary granules: gelatinase and
proteins that kill microbes by interacting with plasminogen activator
microbial membrane. • Capable of diapedesis and
phagocytosis
3. Interferon – serum molecules in which concentrations
increase rapidly at onset of inflammatory disease.
• Alpha IFN – leukemia, nonhodgkin lymphoma,
kaposi’s sarcoma
• Beta IFN – multiple sclerosis
• Gamma IFN – chronic granulomatous disease

4. Acute Phase Reactants – limits spread of viral infections

by blocking translation of proteins
• CRP
• Serum Amyloid A
• Mannose Binding Protein
• Alpha-1-antitrypsin
• Haptoglobin
• Ceruloplasmin
• Fibrinogen
• Haptoglobin 2. Eosinophil
• Complement • 1-3% of circulating WBCs
• Increases in allergic reactions and parasitic
diseases
Characteristics of Acute-Phase Reactants • Neutralizes basophils and mast cells
Protein Response Normal Increase Function • Kills certain parasites
Time (hr) Concentration • Primary granules: acid phosphatase and
(mg/dL)
arylsulfatase
C-reactive 6-10 0.5 100x Opsonization,
• Eosinophil-specific granules:
protein complete eosinophil cationic protein,
activation eosinophil peroxidase and
eosinophil-derived-neurotoxin
Serum 24 3,0 100x Removal of
amyloid A cholesterol
Alpha- 24 200-400 2-5x Protease
3. Basophil
antitrypsin inhibitor • <1% of circulating WBCs
• Involved in immediate
Fibrinogen 24 110-400 2-5x Clot hypersensitivity reactions.
formation • Granules: histamine (contracts
Haptoglobin 24 40-200 2-10x Binds smooth muscle), heparin
hemoglobin
(anticoagulant), eosinophil chemotactic factor
Ceruloplasmin 48-72 20-40 2x Binds copper
and oxidizes
iron 4. Mast Cell
• Resembles basophil but they are
Complement 48-72 60-140 2x Opsonization, connective tissue cells of
C3 lysis
mesenchymal origin.
Mannose- ? 0.15-1.0 ? Complement • Involved in immediate hypersensitivity
binding activation reactions.
• Granules: Acid phosphatase, alkaline phosphatase,
protease.
 5. Monocyte
• Constitutes 4-10% of circulating WBCs
• Largest WBC, ground glass appearance, horse-shoe
shaped nucleus
• Primary granules: peroxidase, ACP,
arylsulfatase
• Secondary granules: lysozyme,
lipase, ß-glucuronidasetype

6. Macrophage
• Differentiated monocyte
• Macrophage names according to
their location:
○ Alveolar – Lung Types of Phagocytosis
○ Kupffer – Liver Indirect Direct
○ Microglial – Brain Via opsonin receptors Via toll-like receptors
○ Histiocyte – Connective Tissue Example:
o CRP
o Complement
7. Dendritic Cell o Antibodies
• Phagocytosed antigen and
present it to T-helper cells
• Most phagocytic cell in the PATHWAYS OF KILLING
tissues PHAGOCYTOSIS
• Resembles nerve cell OXYGEN DEPENDENT OXYGEN INDEPENDENT
dendrites By products of respiratory By lysosomal
• Types of Dendritic Cells burst and by halogenation of antibacterial substance
○ Langerhan cells – skin bacterial proteins catalyzed without the
○ Interdigitating dendritic cells – lymphoid organs by myeloperoxidase. requirement of
○ Follicular dendritic cells – spleen, lymph node respiratory burst
○ Interstitial dendritic cells – lymph node

8. Toll-like receptors
• Additional mechanism that’s discovered on certain
cells is tolllike receptors.
• Toll is a protein originally discovered in fruit fly
Drosophila, in which acts as antifungal immunity in
the adult fly.
• Highest concentration is found on monocytes,
macrophages and neutrophils.

INFLAMMATION
The overall reaction of the body to injury or invasion by an
infectious agent.

CARDINAL SIGNS
1. Redness - Rubor
2. Swelling – Tumor
3. Heat - Calor
PHAGOCYTOSIS 4. Pain – Dolor
1. ADHERENCE: physical contact between the white cell and 5. Loss of function - Functio laesa
the foreign particle
2. PHAGOSOME: formation of a phagosome
3. PHAGOLYSOSOME: fusion with cytoplasmic granules to
form a phagolysosome
4. DIGESTION AND EXCRETION:
Digestion and release of debris to the outside
 Characteristics of Two Types of Adaptive Immunity
Humoral-Mediated Cell-Mediated
Immunity Immunity
Mechanism Antibody mediated Cell mediated
Cell type B lymphocytes T lymphocytes
Mode of Antibodies in serum Direct cell-to-cell
action contact or soluble
products secreted by
cells
Purpose Primary defense Defense against viral
against bacterial and fungal infections,
infection intracellular
organisms, tumor
antigens, and graft
rejection

Comparison of Types of Acquired Immunity

Type Mode of Antibody Duration
Acquisition Produce of
d by Immune
Host Respons
e
Active Natural Infection Yes Long*, ↑

Artificial Vaccination Yes Long*, ↑

Passive Natural Transfer in No Short
vivo or
colostrum
Artificial Infusion of No Short
serum/plas
ma
THE LYMPHOID SYSTEM Secondary Lymphoid Organs
Spleen
LYMPHOCYTES - is the largest secondary lymphoid organ, having a length of
• Lymphocytes represents 20- 40% of the circulating WBCs. approximately 12 cm and weighing 150 g in the adult.
• The typical small lymphocyte is between 7 and 10 μm in
It is located in the upper-left quadrant of the abdomen, just
diameter and has a large rounded nucleus that may be
below the diaphragm and surrounded by a thin connective tissue
somewhat indented. capsule
• These cells are unique, because they arise
• from a hematopoietic stem cell and then are further Splenic tissue can be divided into two main types: red pulp and
differentiated in the primary lymphoid organs. white pulp. The red pulp makes up more than one half of the total
volume, and its function is to destroy old red blood cells.
PRIMARY LYMPHOID ORGAN
Bone Marrow Thymus The white pulp comprises approximately 20 percent of the total
weight of the spleen and contains the lymphoid tissue, which is
All lymphocytes arise from T cells develop their arranged around arterioles in a periarteriolar lymphoid sheath
pluripotential hematopoietic identifying characteristics in in which T Cells are located.
stem cells that appear the thymus, which is a small,
initially in the yolk sac of the flat, bilobed organ found in the Attached to the sheath are primary follicles, which contain B
developing embryo and are thorax, or chest cavity, right cells that are not yet stimulated by antigen
later found in the fetal liver below the thyroid gland and
overlying the heart
It can be considered the
largest tissue of the body, In humans, it weighs an
with a total weight of 1300 to average of 30 g at birth,
1500 g in the adult. reaches about 35 g at puberty,
and then gradually atrophies
In humans, B-cell maturation
takes place within the bone It was first presumed that
marrow itself. In peripheral early in life, the thymus
blood, approximately 10 to 20 produces enough virgin T
percent of all lymphocytes produces to seed the entire
are B cells, 61 to 89 percent immune system, making it
are T cells, and up to 22 unnecessary later on.
percent are NK cells However, new evidence
indicates that although the
thymus diminishes in size, it is
still capable of producing T
lymphocytes until at least the
fifth or sixth decade of life

Lymph Nodes
- are located along lymphatic ducts and serve as central
collecting points for lymph fluid from adjacent tissues.

Lymph fluid arises from passage of fluids and low molecular-

weight solutes out of blood vessel walls and into the interstitial
spaces between cells. Some of this interstitial fluid returns to
the bloodstream through venules, but a portion flows through
the tissues and is eventually collected in thin-walled vessels
known as lymphatic vessels.

Secondary follicles consist of antigen-stimulated proliferating B

cells. The interior of a secondary follicle is known as the
germinal center, because it is here that blast transformation of
the B cells takes place. Plasma cells, which actively secrete
antibody, and memory cells, which are just a step away from
forming plasma cells, are present. Generation of B-cell memory
is a primary function of lymph nodes.
 Mucosal-associated lymphoid tissue
Appendix: located at the junction of the small and large
intestines.

Tonsils: lymphoid tissue found in the mucous membrane lining

of the oral and pharyngeal cavities.

Peyer’s patches: are located at the lower ileum of the intestinal

tract.

Cutaneous-associated lymphoid tissue

Epidermis contains a number of intra epidermal lymphocytes

Within each of these secondary organs, T and B cells are

segregated and perform specialized functions.

Humoral Immunity - B cells differentiate into memory cells and

plasma cells and are responsible for.

Cell-mediated Immunity - T cells play a role in, and as such, they

produce sensitized lymphocytes that secrete.

“Cytokines are small polypeptides that regulate the functions of

lymphocytes and other cells involved in the immune response

SURFACE MARKERS ON LYMPHOCYTES

• Proteins that appear on cell surfaces can be used as
markers to differentiate T cells and B cells.
• 8 Panels of antibodies from different laboratories were
used for analysis, and antibodies reacting similarly with
standard cell lines were said to define clusters of
differentiation (CD). As each antigen, or CD, was found, it
was assigned a number.
STAGES IN B-CELL DIFFERENTIATION
1. Pro-B cells:
First step is the rearrangement of genes that code for the
heavy and light chains of an antibody molecule.

The end result is a B lymphocyte programmed to produce a

unique antibody molecule, which consists of two identical
light chains and two identical heavy chains
- heavy chains are coded on chromosome 14
- Light chains are coded on chromosome 2 and 22
2. Pre-B cell
The first heavy chains synthesized are the μ chains, which
belong to the class of immunoglobulins called IgM.

Pre-B cells also lose the CD43 marker as well as cKit and
TdT.
3. Immature B cells
Rearrangement of genetic sequence coding for light chains STAGES IN T-CELL DIFFERENTIATION
on either chromosome 2 or 22. T lymphocyte
Other surface proteins that appear on the immature. B cell Lymphocyte precursors called enter the thymus from the
include CD21, CD 40, and major histocompatibility complex bone marrow
(MHC) class II molecules.
Within the lobules of the thymus are two main zones
Immature B cells leave the bone marrow and proceed to o Outer cortex
seed the spleen and other secondary lymphoid organs o Inner medulla
4. Mature B cells
In the spleen, immature B cells develop into mature cells A significant selection process occurs as maturation takes
known as marginal zone B cells. place, because it is estimated that approximately 97 percent
of the cortical cells die intrathymically before becoming
These B cells remain in the spleen in order to respond mature T cells.
quickly to any blood-borne pathogens they may come into
contact with.
1. Double-Negative Stage
5. Activated B cells
• Rearrangement of the genes that code for the antigen
Activated B cells exhibit identifying markers that include
CD25, which is found on both activated T and B cells and acts receptor known as TCR begins at this stage.
as a receptor for interleukin-2 (IL-2), a growth factor • Beta-chain rearrangement
produced by T cells. • Signaling by the β chain also triggers the thymocyte to
6. Plasma cells become CD4- positive (CD4+) and CD8-positive (CD8+).
Plasma cells are spherical or ellipsoidal cells between 10 • CD 3→ the complex that serves as the main part of the
and 20 μm in size and are characterized by the presence of T-cell antigen receptor.
abundant cytoplasmic immunoglobulin and little to no
• consists of eight noncovalently associated chains, six of
surface immunoglobulin.
which are common to all T cells
This represents the most fully differentiated lymphocyte, and 2. Double-Positive Stage
its main function is antibody production.
• Rearrangement of alpha chain
• Two selection process:
o Positive selection:
▪ When the CD3-αβreceptor complex (TCR) is
expressed on the cell surface, a positive
selection process takes place that allows only
double-positive cells with functional TCR
receptors to survive.
▪ T cells must recognize foreign antigen in
association with class I or class II MHC
molecules
▪ Any thymocytes that are unable to recognize
self-MHC antigens die without leaving the
thymus
o B. Negative selection
▪ Takes place among the surviving double-
positive T cells.
▪ Strong reactions with self-peptides send a
signal to delete the developing T cell by means
of apoptosis, or programmed cell death.
 ▪ Most T cells that would be capable of an MECHANISM OF CYTOTOXICITY:
autoimmune response are eliminated in this
manner NATURAL KILLER CELLS
▪ This selection process is very rigorous, • There are two main classes of receptors on NK cells that
because only 1 to 3 percent of the double govern this response: inhibitory receptors, which deliver
positive thymocytes in the cortex survive inhibitory signals, and activatory receptors, which deliver
3. Mature T-cells signals to activate the cytotoxic mechanisms.
• The inhibitory signal is based on recognition of MHC class
• CD4+ T cells recognize antigen along with MHC class II I protein, which is expressed on all healthy cells. If NK cells
protein.
react with MHC class I proteins, then inhibition of natural
• T helper cells consist of two subsets. (Th1 and Th2)
killing occurs.
• They each have a different role to play in the immune
response. • If an inhibitory signal is not received at the same time, then
• CD8+ T cells interact with antigen and MHC class I NK cells release substances called perforins and
proteins granzymes
4. ANTIGEN ACTIVATION: • Perforins are pore-forming proteins that polymerize in the
presence of Ca2 and form channels in the target cell
• Antigen must be transported to the T-cell zones of the membrane.
secondary lymphoid tissue. • Granzymes are packets of serine esterase enzymes that
may enter through the channels and mediate cell lysis.
• When antigen recognition occurs, T lymphocytes are
transformed into large activated cells that are
characterized by polyribosome-filled cytoplasm.
Activated T lymphocytes express receptors for IL-2, just
as activated B cells do

• A second method of destroying target cells is also

available to NK cells. They recognize and lyse antibody-
coated cells through a process called antibody-dependent
cell cytotoxicity. Binding occurs through the CD16 receptor
for IgG. Any target cell coated with IgG can be boun dand
destroyed.
• This method is not unique to NK cells, as monocytes,
macrophages, and neutrophils also exhibit such a receptor
and act in a similar manner.

LABORATORY TECHNIQUES TO QUANTIFY AND IDENTIFY

LYMPHOCYTES
• Density Gradient Centrifugation with Ficoll- Hypaque→
technique to separate mononuclear cells from other cells.
• Cell flow cytometry
o Forward light scatter: Cell Size
o Side light scatter: Cell Granularity
• Immunofluoresence microscopy:
• Rosette test/technique:
• Lymphocytes are separated from whole blood
and then mixed with a suspension of sheep red
blood cells
CELL FLOW CYTOMETRY
- An automated system for identifying cells based on the
scattering of light as cells flow in single file through a laser
beam
- Fluorescent antibodies are used to screen of
subpopulation of T and B cells Components:
o sample delivery system
o a laser for cell illumination
o photodetectors for signal detection
o computer based management system

IFA-IMMUNOFLUORESCENCE ASSAY
▪ Direct Immunofluorescence
o Use monoclonal antibodies with a fluorescent tag
fluorescein and phycoerythrin (490nm) rhodamine
(545 nm)
▪ Indirect Immunofluorescence
o Uses unlabeled antibody that first combines with the
antigen by itself and a second antibody that is
complexed with a dye
NATURE OF SLIDESMANI ANTIGENS AND MHC
Immunogen vs Antigen
- The immune response of lymphocytes is triggered by
materials called “immunogens”
- The term antigen refers to a substance that reacts with
antibody or sensitized T cells but may not be able to evoke
an immune response in the first place

FACTORS INFLUENCING THE IMMUNE RESPONSE:

▪ Overall health
▪ Dose
▪ Age
▪ Route of inoculation B cells:
- Surface antibody on B cells may react with both linear
and conformational epitopes present on the surface of
an immunogen.
TRAITS OF IMMUNOGEN Anything that is capable of crosslinking surface
The ability of an immunogen to stimulate a host response immunoglobulin molecules is able to trigger B-cell
depends on the following characteristics: activation.
▪ Macromolecular size
▪ Chemical composition T cells:
▪ Molecular complexity - recognizes an epitope only as a part of a complex
▪ The ability to be processed and presented with MHC formed with MHC proteins on the surface of an antigen-
presenting cell.
molecules
T-cell epitopes are linear

IMMUNOGENICITY
HAPTENS
- Is determined by a substance’s chemical composition and
- The most famous study of haptens was conducted by Karl
molecular complexity
Landsteiner, a German scientist who was known for his
▪ Proteins and polysaccharides are the best
discovery of the ABO blood groups.
immunogens and are powerful immunogens, because
- Some substances are too small to be recognized by
they are made up of a variety of units known as amino
themselves, but if they are complexed to larger molecules,
acids.
they are then able to stimulate a response.
▪ Carbohydrates are somewhat less immunogenic than
protein, because the units of sugars are more limited ○ Examples: Poison Ivy, Drug related
than the number of amino acids in protein.

NATURE OF EPITOPE
- Epitopes are molecular shapes or configurations that are
recognized by B or T cells
- Large molecules may have numerous epitopes, and each
one may be capable of triggering specific antibody
production or a T-cell response

TYPES OF EPITOPE
Linear epitope: or sequential, where amino acids follows one
another on a single chain.

Conformational epitope: results from the folding of one chain

or multiple chains, bringing certain amino acids from RELATIONSHIP OF ANTIGEN TO THE HOST:
different segments of a linear sequence or sequences into AUTOANTIGENS are those antigens that belong to the host.
close proximity with each other so they can be recognized These do not evoke an belong to the host. These do not evoke
together
an immune response under normal circumstances.

ALLOANTIGENS are from other members of the host’s species,

and these are capable of eliciting an immune response. They
are important to consider in tissue transplantation and in blood
transfusions.
HETEROANTIGENS are from other species, such as other GENES CODING FOR MHC MOLECULES (HLA)
animals, plants, or microorganisms. • Alleles are alternate forms of a gene that code for slightly
different varieties of the same product.
HETEROPHILE antigens are heteroantigens that exist in o At each of these loci, or locations, there is the
unrelated plants or animals but are either identical or closely possibility of multiple alleles.
related in structure so that antibody to one will cross-react o The MHC system is described as polymorphic, because
with antigen of the other. there are so many possible alleles at each location.
▪ An example of heterophile antigen this is the human blood
group A and B antigens , which are related to bacterial Number of Alleles:
polysaccharides. It is believed that anti-A antibody, which o HLA-A has at least 580 different alleles○ HLA-B has
is normally found in individuals with blood types other than at least 921 different
A (e.g., type B and type O), is originally formed after o HLA-B has at least 921 different alleles○ HLA-C has at
exposure to pneumococci or other similar bacteria least 312 different alleles

• The probability that any two individuals will express the

same MHC molecules is very low.
ADJUVANTS • An individual inherits two copies of chromosome 6, and
- It is a substance administered with an immunogen that thus there is a possibility of two different alleles for each
increases the immune response. gene on the chromosome, unless that person is
Example: homozygous
o Hepa B Vaccine (aluminum salts) • These genes are described as codominant, meaning that
o Freund’s complete adjuvant, which consists of mineral all alleles that an individual inherits code for products that
oil, emulsifier, and killed mycobacteria (0.5 mg/mL) are expressed on cells.
• Since the MHC genes are closely linked, they are inherited
MAJOR HISTOCOMPATIBILITY COMPLEX together as a package called a haplotype.
- Evidence now indicates that the genetic capability to mount o Thus, each inherited chromosomal region consists of
an immune response is linked to a group of molecules a package of genesfor A, B, C, DR, DP, and DQ.
originally referred to as human leukocyte antigen. • The full genotype would consist of two of each gene at a
- The French scientist Dausset gave them this name, particular locus
because they were first defined by discovering an antibody
response to circulating white blood cells.
- These antigens are also known as, MHC Molecules
because they determine whether transplanted tissue is
histocompatible and thus accepted or recognized as
foreign and rejected.
- Gene coding for the MHC molecules in humans are found
in the short arm of chromosome 6.

GENES CODING FOR MHC MOLECULES (HLA)

- Short arm of chromosome 6 are divided into three
categories or classes.
• Class I molecules are coded for at three different
locations or loci termed A, B, and C.
• Class II genes are situated in the D region, and
there are several different loci DR, DQ, and DP.
• Class III genes are located in between class I and
Class II regions which code for complement
proteins and cytokines such as tumor necrosis
factor
CYTOKINES
- small soluble proteins that regulate the immune system,
orchestrating both innate immunity and the adaptive
response to infection
- Chemical messengers that influence the activities of other
cells.

ACTIONS OF CYTOKINES
• Autocrine –affecting the same cell that secreted it
• Paracrine –affecting a target cell in close proximity
• Endocrine -systemic

ROLES OF CYTOKINES
• Innate immunity
• Adaptive immunity CYTOKINES IN INNATE
• Adaptive immunity 1. Interleukin-1
• Growth and differentiation of immature leukocytes • IL-1 acts as an endogenous pyrogen and induces
fever in the acute phase response through its
FEATURES OF CYTOKINES actions on the hypothalamus
• Pleotropism single cytokine has many different actions 2. Tumor Necrosis Factor-a (TNF-a)
which relates to the widespread distribution of cytokine • were first isolated from tumor cells and were so
receptors on many cell types and the ability of cytokines to named because they induced lysis in these cells.
alter expression of numerous genes 3. Interleukin-6
• Redundancy different cytokines often have very similar • is a single protein produced by both lymphoid and
effects, many cytokines share receptor subunits nonlymphoid cell types.
• Synergy cooperative effect of multiple cytokines 4. Chemokines
• Antagonist inhibition of one cytokine effects by another • are a family of cytokines that enhance motility and
cytokine promote migration of many types of white blood
• Act in networks stimulate the release of other cytokines cells toward the source of the chemokine
Act as growth factors for hematopoietic cells modulate the (chemotaxis)
number and composition of cells 5. TGF- B
• The transforming growth factor beta is composed
of three isoforms: TGF-1, 2, and 3. TGF- was
originally characterized as a factor that induced
growth arrest in tumor cells.
6. Interferon
• viral replication Interferons were originally so
named because they interfere with.
• The type I interferons (alpha and beta) are also
active against certain malignancies and other
inflammatory processes.
 CYTOKINES IN ADAPTIVE
1. Th-1 Cytokines
• IFN-Y (Interferon gamma) is the principal molecule
produced by Th1 cells, and it affects the RNA
expression levels of more than 200 genes.

• Interleukin-2 is also known as the T-cell growth

factor. It drives the growth and differentiation of
both T and B cells and induces lytic activity in NK
cells

• Interleukin-4 is one of the key cytokines regulating

Th2 immune activities and helps drive antibody
responses in a variety of diseases

• Interleukin-10 has anti-inflammatory and

suppressive effects on Th1 cells

CYTOKINES ASSOCIATED WITH T-REGULATORY CELLS

• The third major subclass of CD4 T cells are the T
regulatory (Treg) cells.
• Tregs are CD4+ CD25+ T cells that are selected in the
thymus. They play a key role in establishing peripheral
tolerance to a wide variety of self-antigens, allergens,
tumor antigens, transplant antigens, and infectious
agents.

COLONY STIMULATING FACTORS (CSFS) :

• IL-3
• Erythropoietin (EPO) - regulates RBC production in the
bone marrow but is primarily produced in the kidneys.
• granulocyte, macrophage, and granulocyte-macrophage
colony stimulating factors (G-CSF, M-CSF, and GM-CSF,
respectively).
IMMUNOLOGY & SEROLOGY 311 Lecture
Antibodies Midterms

○ Is important in effector functions of

immunoglobulin molecules, which include
INTRODUCTION
opsonization and complement fixation.
● Fab fragment (fragment antigen-binding)
● When B lymphocytes are stimulated by antigen and undergo ○ Have antigen-binding capacity
differentiation, the end product is antibody or ● Alfred Nisonoff used pepsin to obtain additional evidence for
immunoglobulin. the structure of immunoglobulins.
● Immunoglobulins are glycoproteins found in the serum
portion of the blood.
● They are composed of 82%--96% polypeptide and 2%--14 %
carbohydrate.
● When subjected to electrophoresis at pH 8.6,
immunoglobulins appear primarily in the gamma band.

Serum Electrophoresis
● It uses an electrical field to separate the proteins in the blood
serum into groups of similar size, shape, and charge.
● Blood serum contains two major protein groups:
○ Albumin
○ Globulin

PARTS OF IMMUNOGLOBULINS

A. Light Chains

● Composed of 23,000 daltons and more than 200 amino

acids.
● Common to all immunoglobulin classes.
● Types:
○ Kappa (κ) – 214 amino acids
○ Lambda (λ) – 213 to 216 amino acids
● Gene:
○ chromosome 2 (Kappa)
○ chromosome 22 (Lambda)
● Region:
○ Amino terminal
○ Carboxy terminal
● Determine the serological and physical chain of an antibody.
Tetrapeptide Structure of Immunoglobulins
Bence Jones Protein
● Consists of two large chains called heavy or H chains and ● Found in the urine of patients with multiple myeloma, were in
two smaller chains called light or L chains. fact L chains that were being secreted by the malignant
● The basic structure of immunoglobulins was elucidated in plasma cells.
the 1950s and 1960s by the efforts of two men: Gerald ● Bence-Jones proteins had been discovered in 1845 by Dr.
Edelman, and Rodney Porter. Henry Bence-Jones, who noted the peculiar behavior of
these proteins:
Fragmentation of Antibodies ○ When heated to 60°C, they precipitate from urine
● Degrade immunoglobulin molecules into definable ○ Further heating to 80°C, they redissolve
fragments to facilitate study of their structure.
● Primary agents for fragmentation:
B. Heavy Chains
○ Papain
○ Pepsin
● Fc fragment (fragment crystallizable) ● Composed of 50,000 daltons with 110 amino acids at the
○ Had no antigen-binding ability terminal end.
● Determine serological and physical characteristics of the
antibody.

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 ● Gene: chromosome 14
E. Domains
● Types:
○ IgG – γ
○ IgM – μ ● Are globular regions on polypeptide chain stabilized in
○ IgA – α intrachain disulfide bonds.
○ IgD – δ ● Domains on the heavy chain:
○ IgE – ε ○ VH, CH1, CH2, CH3, CH4
● Region: amino terminal, carboxy terminal ● Domains on light chain:
○ VL, CL

C. Disulfide Bonds

● Bonds that hold the four polypeptide chains in normal

immunoglobulin molecules.
● Types:
○ Interchain bonds – occurs between:
■ Heavy chains (H-H) – hinge region
■ Heavy and light chains (H-L)
■ Light chains (L-L)
○ Intrachain bonds – forms the basis of division of
each immunoglobulin into domains.

D. Regions

F. Hinge Region
● Variable Region (V) – shows a wide variety of amino acid
sequence in the amino terminal portion of the molecule.
● Portion of heavy chain between the CH1 and CH2 domains.
● Areas of high variability:
● In this region, interchain disulfide bonds form between the
○ Variable region of Heavy chain (VH)
arms of Fab fragments
○ Variable region of Light chain (LH)
● High flexible and allows movement of the Fab arms in
relation to others.

TYPES OF IMMUNOGLOBULINS

Immunoglobulin G or IgG

● Predominant Ig among humans comprising 75-80% of the

total Ig pool
● Has 4 major subclasses:
A. Isotypic Variation ○ IgG1, IgG2, IgG3, IgG4
● Refers to the type of heavy chain that is unique to ● Equally distributed in the different fluid compartments with
each immunoglobulin class detectable amounts in CSF and urine
● Isotypes of immunoglobulins are: ● Readily diffusible
○ IgG: have gamma chain ● IgG antibodies response appears later than IgM in primary
○ IgA: have alpha chain response but they form the major antibody of the secondary
○ IgM: have mu heavy chain immune response.
○ IgE: have epsilon heavy chain ● Maternal IgG is actively and selectively transferred across
○ IgD: have delta chain the placenta to the fetus and imparts passive protection to
B. Allotypic Variation the newborn for 6-9 months.
● It refers to the genetic variation in the constant
regions of immunoglobulin molecule. Functions of IgG:
● Certain genetic markers within the constant 1. Provides immunity for the newborn
regions of the heavy chains are different in one or 2. Complement fixation
more 2 amino acids which then distinguish 3. Opsonization
individuals within a species. 4. Neutralization of toxins
C. Idiotypic Variation 5. Participation in agglutination
● Refers to the diversity at the binding site and in 6. Participation in precipitation**
particular relates to the hypervariable segments of
the antibody combining site (Paratope). Increased IgG:
● Infectious diseases, such as hepatitis, rubella, and infectious
mononucleosis.
● Collagen disorders, such as rheumatoid arthritis and
systemic lupus erythematosus

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 ● Hematologic disorders, such as polyclonal gammopathies,
Immunoglobulin D or IgD
monoclonal gammopathies, monocytic leukemia, and
Hodgkin’s disease.
● Heat labile immunoglobulin
● Accounts for less than 1% of the total serum Ig
Immunoglobulin M or IgM ● Anti-idiotypic antibody
● Precise biological action is not known but it may play a role
● The largest of the immunoglobulin molecule, accounting for in antigen-triggered lymphocyte differentiation
5-10% of the total immunoglobulin pool.
● The earliest antibody to appear in the primary immune
Immunoglobulin E or IgE
response but it doesn't persist for long.
● Primitive antibody
● Macroglobulin ● Heat labile Ig, least abundant Ig in the serum accounting for
● Star-shaped in the free state; crab-like in antigen-antibody only 0.002% of the total serum Ig.
reaction. ● Synthesized locally by plasma cell present in the mucous
● Maternal IgM does not cross the placenta. membrane of the GI and respiratory tracts.
● Seen in intravascular hemolysis ● It is unable to fix the complement via the classical pathway
● A powerful agglutinator of particulate antigen ● It is homocytotropic due to its affinity for cells of the host
species, particularly for tissue mast cells and blood
Functions of IgM: basophils
1. Surface receptor for antigens ● Associated with immediate hypersensitivity reactions and
2. Complement fixations with immunity to certain helminthic parasites
3. Opsonization ● Also known as reaginic antibody or nuisance antibody
4. Neutralization of toxins
5. Participate in agglutination***

Increased IgM:
● Infectious diseases, such as subacute bacterial
endocarditis, infectious mononucleosis, leprosy,
trypanosomiasis, malaria, and actinomycosis.
● Collagen disorders, such as scleroderma
● Hematologic disorders, such as polyclonal gammopathies,
monocytic leukemia, and monoclonal gammopathies (e.g.,
Waldenström’s macroglobulinemia)

ANTIBODY RESPONSE
Immunoglobulin A or IgA

● Represents 15-20% of human serum Ig pool. Found in serum

in small amount but predominant in sero-mucous secretions
of the respiratory tract, genito-urinary tract and GI tract
● Found also in saliva, sweat, tears, colostrum, and breastmilk
● Forms of IgA:
○ Serum IgA – can agglutinate motile infectious
agents thus promoting their phagocytosis but they
cannot activate the complement system. a. Primary Antibody Response
○ Secretory IgA – a polymeric form stabilized a short 1. Lag phase – no antibody is detectable
polypeptide chain. It is known as the “antiseptic 2. Log phase – the antibody titer increases logarithmically
paint” of mucous membrane. It can activate the 3. Plateau phase – the antibody titer stabilizes
bacteriolytic activity through the alternate pathway 4. Decline phase – the antibody is catabolized
of complement system and only the presence of
lysozyme. b. Secondary Antibody Response
● An anamnestic response differs from a primary response as
Increased IgA: followed:
● Infectious diseases, such as tuberculosis and actinomycosis 1. Time – a secondary response has a shorter lag
● Collagen disorders, such as rheumatoid arthritis phase, longer plateau, and more gradual decline
● Hematologic disorders, such as polyclonal gammopathies, 2. Type of antibody – IgM-type antibodies are the
monocytic leukemia, and monoclonal gammopathy (e.g., IgA principal class formed in the primary response.
myeloma) Although some IgM antibody is formed in a
● Liver disease, such as Laennec’s cirrhosis and chronic active secondary phase, the IgG class is the predominant
hepatitis type formed.
3. Antibody titer – in a secondary response, antibody
levels attain a higher titer. The plateau levels in a
secondary response are typically 10-fold or greater
than the plateau levels in the primary response.

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 THEORIES OF ANTIBODY PRODUCTION

Side Chain Theory – Paul Ehrlich

● Certain cells has had specific surface receptors for antigen
that were present before contact with antigen occurred. Uses of Monoclonal Antibodies:
● Once antigen was introduced, it would select the cell with ● Identifying and quantifying hormones
proper receptors. Combination wouldn’t take place and then ● Typing tissue and blood
receptors will break off and enter the circulation as antibody ● Identifying infectious agents
molecules. ● Identifying clusters of differentiation for the classification of
● New receptors will be formed in place of those broken off leukemias and lymphomas and follow-up therapy
and this process could be repeated. ● Identifying tumor antigens and autoantibodies
● Delivering immunotherapy.
The Template Theory – Breinl and Haurowitz
● Antibody-producing cells are capable of synthesizing a
generalized type of antibody, and when contact with an
antigen occurs.
● The antigens serves as a mold or template and alters protein
synthesis so that antibody with a specific fit is made.
● The “molded” antibody then enters the circulation, while the
antigen remains behind to direct further synthesis.

Selective Theory
● Assumes that antibodies are synthesized in a manner similar
to that of other proteins.
● Instructions for their synthesis are provided by genetic
elements in the nucleus of the cells rather from the antigen.

Clonal Selection Theory – Niels Jerne and Macfarlane Burnet

● Individual lymphocytes are genetically pre-programmed to
produce one type of immunoglobulin, and that specific
antigen finds or selects those particular cells capable of
responding to it, causing these to proliferate.
● Repeated contact with the antigen would continually
increase a lymphocyte pool.

MONOCLONAL ANTIBODIES

● Monoclonal antibodies are purified antibodies cloned from a

single cell.
● These antibodies exhibit exceptional purity and specificity
and are able to recognize and bind to a specific antigen.

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IMMUNOLOGY & SEROLOGY 311 Lecture
Complement System Midterms

COMPLEMENT

● It is a complex series of more than 30 soluble and cell-bound

proteins that interact in a very specific way to enhance host
defense mechanisms against foreign cells.
● Originally recognized in the 1890s as a heat-labile substance
present in normal non immune serum, complement was
coined by Paul Erhlich.
● Jules Bordet was awarded the Nobel Prize in 1919 for his
role in elucidating the nature of complement.

Effects of Complement Activation

Physiologic consequences Cellular consequences

● Blood vessel dilation ● Cell activation, such as

● Increased vascular production of
permeability inflammatory mediators.
● Cytolysis or hemolysis, if
the cells are
erythrocytes. The most
important biologic role
of complement in blood
group serology is the
production of cell
membrane lysis of
antibody-coated targets.

Complement Levels

Elevated Decreased

● Inflammatory conditions ● Complement has been

● Trauma excessively activated
● Acute illness, such as recently.
myocardial infarction ● Complement is currently
being consumed
● A single complement is
absent because of a
genetic defect.

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 The alternative pathway. C3 is hydrolyzed by water to produce a C3b sometimes called iC3. This
PATHWAY molecule can bind factor B. When factor B is bound to C3b, B is cleaved to form C3Bb, an
enzyme with C3 convertase activity. More C3 is cleaved to form more C3bBb. This enzyme is
stabilized by properdin, and it continues to cleave additional C3. If a molecule of C3 remains
attached to the C3bBbP enzyme, the convertase now has the capability to cleave C5. The C5
Classical Pathway convertase thus consists of C3bBb3bP. After C5 is cleaved, the pathway is exactly the same as
the classical pathway.

● The classical pathway or cascade is the main antibody

directed mechanism for triggering complement activation. Lectin Pathway
● Initiators:
○ Immune complexes
● The lectin pathway represents another means of activating
○ Apoptotic cells
complement without antibody being present.
○ Certain viruses and gram-negative bacteria
● Lectins are proteins that bind to carbohydrates.
○ C-reactive protein bound to ligand
● This pathway provides an additional link between the innate
and acquired immune response, because it involves
nonspecific recognition of carbohydrates that are common
constituents of microbial cell walls and that are distinct from
those found on human cell surfaces.
● Initiators: Mannose-binding lectin
○ Microbes with terminal mannose groups

The classical complement cascade. C1qrs is the recognition unit that binds to the FC portion of
two antibody molecules. C1s is activated and cleaves C4 and C2 to form C4b2a, which is known
as C3 convertase. C3 convertase cleaves C3 to form C4b2a3b, known as C5 convertase. The
combination of C4b2a3b is the activation unit. C5 convertase cleaves C5. C5b attracts C6, C7,
C8, and C9, which bind together, forming the membrane attack complex. C9 polymerizes to
cause lysis of the target cell.

Alternative Pathway

IMMUNOLOGIC ASSAYS
● Pathogens can be destroyed in the absence of antibody by
means of the alternate pathway, which acts as part of innate
or natural immunity. Individual Components
● This pathway is important as an early defense against
pathogens.
Radial Immunodiffusion
● Phylogenetically, this represents the oldest of the C3
activating pathways.
● Initiators:
○ Various bacteria, fungi, viruses, or tumor cells.

● It uses agarose gel into which specific antibody is

incorporated. Serum serves as the antigen and is placed in
wells that are cut in the gel. Diffusion of the antigen from the
well occurs in a circular pattern. The radius of the resulting
circle can be related to antigen concentration
● This is a sensitive technique when performed correctly, but it
takes at least 24 hours before test results are available

Nephelometry
● It measures concentration according to the amount of light
scattered by a solution containing a reagent antibody and a
measured patient sample.
● Generally, the more antigen–antibody complexes that are
present, the more a beam of light will scatter as it passes
through the solution

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 ● To destroy any complement present, patient serum must be
Classical Pathway
heated to 56ºC for 30 minutes prior to testing.

Hemolytic titration (CH50) assay

● This measures the amount of patient serum required to lyse
50 percent of a standardized concentration of
antibody-sensitized sheep erythrocytes. Because all proteins
from C1 to C9 are necessary for this to occur, absence of any
one component will result in an abnormal CH50, essentially
reducing this number to zero

Radial Hemolysis in Agarose Plates

● Rabbit red blood cells that have been sensitized with

antibody are implanted in agarose, and patient serum is
added to wells punched in the gel. Lysis appears as a clear
zone around each well, and if complement standards are run,
the size of the zone can be related to complement
concentration.

ELISA
● It have been designed as another means of measuring
activation of the classical pathway. Solid-phase IgM
attached to the walls of microtiter plates is used to initiate
complement activation.

Alternative Pathway

AH50 Assay
● It can be performed in the same manner as the CH50, except
magnesium chloride and ethylene glycol tetraacetic acid are
added to the buffer, and calcium is left out.
● This buffer chelates calcium, which blocks classical pathway
activation. Rabbit red cells are used as the indicator, because
these provide an ideal surface for alternative pathway
activation.

ELISA
● One such test can detect C3bBbP or C3bP complexes in very
small quantities. Microtiter wells are typically coated with
bacterial polysaccharide to trigger activation of the
alternative pathway

COMPLEMENT FIXATION TESTING

● Complement itself can actually be used as a reagent in the

test known as complement fixation. Because complement
fixation occurs after the binding of antigen and antibody,
uptake of complement can be used as an indicator of the
presence of either specific antigen or antibody.
● This technique has been used in the detection of viral, fungal,
and rickettsial antibodies.
● The test involves a two-stage process:
1. a test system with antigen and antibody, one of
which is unknown, and
2. an indicator system consisting of sheep red blood
cells coated with hemolysin, which will lyse in the
presence of complement.

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Lecture
Hypersensitivity Reactions Midterms

Anaphylaxis
INTRODUCTION
● Anaphylaxis is the clinical response to immunologic
Hypersensitivity formation and fixation between a specific antigen and a
● It is a heightened state of immune responsiveness. Typically, tissue-fixing antibody. This reaction is usually mediated by
it is an exaggerated response to a harmless antigen that IgE antibody and occurs in the following three stages:
results in injury to the tissue, disease, or even death 1. The offending antigen attaches to the IgE antibody
fixed to the surface membrane of mast cells and
Immunization or Sensitization basophils. Cross-linking of two IgE molecules is
● It describes an immunologic reaction dependent on the necessary to initiate mediator release from mast
host’s response to a subsequent exposure of antigen. Small cells.
quantities of the antigen may favor sensitization by 2. Activated mast cells and basophils release various
restricting the quantity of antibody formed. mediators.
● An unusual reaction, such as an allergic or hypersensitive 3. The effects of mediator release produce vascular
reaction that follows a second exposure to the antigen, changes and activation of platelets, eosinophils,
reveals the existence of the sensitization. neutrophils, and the coagulation cascade.
● It is believed that physical allergies (e.g., to heat, cold,
Allergy ultraviolet light) cause a physiochemical derangement of
● Our basic understanding of allergy has evolved from the proteins or polysaccharides of the skin and transform them
discovery in 1967 of a previously unknown antibody, into autoantigens.
immunoglobulin E (IgE). The most significant property of IgE
antibodies is that they can be specific for hundreds of Anaphylactoid Reactions
different allergens. ● Anaphylactoid reactions (anaphylaxis like) are clinically
● Antigens that trigger allergic reactions are called allergens. similar to anaphylaxis and can result from immunologically
● Allergens: Animal dander, pollens, foods, molds, dust, metals, inert materials that activate serum and tissue proteases and
drugs, and insect stings. the alternate pathway of the complement system.
● Anaphylactoid reactions are not mediated by
antigen-antibody interaction; instead, offending substances
act directly on the mast cells, causing release of mediators,
or on the tissues, such as anaphylotoxins of the complement
cascade (e.g., C3a, C5a)

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Lecture
Autoimmunity Midterms

INTRODUCTION

Autoimmunity – it represents a breakdown of the immune system’s

ability to discriminate between self and nonself.

Autoimmune Disorders – these are conditions in which damage to

● Warm-reactive autoantibodies – erythrocytes are coated
organs or tissues results from the presence of autoantibody or
with both IgG and complement
autoreactive cells.
● Cold-reactive autoantibodies – erythrocytes are coated
● When demonstrable immunoglobulins (autoantibodies) or
with both IgM and complement, acute form is secondary
cytotoxic T cells display specificity for self-antigens, or
to Mycoplasma pneumoniae infection of
autoantigens, and contribute to the pathogenesis of the
lymphoproliferative disorder
disorder.
● Paroxysmal cold hemoglobinuria – Previously associated
with syphilis, paroxysmal cold hemoglobinuria is now
Self-tolerance – under normal circumstances, the immune response
seen more often as an acute transient condition
was held in check so that self-antigens were not destroyed.
secondary to viral infection.
● Drug-induced hemolysis – Coating of RBCs
Central tolerance – during the maturation process of T cells, the great
demonstrated by a positive direct anti–human globulin
majority of undifferentiated lymphocytes that are processed through
test (DAT) result may be drug induced and accompanied
the thymus do not survive.
by hemolysis.
● This destruction of potentially self-reactive
lymphocytes.
b. Immune Thrombocytopenia Purpura
Peripheral tolerance – in secondary lymphoid organs, it is maintained
by a delicate balance between the T helper cell type 1 (Th1) and T
helper cell type 2 (Th2) populations.

Factors influencing development of Autoimmunity:

1. Age
2. Genetic factors ● Also known as idiopathic thrombocytopenia purpura is a
3. Exogenous factors (UV radiation, drugs, viruses, and chronic bleeding disorder in which the immune system destroys
infectious disease) platelets, which are necessary for normal blood clotting.
4. Release of sequestered antigens – trauma to the tissue by People with the disease have too few platelets in the
means of an accident or infection may introduce such blood.
antigens to the general circulation.
5. Molecular mimicry – refers to the fact that many individual Endocrine Disorders:
viral or bacterial agents contain antigens that closely
resemble self-antigens. a. Grave’s Disease
6. Polyclonal B-cell activation – B-cell defects include the
abnormal expression or function of key signaling molecules,
dysregulation of cytokines, and changes in B-cell
developmental subsets.

TYPES OF AUTOIMMUNITY

Organ Specific Autoimmune Diseases

Hematologic Disorders:

a. Autoimmune Hemolytic Anemia

● is a group of disorders characterized by a malfunction of
the immune system that produces autoantibodies, which ● also known as hyperthyroidism . The disease is
attack red blood cells as if they were substances foreign manifested as thyrotoxicosis, with a diffusely enlarged
to the body goiter that is soft instead of rubbery.
● Clinical symptoms include nervousness, insomnia,
depression, weight loss, heat intolerance, sweating, rapid
heartbeat, palpitations, breathlessness, fatigue, cardiac
dysrhythmias, and restlessness.

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 b. Hashimoto’s Thyroiditis Renal Disorders:

a. Goodpasture’s Syndrome

● also known as chronic autoimmune thyroiditis.

● Patients develop a combination of goiter (or enlarged
● Is characterized by the presence of autoantibody to
thyroid), hypothyroidism, and thyroid autoantibodies. The
glomerular, renal tubular, and alveolar basement
goiter is irregular and rubbery, and immune destruction of
membranes, resulting primarily in injury to the glomerulus
the thyroid gland occurs.
that can rapidly progress to renal failure.
● Symptoms of hypothyroidism include dry skin, decreased
sweating, puffy face with edematous eyelids, pallor with a
Gastrointestinal Disorders:
yellow tinge, weight gain, and dry and brittle hair.
a. Pernicious Anemia
c. Type 1 Diabetes

● also known as insulin dependent diabetes ● is a type of vitamin B12 anemia. The body needs vitamin
● It is characterized by insufficient insulin production B12 to make red blood cells. You get this vitamin from
caused by selective destruction of the beta cells of the eating foods such as meat, poultry, shellfish, eggs, and
pancreas. Beta cells are located in the pancreas in dairy products. A special protein, called intrinsic factor
clusters called the islets of Langerhans (IF), binds vitamin B12 so that it can be absorbed in the
intestines.
Neuromuscular Disorders:
b. Primary Biliary Cirrhosis
a. Myasthenia Gravis

● is an autoimmune disease that affects the neuromuscular

junction. It is characterized by weakness and fatigability
of skeletal muscles.

b. Multiple sclerosis

● Primary biliary cholangitis (PBC), formerly known as

primary biliary cirrhosis, is a chronic disease of the liver,
presumably autoimmune in nature, that leads to
progressive cholestasis and often end-stage liver disease.
● In people with PBC, the bile ducts become injured, then
inflamed, and eventually permanently damaged. The bile
ducts are small tubes in the liver that carry bile (a
substance needed to digest food) from the liver to other
● It is an inflammatory autoimmune disorder of the central parts of the digestive system.
nervous system.
● It is characterized by the formation of lesions called
plaques in the white matter of the brain and spinal cord,
resulting in the progressive destruction of the myelin
sheath of axons.

Maekaella R. Roca – OLFU Laguna

 Systemic Diseases 2. Fluorescent Antinuclear Antibody (FANA) test

1. Rheumatoid Arthritis

● Screening test o (+) green gold fluorescence

● Anti-nuclear antibodies – are heterogenous
group of antibodies that have different
antigenic specificities

● RA can be characterized as a chronic, symmetric, and

erosive arthritis of the peripheral joints that can also
affect multiple organs such as the heart and the lungs
● Affects the synovial lining of joints; necrotic areas are
surrounded by granulation tissue that eventually leads to
joint disintegration.
● The main immunologic finding is the presence of an
antibody called Rheumatoid Factor RF [ IgM ] directed
against IgG , - Anti cyclic citrullinated peptide [ Anti - CCP ]
– more specific
● Diagnosis of RA: Clinical Manifestations, (+) RF Test
Result (LATEX TEST), (+) anti- CCP test
3. Indirect immunofluorescence
2. Systemic Lupus Erythematosus ● Utilize Crithidia lucillea – a hemoflagellate
(trypanosome with circular dsDNA in the
kinetoplast) as a substrate to detect
anti-dsDNA

3. Polymyositis

● represents the prototype of human autoimmune diseases. ● Polymyositis is an idiopathic inflammatory myopathy that
It is a chronic systemic inflammatory disease marked by causes symmetrical, proximal muscle weakness; elevated
alternating exacerbations and remissions. skeletal muscle enzyme levels
● Is a systemic rheumatic disorder that is characterized by
the presence of circulating immune complexes.
● Associated with HLA DR3
● It is most commonly seen in women and persons of
African.
● Causes: Idiopathic, Drug - induced [ procainamide,
quinidine, hydralazine, isoniazid, methyldopa ]
● Symptoms: Rash (Butterfly) or other kind abnormalities ,
Myocarditis, Lymphadenopathy, Glomerulonephritis,
Serositis

Laboratory Tests:

1. LE cell test

● Neutrophil that engulfed the antibody-coated

nucleus of another neutrophil

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Lecture
Immunodeficiency Diseases Midterms

DEFICIENCIES Combined Deficiencies

● Defects in both humoral (B cell) and cell-mediated (T cell)

Deficiencies of B-cell immunity can be caused by a defect that affects
development of both types of lymphocytes or a defective
interaction between the two limbs of the immune system.
● Deficiencies of immunoglobulins have been termed
agammaglobulinemia. 1. Severe combined immunodeficiency (SCID)
● The mechanisms of the agammaglobulinemia include ● The abnormal gene involved codes for a protein chain
genetic defects in B-cell maturation or mutations leading to called the common gamma chain, which is common to
defective interactions between B and T cells. receptors for interleukins- 2, 4, 7, 9, 15, and 21.
● The gene is referred to as the IL2RG gene and is located
on the X chromosome. Normal signaling cannot occur in
Transient Hypogammaglobulinemia
cells with defective receptors, thus halting natural
● All infants experience low levels of immunoglobulins at
maturation
● approximately 5 to 6 months of age, but in some babies, the
low levels persist for a longer time. 2. Wiskott-Aldrich syndrome (WAS)
● Immunoglobulin levels usually normalize spontaneously, ● It is defined by the triad of immunodeficiency, eczema,
often by 9 to 15 months of age. and thrombocytopenia. WAS is usually lethal in childhood
because of infection, hemorrhage, or malignancy.

3. Ataxia-telangiectasia (AT)
● is a rare autosomal recessive syndrome characterized by
cerebellar ataxia and telangiectasias, especially on the
earlobes and conjunctiva. Patients with AT have a defect
in a gene that is apparently essential to the recombination
process for genes in the immunoglobulin superfamily.

Deficiencies of T-cell

● Defects in cell-mediated immunity can result from

abnormalities at many different stages of T-cell
development. Phagocytic Cell Deficiency
● Many different molecular defects can result in a similar
clinical picture (as in severe combined immunodeficiency 1. Chronic granulomatous disease
[SCID]). ● is a group of disorders inherited as either an X-linked or
● Note: autosomal recessive gene that affects neutrophil
○ In general, defects in cellular immunity are more microbicidal function.
difficult to manage than defects in humoral ● Symptoms: recurrent suppurative infections, pneumonia,
osteomyelitis, draining adenopathy, liver abscesses,
immunity. When immunoglobulin production is
dermatitis, and hypergammaglobulinemia.
deficient, replacement therapy is often very
effective. ● The process of generating partially reduced forms of
oxygen by stimulated neutrophils was first detected as an
increase in oxygen consumption. Therefore, this response
was originally termed the neutrophil “respiratory burst.” A
more correct term is oxidative burst.

Maekaella R. Roca – OLFU Laguna

 Microbicidal Defects

1. Neutrophil glucose-6-phosphate dehydrogenase deficiency

● leads to an inability to generate enough NADPH to supply
reducing equivalents to the NADPH oxidase system. This
leads to a defect in hydrogen peroxide production and a
clinical picture similar to that of CGD.

2. Myeloperoxidase deficiency
● is relatively common, occurring in about 1 in 3000
persons in the United States. Deficient patients may have
recurrent candidal infections.

3. Leukocyte Adhesion Deficiency

● a protein (CD18) that is a component of adhesion
receptors on neutrophils and monocytes (with CD11b or
CD11c) and on T cells (with CD11a) is defective. This
defect leads to abnormal adhesion, motility, aggregation,
chemotaxis, and endocytosis by the affected leukocytes.

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Lecture
Tumor Immunology Midterms

TYPES OF TUMORS
INTRODUCTION

Benign
Tumor Immunology
● is the study of the antigens associated with tumors, the
immune response to tumors, the tumor’s effect on the host’s ● Benign tumors are often named by adding the suffix -oma to
immune status, and the use of the immune system to help the cell type (e.g., lipoma), but there are exceptions (e.g.,
eradicate the tumor. lymphomas, melanomas, hepatomas).
● Oncology is that branch of medicine devoted to the study ● Benign tumors arising from glands are called adenomas;
and treatment of tumors. those from epithelial surfaces are termed polyps or
● Tumor is commonly used to describe a proliferation of cells papillomas.
that produces a mass rather than a reaction or inflammatory ● Benign tumors are characterized by the following:
condition ○ Usually are encapsulated
○ Grow slowly
Causative Factors: ○ Usually are non spreading
1. Environmental factors ○ Have minimal mitotic activity
(chemical and ○ Resemble the parent tissue
radiation)
2. Host factors and Malignant
disease associations
3. Viruses
● A malignant neoplasm of epithelial origin is referred to as
Proto-oncogenes carcinoma, or cancer.
● act as central ● Those arising from squamous epithelium (e.g., esophagus,
regulators of the lung) are called squamous cell carcinomas, those arising
growth in normal cells from glandular epithelium (e.g., stomach, colon, pancreas)
that code for proteins are called adenocarcinomas, and those arising from
involved in growth transitional epithelium in the urinary system are called
and repair processes transitional cell carcinomas.
in the body ● Malignant tumors are characterized by the following:
○ Increase in the number of cells that accumulate
p53 gene (tumor suppressor ○ Usually, invasion of tissues
gene) ○ Dissemination by lymphatic spread or by seeding
● is located on within a body cavity
chromosome 17 and ○ Metastasis
produces a protein ○ Characteristic nuclear cellular features
that downregulates ○ Receptors for integrin molecules (e.g., fibronectin),
the cell cycle. which help malignant cells adhere to extracellular
matrix
The process of cancer:
Cancer is a multistep process involving the following: TUMORS MARKERS
● Initiation – irreversible mutations involving proto-oncogenes
● Promotion – growth enhancement to pass on the mutation
● are substances present in or produced by tumors that can be
to other cells.
used to detect the presence of cancer based on their
● Progression – e.g. development of tumor heterogeneity for
measurement in blood, body fluids, cells, or tissue
metastasis, drug resistance
● tumor-specific antigens (TSAs)

Tumor immunity has the following general features:

1. Tumors express antigens that are recognized as foreign by
the immune system of the tumor-bearing host.
2. The normal immune response frequently fails to prevent the
growth of tumors.
3. The immune system can be stimulated to kill tumor cells and
rid the host of the tumor.

Host defense mechanisms against tumors:

● T lymphocytes
● Natural killer cells
● Macrophages
● Antibodies

Maekaella R. Roca – OLFU Laguna

 Laboratory test for Tumor Markers

● Cytogenetic studies
● Nucleic acid amplification techniques:
○ Polymerase chain reaction (PCR) and its variants
increase the inherent level of DNA or RNA,
allowing the detection of small populations of
cancer cells
● Fluorescent in situ hybridization (FISH):
○ Nucleic acid probes capable of binding to
sequences of interest are tagged with fluorophors
and applied to cells.

Immunotherapy

● Passive Immunotherapy
○ Passive transfer of allogeneic cellular immunity
from one person to another to fight cancer has
many barriers because of possible recipient
rejection of foreign cells, graft-versus host disease
(GVHD), and the fragility of live cells, although
research models are being studied
● Active Immunotherapy
○ The goal of active immunotherapy is to have the
patient develop an immune response that will help
eliminate the tumor. Nonspecific stimulation by
adjuvants such as Bacillus Calmette Guerin (BCG)
was first attempted, and superficial bladder cancer
is still treated with BCG.

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Lecture
Immunoproliferative Disease Midterms

● Types of ALL:
○ CALLa (CD10)-expressing immature B
INTRODUCTION
cell ALL – most common
○ pre-B cell ALL without CALLa (CD10) –
● Leukemias – the malignant cells are primarily present in the 2nd common
bone marrow and peripheral blood. ○ T-cell ALL
● Lymphomas – the malignant cells arise in lymphoid tissues, ○ B-cell ALL – rarest
such as lymph nodes, tonsils, or spleen.
● Plasma Cell Dyscrasias – these commonly involve the bone 2. Chronic lymphoid leukemia/lymphoma
marrow, lymphoid organs, and other nonlymphoid sites. They
are considered biologically distinct and not classified as
either leukemias or lymphomas.

Excess accumulation of cells:

● are a group of diseases almost exclusively of
1. Rapid proliferation
B-cell origin. They include chronic lymphocytic
2. Clonal proliferation
leukemia (CLL), small lymphocytic lymphoma
3. Chromosomal mutations malignant cells do not develop into
(SLL), prolymphocytic leukemia, and hairy cell
mature cells.
leukemia.
● CLL/SLL a single disease with different clinical
Lymphomas presentations. Both reveal B-cell marker CD19 but
weakly express CD20.
1. Hodgkin’s Lymphoma ● Hairy Cell Leukemia, the malignant cells strongly
express B-cell markers CD19, CD20, and CD22.

Plasma Cell Dyscrasias

1. Multiple Myeloma

● Hodgkin’s lymphoma (HL) is a highly treatable and ● is a malignancy of

often curable lymphoma that occurs both in young mature plasma cells
adults and in the elderly. that accounts for about
● It is characterized by the presence of Reed- 10 percent of all
Sternberg (RS) cells in affected lymph nodes and hematologic cancers. It
lymphoid organs. RS cells are typically large with a is the most serious and
bilobed nucleus and two prominent nucleoli. common of the plasma cell dyscrasias
● Patients with multiple myeloma typically have
2. Non-Hodgkin’s Lymphoma excess plasma cells in the bone marrow, a
● The most common is diffuse large B-cell monoclonal immunoglobulin component in the
lymphoma, which accounts for 30 to 40 percent of plasma and/or urine, and lytic bone lesions.
NHL. The next most common type is follicular ● Malignant plasma cells phenotypically express
lymphoma, characterized by a much more CD38, CD56, and CD138.
aggressive course than diffuse large B-cell ● Bence Jones protein is the name given to free
lymphoma. immunoglobulin light chains (κor λ) excreted in the
urine.

Leukemia 2. Waldenström’s Macroglobulinemia

1. Acute lymphoblastic leukemia (ALL) ● is a malignant proliferation of

IgM-producing lymphocytes
● is characterized by the and corresponds to
presence of very poorly lymphoplasmacytoid
differentiated precursor cells lymphoma.
(blast cells) in the bone marrow ● The malignant cells are more
and peripheral blood. These immature than plasma cells
cells can also infiltrate soft and have a microscopic
tissues, leading to organ appearance somewhere between that of small
dysfunction. lymphocytes and plasma cells. These cells
● ALL is usually seen in children, between 2 and 10 produce the pan-B-cell markers CD19, CD20, CD22,
years of age, and is the most common form of and FMC7.
leukemia in this age group. ● overproduction of monoclonal IgM

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Lecture
Basic Immunologic Procedures Finals

INTRODUCTION

Serology
● Is a medical science dealing with blood serum especially
in regard to its immunological reactions and properties

Blood Specimen Preparation Agents for Erroneous Results

● Plain evacuated tube ● Icteric sample
● No anticoagulant ● Turbid
● Allow to clot ● Bacterial
● Centrifuge for 10 contamination
minutes ● Chyle
● Perform test ● Contamination with
immediately alkali or acid
● <72 hrs = refrigerate ● Incorrect time of
● >72 hrs = freeze at collection Types of Agglutination Reactions
–20°C

Complement activation 1. Direct Agglutination

● Inactivation – is the process that destroys complement ● Occurs when antigens are found naturally on the surface
of a particle.
activity
● If there is a dark red, smooth button at the bottom of the
● Complement – is known to interfere with the reactions of microtiter well, the result is negative.
certain syphilis tests and complement components (e.g. ● A positive result will have cells that are spread across the
C1q). well’s bottom, usually jagged pattern with an irregular
○ Latex passive agglutination assays – false positive edge.
○ Hemagglutination assay – false negative ● Test tubes also can be centrifuged and then shaken to
see if the cell button can be evenly resuspended. If it is
resuspended with no visible clumping, then the result is
Note: negative.
● Inactivated by heating to 56°C for 30 minutes ● Hemagglutination – is agglutination reactions involving
● Re-inactivated by heating it to 56°C for 10 minutes after red blood cells.
more than 4 hrs since inactivation ○ Example:
■ ABO Rh blood typing
■ Hepatitis A virus (HAV)
■ Hepatitis B virus (HBV)
I. AGGLUTINATION REACTIONS ■ Hepatitis C virus (HCV)
■ Human immunodeficiency virus (HIV) I and II.
● It is a process whereby specific antigens (e.g, red blood
cells) aggregate to form larger visible clumps when the 2. Passive or Indirect Agglutination
corresponding specific antibody is present in the serum. ● Passive, or indirect, agglutination employs particles that
are coated with antigens not normally found on their
● Antibodies that produce such reactions are often called
surfaces.
agglutinins ● Latex particles – inexpensive, are relatively stable, and
● Particles initiate agglutination: are not subject to cross-reactivity with other antibodies.
○ Erythrocytes ● Singer and Plotz – found by happenstance that IgG was
○ Bacterial cells naturally adsorbed to the surface of polystyrene latex
○ Inert carriers such as latex particles particles.
● Used to detect:
○ rheumatoid factor (RF)
Steps in Agglutination
○ antinuclear antibody (ANA)
1. Sensitization ○ antibodies to group A streptococcus antigens
● involves antigen–antibody combination through ○ antibodies to Trichinella spiralis
single antigenic determinants on the particle ○ antibodies to viruses such as cytomegalovirus,
surface and is rapid and reversible. rubella, varicella-zoster, and HIV-1/ HIV-2
2. Lattice formation
● The sum of interactions between antibody and
multiple antigenic determinants on a particle.

Maekaella R. Roca – OLFU Laguna

 which naturally adsorbs the fragment crystallizable (FC)
portion of antibody molecules
● Coagglutination reagents have been used in identification
of streptococci, Neisseria meningitidis, Neisseria
gonorrhoeae, Vibrio cholera 0139, and Haemophilus
influenzae

3. Reverse Passive Agglutination

● Antibody rather than antigen is attached to a carrier
particle.
● In all of these reactions, rheumatoid factor will cause a
false positive as it reacts with any IgG antibody. Antihuman Globulin Test / Coomb’s Test
● Used in tests for:
○ Group B streptococcus
○ Staphylococcus aureus Direct antiglobulin test is used Indirect antiglobulin test or
○ Neiserria meningitidis to demonstrate in vivo indirect Coombs test, is
○ Streptococcal groups A and B attachment of antibody or used to determine the
○ Haemophilus influenzae complement to an individual’s presence of a particular
○ Rotavirus red blood cells. antibody in a patient, or it
○ Cryptococcus neoformans can be used to type patient
○ Vibrio cholera 01 This test serves as an indicator red blood cells for specific
○ Leptospira of autoimmune hemolytic blood group antigens.
○ Therapeutic drugs, hormones and plasma proteins, anemia, hemolytic disease of
such as haptoglobin and CRP the newborn, sensitization of red
blood cells caused by the
presence of drugs, or a
transfusion reaction.

4. Agglutination Inhibition
● based on competition between particulate and soluble
antigens for limited antibody-combining sites
● Lack of agglutination is an indicator of a positive reaction
● Hemagglutination inhibition – reactions use the same
principle, except red blood cells are the indicator
particles.
● This type of testing has been used to detect antibodies to
certain viruses, such as rubella, mumps, measles,
influenza parainfluenza HBV, herpes virus, respiratory
syncytial virus, and adenovirus.

5. Coagglutination
● Uses bacteria as the inert particles to which antibodies
are attached
● Staphylococcus aureus is most frequently used, because
it has a protein on its outer surface, called protein A,

Maekaella R. Roca – OLFU Laguna

 False-Positive Reactions in Agglutination Testing Incorrect incubation Check temperature at which test
temperature: too low a is carried out
Cause Correction temperature may result in the
lack of association of antigen
Over centrifugation: Button is Regulate centrifuge to proper and antibody.
packed too tight and difficult to speed and time.
resuspend. Insufficient incubation time: Follow instructions carefully
antigen and antibody may not
Contaminated glassware, slides, Keep supplies covered in have time for association
or reagents: Contaminants (dust, storage and handle with care.
dirt, or fingerprints) may cause Prozone phenomenon: too If this is suspected, dilute
cells to clump. much patient antibody for antibody and repeat the test
amount of test
Autoagglutination: Test cells Use a control with saline and
clump without specific antibody no antibody present. If the Failure to add antiglobulin Add check cells that are
present; mainly a problem with red control is positive, test is reagent: this occurs mainly in antibody-coated to see if
cells. invalidated. direct and indirect antiglobulin agglutination occurs after a
testing. negative test. If there is still no
agglutination, disregard the
Saline stored in glass bottles: Store saline in plastic results and repeat.
Colloidal silica may leach out and containers.
cause agglutination.
II. PRECIPITATION REACTIONS
Presence of cross-reactivity. Use purified antigen
reparations and specific
● Involves combination of soluble antigen with soluble
monoclonal antibody
whenever possible. antibody to produce insoluble complexes that are visible.
● Affinity – the initial force of attraction that exists between a
Presence of rheumatoid factor Test specifically for single Fab site on an antibody molecule and a single epitope
rheumatoid factor to rule out or determinant site on the corresponding antigen.
its presence. If rheumatoid ● Avidity – represents the sum of all the attractive forces
factor is present, agglutination between an antigen and an antibody.
results must be interpreted
carefully.

Presence of heterophile antibody: Preabsorb serum with red

this occurs mainly when red blood blood cells without specific
cells are used as the carrier antigen, or pretreat red blood
particle. cells to remove other
antigens.

Delay in reading a slide: dried out Follow directions and rad

antigen may look like reactions immediately after
agglutination incubation

False-Negative Reactions in Agglutination Testing

Cause Correction

Under centrifugation: if sample Regulate centrifuge proper

is under centrifuged, cells may speed and time Precipitation Curve
not be close enough to interact
Zone of Equivalence
Inadequate washing of cells, Wash cells thoroughly,
● Optimum precipitation occurs in the zone of equivalence, in
especially in antiglobulin according to the procedure
testing: unbound being followed. Use control cells which the number of multivalent sites of antigen and
immunoglobulins may neutralize on negative reactions antibody are approximately equal.
the antihuman globulin
a. Prozone phenomenon
Reagents not active: this may Refrigerate antisera, but do not ● In the case of antibody excess, the prozone
be caused by improper storage freeze because loss of activity phenomenon occurs, in which antigen combines
may occur. with only one or two antibody molecules, and no
cross-linkages are formed.
Delays in testing procedures: Once a procedure is begun,
b. Postzone phenomenon
this especially pertains to follow through to the end
antiglobulin testing. Antibody without delay. ● where there is antigen excess, the postzone
may be eluted from red blood phenomenon occurs, in which small aggregates
cells. are surrounded by excess antigen, and again no
lattice network is formed.

Maekaella R. Roca – OLFU Laguna

 A. Precipitation in a Fluid Medium
C. Precipitation by Electrophoretic Techniques

1. Turbidimetry
● Electrophoresis separates molecules according to
● Is a measure of the turbidity or cloudiness of a solution.
differences in their electric charge when they are placed in
an electric field.
2. Nephelometry
● Measures the light that is scattered at a particular angle ● A direct current is forced through the gel, causing antigen,
from the incident beam as it passes through a antibody, or both to migrate.
suspension.

1. Rocket
immunoelectrophoresis
B. Precipitation by Passive Immunodiffusion ● The end result is a
precipitin line that is
● Reactants are added to the gel, and antigen–antibody conical in shape,
resembling a rocket, hence
combination occurs by means of diffusion.
the name rocket
● No electrical current is used to speed up the process. immunoelectrophoresis.
● The rate of diffusion is affected by the size of the particles,
the temperature, the gel viscosity, and the amount of
hydration.

1. Radial Immunodiffusion
2. Immunoelectrophoresis
a. Mancini/Endpoint Method ● A double-diffusion technique that
● Antigen is allowed to diffuse to incorporates electrophoresis current
completion and when equivalence to enhance results
is reached, there is no further
change in ring diameter
b. Fahey and McKelvey/Kinetic Method
● uses measurements taken before
the point of equivalence is 3. Immunofixation Electrophoresis
reached ● Similar to immunoelectrophoresis except that after
● diameter is proportional to the log electrophoresis takes place, antiserum is applied directly
of the concentration. to the gel’s surface rather than placed in a trough.

2. Ouchterlony Double Diffusion

● Both antigen and antibody diffuse independently through
a semisolid medium in two dimensions, horizontally and
vertically

III. COMPLEMENT FIXATION

● The test involves a two-stage process:

1. A test system with antigen and antibody, one of
which is unknown.
2. An indicator system consisting of sheep red blood
cells coated with hemolysis, which will lyse in the
presence of complement.

Maekaella R. Roca – OLFU Laguna

 IV. FLOCCULATION

● The formation of downy masses of precipitate that occurs

over a narrow range of antigen concentration.
● For antibody detection are based on the interaction of
soluble antigen with antibody, which results in the formation
of a precipitate of fine particles.

V. NEUTRALIZATION

● Depend on the capacity of an antibody to neutralize the

infectious properties of the infectious organisms.
● Used in ASO, Anti-Dnase, Hepatitis, Rubella, Mumps,
Arbovirus.

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Lecture
Labeled Immunoassays Finals

● Radioactive labels
○ Iodine-135
INTRODUCTION
○ Iodine-125 → most popular, half life of 60 days
○ Tritiated hydrogen, Hydrogen-3
● Labeled immunoassays are designed for antigens and
antibodies that may be small in size or present in very low
concentrations
● The substance to be measured is known as the analyte
○ Examples: bacterial antigens, hormones, drugs,
tumor markers. Specific immunoglobulins

Characteristics of Labeled Assays

Competitive Immunoassay Noncompetitive Immunoassay

● All the reactants are ● Antibody, often called
mixed together capture antibody.
simultaneously, and ● It is first passively absorbed
labeled antigen to a solid phase.
competes with ● Unknown patient antigen is
unlabeled patient then allowed to react with
antigen for a limited and be captured by the
number of antibody. After washing to
antibody-binding sites. remove unbound antigen, a
second antibody with a label
is added to the reaction.

Advantages Disadvantages

● RIA is an ● Health hazard

extremely ● More difficult and
sensitive and expensive to maintain a
precise technique license and to comply
for determining with federal regulations
trace amounts of ● Disposal problems
analytes that are ● Short shelf life
small in size ● Need for expensive
equipment

ENZYME IMMUNOASSAY

● Also known as enzyme-linked immunosorbent assay

Immunoassay Formats
(ELISA)
● It is designed to detect antigens or antibodies by producing
Heterogeneous Immunoassay Homogeneous Immunoassay
an enzyme-triggered color change
● Involve a solid phase ● Consist only of a
(microwell, bead) and liquid phase and do ● To be used in an EIA, an enzyme must fulfill the following
require washing steps not require washing criteria:
to remove unbound steps. ○ High degree of stability
antigens or ● Are faster and easier ○ Extreme specificity
antibodies. to automate than ○ Absence from the antigen or antibody
● Can have competitive heterogeneous
○ No alteration by inhibitor within the system
or non-competitive ● Competitive

RADIOIMMUNOASSAY (RIA)

● Pioneered by Yalow and Berson

● It was used to determine the level of insulin–anti-insulin
complexes in diabetic patients.
● Radioactive elements have nuclei that decay spontaneously,
emitting matter and energy.

Maekaella R. Roca – OLFU Laguna

 ● It does not require sample radiation and nonselective
Advantages Disadvantages
excitation and source instability are eliminated
● Most chemiluminescent reagents and conjugates are stable
● Have achieved a ● Some specimens may
sensitivity similar to contain natural and relatively nontoxic.
that of RIA without inhibitors
creating a health ● The size of the
hazard or causing enzyme label may be
disposal problems a limiting factor in the
● Shorter incubation design of some
times than the original assays.
RIAs. ● Nonspecific protein
● There is no need for binding is another
expensive difficulty encountered
instrumentation, with the use of
because most assays enzyme labels.
can be read by
spectrophotometry or
by simply noting the
presence or absence
of color.
Chemiluminescent labels: Direct labels:
● Reagents are
1. Luminol 1. Luminol
inexpensive and have
a long shelf-life 2. Acridinium esters 2. Acridinium Ester
3. Peroxyoxalates 3. Electrogenerated
4. Dioxetanes luminescent chelate from
Enzyme Immunoassays 5. tris(2,2'-bipyridyl)-ruthenium ruthenium and
tripropylamine (TPA)
Borrelia burgdorferi (IgG and IgM) Hepatitis delta virus (total Ab) complex [Ru(bpy)3+)
Cytomegalovirus (IgG and IgM) HIB Ab
Cytomegalovirus (Ag) HIV Ag
Hepatitis A virus (total Ab) HTLV-I Ab Chemiluminescent Enzyme Wavelength
Hepatitis B virus (HBV) HTLV-II Ab
● Anti-HBs Human B-lymphotropic virus
Isoluminol Hydrogen peroxide 425 nm
● Anti-HBc Ab
● Anti-HBe Rubella virus (IgG and IgM)
● Anti-HBc (IgM) Toxoplasma gondii (IgG and Acridinium ester Hydrogen peroxide 429 nm
● HBs Ag IgM)
● HBe Ag Aequorea (jellyfish) Coelenterazine -- 469 nm
calcium chloride

Enzymes used in Enzyme immunoassays

Advantages Disadvantages
Enzyme Source
● Excellent sensitivity, ● False results may be
Acetylcholinesterase Electrophorous electricus comparable to EIA obtained if there is
and RIA lack of precision in
● Reagents are stable injection of the
Alkaline phosphatase Escherichia coli
and relatively nontoxic hydrogen peroxide
● Inexpensive to ● Urine or plasma cause
β-Galactosidase Escherichia coli perform quenching of the light
● Faster turnaround emission.
Glucose oxidase Aspergillus niger time

Glucose-6-phosphate Leuconostoc mesenteroides

dehydrogenase (G6PD) IMMUNOFLUORESCENCE

Lysozyme Egg white

● In 1942, Albert Coons demonstrated that antibodies could be
Malate dehydrogenase Pig heart labeled with molecules that fluoresce.
● These fluorescent compounds are called fluorophores or
Peroxidase Horseradish fluorochromes
● Fluorescein absorbs maximally at 490 to 495 nm and emits
a green color at 517 to 520 nm. It has a high intensity, good
CHEMILUMINESCENCE photostability, and a high quantum yield.
● Tetramethylrhodamine absorbs at 550 nm and emits red
● Refers to light emission produced during a chemical light at 580 to 585 nm. Because their absorbance and
reaction, it is used extensively in automated immunoassays. emission patterns differ, fluorescein and rhodamine can be
● This methodology has excellent sensitivity and dynamic used together.
range

Maekaella R. Roca – OLFU Laguna

 Advantages Disadvantages

● Has the potential for ● Nonspecific binding to

high sensitivity and substances in serum can cause
versatility quenching or diminishing of the
● Fairly simple, and signal and change the amount
there is no need to of fluorescence generated
deal with and ● Separation of the signal on the
dispose of label from autofluorescence
hazardous produced by different organic
substances. substances normally present in
serum.

A. Direct Immunofluorescent Assay Type of Immunoassays

Type Antibody Comments

● Antibody that is conjugated with a fluorescent tag is added
directly to unknown antigen that is fixed to a microscope Enzyme Enzyme-labeled antibody Competitive ELISA
slide. immunoassay (EIA; (e.g., horseradish
enzyme-linked peroxidase) Noncompetitive (e.g.,
● Examples of antigens detected by this method: immunosorbent direct ELISA, indirect
○ Legionella pneumophila assay, ELISA) ELISA)
○ Pneumocystis carinii
○ Chlamydia trachomatis
Chemiluminescence Chemiluminescent Competitive or
○ Respiratory syncytial virus (RSV) molecule–labeled sandwich
antibody (e.g., isoluminol or immunoassay
acridinium
B. Indirect Immunofluorescent Assay ester labels)

Electrochemilumine Electrochemiluminescent
● Involve two steps: scence molecule–labeled antibody
1. Incubation of patient serum with a known antigen (e.g., ruthenium label)
attached to a solid phase.
2. The slide is washed, and then an antihuman Fluoroimmunoassay Fluorescent Heterogeneous (e.g.,
molecule–labeled antigen time-resolved
immunoglobulin containing a fluorescent tag is
(e.g., europium or immunofluoroassay)
added. fluorescein label)
● Used to detect treponema, antinuclear, chlamydial, and Homogeneous (e.g.,
fluorescence
toxoplasma antibodies, as well as antibodies to such viruses
polarization
such as herpes simplex, Epstein-Barr, and cytomegalovirus immunoassay)

OTHER LABELED IMMUNOASSAY

Tyramide signal amplification (TSA)

● Can be used in various fluorescent and colorimetric
detection applications. TSA provides a messenger RNA
(mRNA) in situ hybridization protocol that is effective in
detecting B cell clonality in plastic embedded tissue
specimens.

Magnetic labeling technology

OTHER TYPE OF IMMUNOFLUORESCENCE ● Magnetic labeling technology is an application of the
high-resolution magnetic recording technology developed
for the computer disk drive industry. Increased density of
● Quantitative fluorescent immunoassays (FIAs) can be
microscopic, magnetically labeled biological samples
classified as heterogenous or homogenous, corresponding (e.g. nucleic acid on a biochip) translates directly into
to similar types of enzyme immunoassays reduced sample-processing times.
● Fluorescence polarization immunoassay (FPIA), which is
based on the change in polarization of fluorescent light Time-resolved fluoroimmunoassay
emitted from a labeled molecule when it is bound by ● In a time-resolved assay, fluorescence is measured after
antibody. a certain period to exclude background interference
fluorescence. This form of immunoassay is
heterogeneous with a direct format (sandwich assay),
similar to direct ELISA.

Quantum dots (Q dots)

● An advanced labeling technique, quantum dots are
semiconductor nanocrystals used as fluorescent labeling
reagents for biological imaging.

Maekaella R. Roca – OLFU Laguna

 SQUID technology
● A novel method of labeling is to tag antibodies with
superparamagnetic particles, allow the tagged antibodies
to bind with the target antigen, and use a superconducting
quantum interference device (SQUID) to detect the tagged
antigen-antibody complex.

Luminescent Oxygen-Channeling immunoassay

● This novel detection technology is based on two different
200 nm latex particles, a sensitizer particle that absorbs
energy at 680 nm with the generation of singlet oxygen
(donor bead) and a chemiluminescer molecule that shifts
the emission wavelength to 570 nm (receptor bead)

Fluorescence Polarization Immunoassay

● A homogeneous competitive fluoroimmunoassay, the
polarization of the fluorescence from a
fluorescein-antigen conjugate is determined by its rate of
rotation during the lifetime of the excited state in solution.

Fluorescence in situ hybridization

● Uses fluorescent molecules to brightly “paint” genes or
chromosomes. The rapid expansion in the availability of
polyclonal and monoclonal antibodies has fostered
dramatic increase in light microscopic
immunohistochemistry (IHC) and in situ hybridization.

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Lecture
Immunology and Serology of Bacterial Infections Finals

3. Latent Syphilis
1. SYPHILIS ● Characterized by lack of
clinical symptoms.
● Patients are non-infectious
at this time, except
pregnant women who can
pass the infection to the
fetus.
● Diagnosis:
○ Made only by
● Also known as French disease, Spanish disease, great pox serologic methods
or evil pox.
● Genus Treponema contains four species of pathogenic 4. Tertiary Syphilis or Late Syphilis
organisms. ● Characterized by the appearance of lesions called
gummas.
● Congenital syphilis occurs
Treponema pallidum subspecies pallidum Human syphilis
● Diagnosis:
Treponema pallidum subspecies pertenue Yaws ○ reactive serological test
○ reactive spinal fluid test (neurosyphilis)
Treponema pallidum subspecies endemicum Nonvenereal endemic
syphilis
★ LABORATORY DIAGNOSIS
Treponema carateum Agent of pinta

Treponema cuniculi Rabbit syphilis

Syphilis Antigen:
1. Wasserman antigen
Mode of Transmission ● Made up of cardiolipin which is isolated from cardiac
muscle.
● Sexual transmission
● A hapten
● Parenteral exposure through contaminated needles and 2. Treponemal antigen
blood ● Reiter treponeme
● Congenital infections during pregnancy ○ Non-virulent, cultivable variant of T. pallidum
● Blood transfusion ○ Commonly used in complement fixation
tests and treponema pallidum
immobilization
★ STAGES OF SYPHILIS ● Nichol’s treponeme
○ Virulent strain of T.pallidum
○ Used in fluorescent treponemal antibody
1. Primary Syphilis (Early Syphilis) absorption (FTA-ABS)
● Patient develops a characteristic,
primary inflammatory lesion
called chancre at the point of I. Direct Observation of Organism by Dark field Microscopy
initial inoculation and
multiplication of the spirochetes. ● For symptomatic patients with
● Serological tests – gives non primary syphilis, darkfield
reactive result (negative) at the microscopy is the test of choice.
time. ● A darkfield examination is also
suggested for immediate results
2. Secondary Syphilis in cases of secondary syphilis,
● It is usually observed 1 to 2 with a titer follow-up test.
months after the disappearance
of chancre.
II. Serologic Tests
● Systemic dissemination of the
organism occurs.
● Patients may develop A. Non Treponemal Tests
condylomata lata (flat wart-like ● Detect reagin antibodies
lesions). ● Not specific for syphilis
● Diagnosis: ● Inexpensive to carry out
○ Dark field microscopy of the fluid of the rash ● Reagent: Cardiolipin Lecithin Cholesterol
● Disadvantage
○ Serological tests
○ reagin disappears soon after syphilis is cured.

Maekaella R. Roca – OLFU Laguna

 ● False-positive reactions 3. ● Treponema pallidum Agglutination (TPA)
○ Transient: hepatitis, IM, VAricella, herpes, measles, Agglutination ● T. pallidum Hemagglutination (TPHA)
malaria, TB and pregnancy test ● Microhemagglutination-Treponema pallidum
○ Sustained: SLE, leprosy, IV drug use, autoimmune (MHA-TP)
arthritis ● Hemagglutination Treponemal Test for Syphilis
● VDRL (HATTS)
○ Principle: flocculation
4. Immuno- ● Fluorescent antibody test
1.Qualitative ● 50 uL or 0.05 mL serum fluorescence ● Indirect fluorescent antibody test
Serum VDRL ● 56°C for 30 mins to inactive complement ● Test of choice to run with dark field microscopy
● One drop of antigen (1/60 mL) of VDRL antigen is ● Earliest test to be positive
added to the ring ● Patient serum is heat inactivated and made with a
● Rotated for 4 minutes at 180 rpm sorbent consistent of non-pathogenic Treponemes
● Determine the presence of flocculation (Reiter strain) which removes cross-reactivity with
○ Non-reactive (NR) Treponemes other than T. pallidum.
○ No flocculation or no clumping ● Nichol’s strain of T. Pallidum have been fixed to slides
○ Weakly reactive (WR) for the test.
○ Slight flocculation (small clumps)
○ Reactive
○ Definite flocculation (medium and large clumps)
2. GROUP A STREPTOCOCCAL INFECTION

2. ● Needle used shall deliver 75 drops of antigen

Quantitative suspension per 1 mL. ● Caused by Streptococcus pyogenes
Serum VDRL ● 100 drops of saline per 1 mL ● Major sites of infection are the upper
● The highest serum dilution that gives reactive result is respiratory tract and skin with pharyngitis
reported as the endpoint titer. and impetigo being the most common
● Often used as a therapeutic index in the treatment of
● May lead to rheumatic fever and
syphilis
● Diagnostic for syphilis if reactive in dilution 1:16 or
post-streptococcal glomerulonephritis.
higher.
Streptococcal Antigens
A. Streptolysin O antigen
3. CSF VDRL ● Boerner agglutination slide which has concave wells ● Oxygen-labile hemolysin active against human and
16mm in diameter and 1.75 mm deep is used rabbit RBC
● The serum is not heated before testing ● Its reduced form, capable of lysing human or rabbit rbc
● The antigen must be diluted before use with 10% ● It unites with an antibody (ASO) and thereby loses its
saline solution lytic activity.
● Antigen delivery: 21 or 22 g (1/100 mL) B. Hyaluronidase
● Rotation: C. Streptokinase
○ VDRL serum: 180 rpm for 4 mins
○ VDRL CSF: 180 rpm for 8 mins

★ DIAGNOSTIC TESTS FOR STREPTOCOCCAL INFECTION

B. True Treponemal Tests
● Specific treponemal antigens
● Used to detect treponemal antibodies developed in response to
treponemal infections. 1. Anti-Streptolysin Titer
● Highly specific and sensitive ● Based on neutralization of the hemolytic activity of
● Used as verifications procedures Streptolysin O
● Normal: titer of 166 Todds units or below
● Moderately elevated: if the titer is at least 250 Todd units in
1. ● The complement fixation is a blood test that can
an adult and 320 in child.
Complement determine the presence of antigen-specific antibodies
● ASO increases within 1-2 weeks after infection and peak
fixation tests by incubating patient serum with antigen and
between 3 to 6 weeks following initial symptoms.
complement.
● Involves dilution of the unknown serum to which a
● This assay takes advantage of the requirement for
measured amount of Streptolysin O antigen is added
complement to be activated by the combination of
● 5% suspension rabbit or human RBC are added as indicator
antigen-antibody complexes.
● The tube containing the least amount of serum (one with
● For example, the Wasserman reaction is an example
highest dilution) which demonstrate no hemolysis is the
of a diagnostic complement fixation test to detect
ASO titer.
antibodies to the syphilis organism Treponema
○ Test tube 13: RBC control; no hemolysis
○ A positive reaction indicates the presence of
○ Test tube 14: ASO control; complete hemolysis
antibodies → syphilis infection

2. Anti-Dnase B Testing
2. Treponemal ● Considered as standard or reference to which all ● Measurement is based on neutralization
immobilizatio other treponemal tests are evaluated. ● If anti-DNAse B antibodies are present they will neutralize
n test ● Involve mixing of the patient serum with live, actively the reagent DNAse B.
motile T. pallidum extracted from testicular chancre ● Presence of DNAse is measured by its effect on
of a rabbit and complement. DNA-methyl green conjugate.
● Test is considered positive if >50% Treponemas are ● The conjugate is in its intact form, but when hydrolyzed by
immobilized. DNAse, the methyl green is reduced and becomes
● Positive: if 50% or more treponemas are immobilized. colorless.
● Negative: if fewer than 20% are immobilized. ● Tubes are graded for color:
● “Doubtful result”: range of 20-50% ○ 4+ = intensity of color is unchanged.

Maekaella R. Roca – OLFU Laguna

 ○ 0 = total loss of color
★ ROCKY MOUNTAIN SPOTTED FEVER (RMSF)
3. Streptozyme testing
● Slide agglutination screening test for detection of
● It is caused by Rickettsia rickettsii
antibodies to several streptococcal antigens.
● Sheep RBC’s are coated with streptolysin, streptokinase, ● Transmitted by the bite of a tick.
hyaluronidase, DNAse and NADase so that antibodies to ● Causes endothelial cell damage
any of the streptococcal antigens can be detected. leading to increased vascular
permeability, which then results in
edema, hypovolemia, and
3. HELICOBACTER PYLORI hypotension.
● Various cytokines are released and
damage to the host may have a fatal outcome if therapy is
● It is the leading cause of gastric
not initiated in a timely fashion.
and duodenal ulcers and is
● The gold standard for the serological diagnosis of RMSF is
associated with gastric carcinoma
the indirect immunofluorescence assay (IFA) with R.
(stomach cancer).
rickettsii antigen performed on two paired serum samples to
● H pylori produces a large amount of
demonstrate a significant (four-fold) rise in antibody titers.
urease that protects the organism
from the acidic environment.
● Serological testing is the primary ★ LABORATORY DIAGNOSIS
screening method of detecting H.
pylori infection.
● Testing for urease is also used to detect and diagnose an Disease Weil-felix
infection with H pylori.
OX-19 OX-2 OX-K
● Detection of H pylori antigen in stool samples can be used to
monitor the effectiveness of treatment for H pylori Rocky mountain + + –
infections. spotted
fever/Spotted
fever group
4. RICKETTSIAL INFECTION
Rickettsial pox – – –

● The rickettsiae are short rods, or coccobacilli, that are Epidemic typhus + – –
obligate, intracellular, gram-negative bacteria.
● The genus Rickettsia is made up of two distinct groups: Endemic typhus + – –
○ the spotted fever group (SFG)
○ the typhus group (TG) Scrub typhus – – +

Etiology and Ecology of Rickettsial Diseases 5. CHLAMYDIA

Organism Geographical location Disease Mode of

association transmission ● Chlamydia is a common STD caused by
infection with Chlamydia trachomatis. It
Spotted Fever Group (SFG)
can cause cervicitis, urethritis, and
proctitis.
R. rickettsii Western Hemisphere Rocky Mountain Tick bite
spotted fever ● Lymphogranuloma venereum (LGV) is
another type of STD caused by C.
R. japonica Japan Japanese Tick bite trachomatis. LGV is the cause of recent
spotted fever proctitis outbreaks among gay, bisexual, and other men who
have sex with men (MSM) worldwide.
R. felis North and South Flea-borne Unknown
America, Europe, Africa spotted fever ● Anyone with the following genital symptoms should not have
sex until they see a healthcare provider:
R. akari United States, Ukraine, Rickettsial pox Mite bite ○ A discharge
Croatia, Korea ○ A burning sensation when peeing
○ Unusual sores, or a rash
Typhus Group (TG)

R. typhi Worldwide Endemic typhus Flea feces 6. MENINGOCOCCUS

Worldwide Epidemic Louse feces

typhus ● Meningococcal disease is any type of
R. infection that's caused by the bacteria
prowazekii Worldwide Recrudescent Years after Neisseria meningitidis, which is also
typhus epidemic
called meningococcus.
typhus
● The bacteria can infect the meninges
and the blood. These infections can be
serious, even fatal.

Maekaella R. Roca – OLFU Laguna

 ● They're called meningococcal meningitis and
meningococcal septicemia.
● If a doctor suspects meningococcal disease, they will collect
samples of blood or cerebrospinal fluid (fluid near the spinal
cord).

7. HAEMOPHILUS

● Haemophilus influenzae is a
Gram-negative, opportunistic
pathogen that remains a significant
human pathogen.
● H. influenzae utilizes phase variation
of its surface antigens to evade the
host immune response.
● H. influenzae establishes an infection
by first colonizing the upper
respiratory tract. When the host is compromised, the
pathogen can disseminate and cause otitis media,
meningitis, pneumonia, epiglottitis, and other infections.

Maekaella R. Roca – OLFU Laguna

 IMMUNOLOGY AND SEROLOGY
IMSE311 - LECTURE

IMMUNOLOGY AND SEROLOGY OF VIRUS

- IgG antibodies indicates previous
exposure to a virus or immunity as a result
of vaccination.
VIRAL SEROLOGY - Culture, antigen detection, and molecular
● Innate, humoral, and cell-mediated defenses methods for viral nucleic acid can be used
protect against viruses to directly identify viruses
● Cells infected by virus produce interferon, which
enhances activity of NK cells
● Antibodies produced and involved in
- Viral neutralization HEPATITIS VIRUSES
- ADCC (Antibody-dependent cell-mediated
toxicity) ● The term hepatitis refers to inflammation of the
- Activation of complement liver.
● Ways viruses avoid the immune system : ● The viral agents of acute hepatitis can be divided
- Fast replication: Evolve and mutate quickly into two major groups, as follows:
- Alter the function of immune system cell - Primary hepatitis viruses: A, B, C, D, E,
by inserting their genetic material into a and GB virus C
host cell - Secondary hepatitis viruses: Epstein-Barr
virus (EBV), cytomegalovirus (CMV),
TORCH testing is a panel of serologic procedures used to herpesvirus, and other
detect a group of viruses and other organisms that cause
congenital disease

- Toxoplasma
- Other (viruses)
- Rubella
- Cytomegalovirus (CMV)
- Herpes simplex virus

VIRUSES
● are obligate intracellular pathogens
● can exist as either free infectious virions or Hepatitis Type/Family Transmission
intracellular particles in infected host cells. Viruses
● Interferons to inhibit viral replication
● NK cells release cytotoxic proteins that destroy Hepatitis A Virus Picornavirus Fecal - Oral
virus-infected host cells (HAV)
● Antibodies directed against specific viral antigens
can prevent the spread of viral infection by
Hepatitis B Virus Hepadnavirus Parenteral,
neutralizing a virus and preventing it from binding to
(HBV) Sexual, Perinatal
host cells, opsonizing a virus to make it more likely
to be phagocytized, activating
Hepatitis C Virus Flavivirus Parenteral
complement-mediated mechanisms of destruction,
(HCV)
and agglutinating viruses.
● Viruses have evolved in several ways to escape the
host's defenses such as: Hepatitis D Parenteral
- Frequent genetic mutations to produce (Delta Virus)
new viral antigens;
- Evading the action of interferons, Hepatitis E Calicivirus Fecal - Oral
- complement, or other components of the (HEV)
immune system;
- suppressing the immune system.

HEPATITIS A
● Diagnosis ● Known as infectious hepatitis
- IgM indicates a current or recent infection ● Short incubation hepatitis (ave of 28 days)
or a congenital infection, if present in ● HAV Antigens:
infant serum. - Shed in feces of infected individual
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 WEEK 16: IMMUNOLOGY AND SEROLOGY OF VIRUS

● HAV Antibodies: - Also produced after immunization with hepatitis B

vaccine
1. IgM Anti- HAV

- Marker of acute hepatitis A

- Peak during 1st month of illness and decline within
6-12 months
- Routinely detected by solid-phase antibody capture
ELISA

2. IgG Anti-HAV

- Produced as a result of natural infection or

immunization
- Indicate immunity to HAV
- Detected by competitive inhibition ELISA Tests

HEPATITIS B
● Serum Hepatitis
● Dane particle: complete HBV that causes infection
● HBV antigens:

1. Hepatitis B surface antigen (HBsAg)

- First marker to appear

- Indicator of active infection or chronic infection
- Important marker in screening blood donors

2. Hepatitis B Envelope Antigen (HBeAg)

- Present during periods of active replication of the

virus
- Indicates a high degree of infectivity when present
- High vertical transmission

3. Hepatitis B Core Antigen (HBcAg)

- Not detectable in serum because of the viral

envelope that masks it
- Detected only through biopsy of the infected liver
- Not considered as a serologic marker

● HBV antibodies:

1. IgM Anti-HBc HEPATITIS C

- 1st antibody to be produced ● Previously classified as "non A -non B" hepatitis
- Indicator of current or recent infection ● Major cause of post-transfusion hepatitis
- Useful in detecting infection during the "core
window" period

2. IgG Anti-HBc HEPATITIS D

- Life-long marker of HBV infection ● Unclassified, single-stranded RNA virus
● Incomplete virus
3. Anti-Hbe ● Parenterally transmitted infection that can only
- Indicates that the patient is recovering from HBV occur in the presence of hepatitis B
infection - Infection with two virus occur either:
- Marker of convalescence and favorable prognosis ★ Simultaneously as → Confection
★ Sequentially → Superinfection in chronic
4. Anti-HBs HBV carriers
● Co-infection: Where a person who is susceptible to
- Appears during the recovery period of acute HBV is exposed to someone who is co-infected with
hepatitis B, weeks to months after HBsAg produced HBV and delta virus, this results in acute
- Persists for years and provide protective immunity co-infection with both the viruses at the same time.

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 WEEK 16: IMMUNOLOGY AND SEROLOGY OF VIRUS

● Super-infection: When an HBV carrier is exposed to 3. Pol (polymerase)

infected blood from co-infected patients then the
exposure results in super-infection of the existing - Reverse transcriptase
HBV infection with delta virus; this may result in - Integrase
development of acute hepatitis (due to delta virus) - RNAse
in an HBV chronic carrier. - Protease

HEPATITIS E
● Usually presents as an acute, self-limiting hepatitis
without progression to a chronic carrier state
● Associated with a high rate of mortality in pregnant
women
● "water-borne hepatitis"

● HEV Antibodies
- IgM anti-HEV is typically present during
the acute infection but rapidly declines in
the early recovery period
- ELISA, Western blot and fluorescent
antibody blocking assay
● HEV RNA
- Detected in feces of most patients for
about 2 weeks after the onset of illness,
but may persist longer in some cases
- Identified by means of PCR

HUMAN IMMUNODEFICIENCY VIRUS

● Etiologic agent of the Acquired Immunodeficiency
Syndrome (AIDS)

● HIV-1
- Formerly called human T cell
Lymphotropic virus-type III (HTLV-I),
Lymphadenopathy-associated Virus (LAV),
and AIDS-associated retrovirus (ARV)
- Causes AIDS in US, Europe

● HIV-2
- Endemic in West Africa
- Less pathogenic and has a lower rate of
transmission

● Main Structural Genes:

1. Env (envelope)

- gp120, gp41
- Attachment and fusion to CD4 cells
● Laboratory testing for HIV Infection

2. Gag (group antigen) gene A. SCREENING

- p55, р15, р17, р24 1. ELISA

2. Agglutination Tests
3. Dot-Blot Testing
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 WEEK 16: IMMUNOLOGY AND SEROLOGY OF VIRUS

B. CONFIRMATORY
INFECTIOUS MONONUCLEOSIS
1. Western Blot Testing
2. Immunofluorescent Assay ● Heterophile antibodies are antibodies capable of
reacting with similar antigens from two or more
unrelated species
● Infectious Mononucleosis
WESTERN BLOT - Caused by Epstein Barr virus
- Target: B cells (CD21)
- It is based on the recognition of the major HIV - Atypical lymphocyte: T cells reacting to B
proteins (p24, gp41, gp120/160) by fractionating cells infected with EBV
them according to their weight by electrophoresis
and then visualizing their binding with specific
antibodies over nitrocellulose sheets.
- A positive result: presence of any two of the ● Laboratory Diagnosis
following bands-p24, gp41, and gp120/160
- If the test is positive for bands gp41 and/or p24 in 1. Paul- Bunnell test
conjunction with a positive ElA test result, it is
- Detect heterophile antibodies in patient serum
regarded as a confirmatory test.
when mixed with antigen-bearing sheep
- A negative result demonstrates the absence of
erythrocytes.
bands
- Dilutions of inactivated patient serum are mixed
with sheep erythrocytes, incubated, centrifuged,
and macroscopically examined for agglutination.
Immunofluorescence Assay - Positive reactions are preliminary

● IFA can provide a definitive diagnosis in samples 2. Davidson Differential test

that test indeterminate with other confirmatory tests.
● IFA is used to locate the HIV-1 antigen in infected - Distinguishes between the heterophile antibodies
cells. that agglutinate the antigen-bearing erythrocytes of
● Infected cells are treated with polyclonal or sheep
monoclonal antibody against p17 or р24. - Performed only if the preliminary Paul-Bunnell test
● After being washed, the cells are incubated with is positive in a titer of 1:56 or greater
fluorescein isothiocyanate or rhodamine conjugate
3. Monospot test
as a secondary antibody and then washed,
mounted, and examined using a fluorescence - based on the agglutination of horse erythrocytes by
microscope. heterophile antibody present in infectious
● The limitations of this technique include the need mononucleosis.
for expensive equipment and the fact that - Horse red blood cells (RBCs) exhibit antigens
fluorescence fades quickly directed against both Forssman and infectious
mononucleosis antibodies, a differential absorption
of the patient's serum is necessary to distinguish
the specific heterophile antibody from those of the
Forssman type.

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 WEEK 16: IMMUNOLOGY AND SEROLOGY OF VIRUS

CYTOMEGALOVIRUS VARICELLA-ZOSTER
● The infection is asymptomatic in most healthy ● Initial disease is chickenpox
individuals but may cause a mononucleosis-like ● Causes a rash that blisters and forms scabs in
syndrome, disseminated infection in organ recovery
transplant recipients and patients with HIV/AIDS, ● Severe cases can lead to varicella-zoster
and congenital abnormalities in infants born to pneumonia
infected mothers. ● After initial infection, virus lies dormant
● CMV infection is best detected by molecular assays - Possible reactivation years later as
for CMV DNA, CMV antigenemia assays for pp65 shingles
antigen, or shell vial culture ● Diagnosis is made by testing for antigen (virus) by
● Serological assays for CMV antibody are most immunofluorescence
helpful in documenting a past infection in potential ● Antibody testing: Rise in antibody titer using two
blood and organ donors. specimens weeks apart is diagnostic

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 WEEK 16: IMMUNOLOGY AND SEROLOGY OF VIRUS

HERPES SIMPLEX VIRUS RUBELLA

● Cause sores on and around the mouth and genital ● Togaviridae family
areas ● Also known as German measles
● Can be passed congenitally to infant ● Causes systemic rash in children
● If untreated, progresses to disseminated disease ● Severe complications if congenital
- Can be fatal for infant ● Vaccination to rubella is important in reducing the
● Serology: Currently only method approved by U.S. number of infants born with congenital rubella
Food and Drug Administration for diagnosing ● Antibody titers are performed to determine
herpes simplex virus infections immunity
● Titers are compared to a standard provided by the
manufacture of the test kit

RUBEOLA
● Not to be confused with rubella
● Also known as measles
● Caused by a virus in the Paramyxoviridae family
● Diagnosis is made typically by ElA for IgM antibody
or rise in titer in subsequent specimens from the
same patient

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 WEEK 16: IMMUNOLOGY AND SEROLOGY OF VIRUS

COVID-19
● Coronavirus disease (COVID-19) is an infectious
disease caused by the SARS-CoV-2 virus.
● They found that COVID-19 patients may have
persisting symptoms for weeks after acute
SARS-CoV-2 infection, including dyspnea, fatigue,
myalgia, insomnia, cognitive and olfactory disorders
● Once inside the body, the virus binds to host
receptors and enters host cells through endocytosis
or membrane fusion.

Laboratory Diagnosis:

- NAATs, such as PCR-based tests, are most often

performed in a laboratory. They are typically the
WEST NILE VIRUS most reliable tests for people with or without
● A flavivirus symptoms. These tests detect viral genetic
● Causes respiratory illness with possible material, which may stay in your body for up to 90
encephalitis and fatality days after you test positive. Therefore, you should
● Fastest diagnosis is through serologic testing for not use a NAAT if you have tested positive in the
IgM or IgG in the cerebrospinal fluid, if nervous last 90 days.
system involvement is suspected - Antigen tests* are rapid tests which produce results
in 15-30 minutes. They are less reliable than
NAATs, especially for people who do not have
symptoms. A single, negative antigen test result
does not rule out infection. To best detect infection,
a negative antigen test should be repeated at least
48 hours apart (known as serial testing).

7 | Cyrille nicole bathan| BSMLS3-YA-1

 IMMUNOLOGY AND SEROLOGY
IMSE311 - LECTURE

IMMUNOLOGY AND SEROLOGY OF PARASITE AND FUNGI

Parasitic Immunology
● Parasites are microorganisms that survive by living
off of other organisms, referred to as hosts.
● Three types of organisms may cause parasitic
infections: protozoa, helminths, and ectoparasites.
● The immune response to parasitic infections differs
from that associated with bacterial infections,
mostly because of the multicellular nature of
parasites.

● It is described that general concepts need to be

considered in relation to host immune responses to
parasites:

(1) Heterogeneity with respect to life cycles and antigenic IgE Antibodies and Parasites
expression is a key feature of parasitic agents.
● IgE antibodies are best known for their role in
(2) Many parasitic infections are chronic in nature. allergic reactions.
● Play an important role in the defense against
(3) The mechanisms of immune evasion are significantly parasites such as helminths, which are too large to
different from those of bacterial infections. be phagocytized.
● Killing of the parasites is accomplished by ADCC
(4) Many parasites develop significant genetic and antigenic
(Antibody-dependent cellular cytotoxicity).
variation in a relatively short period.
● In this mechanism, the Fc portions of the
(5) The innate immunity in the natural hosts may be parasite-specific IgE antibodies bind to specific
genetically determined. receptors on the surface of eosinophils, which are
then stimulated to release enzymes from their
(6) Humans, as well as animals, differ widely in their ability to granules that destroy the parasite.
handle the complex antigens found in parasites. ● The concentration of IgE and the number of
eosinophils in the peripheral blood are increased,
indicating their importance in defense against
parasitic infections.
Immune Responses to Parasites
● Defenses to parasitic infection involve both innate
and acquired (adaptive) immune mechanisms.
● The innate or nonspecific immune response may
result in the destruction and removal of the
parasite, thus preventing establishment of an
infection.
● The nonspecific immune defenses can include
activation of cells that may destroy the parasite by
phagocytosis, release of cytokines such as: TNF-a,
IL-1, IL-10, L-12, type I interferons, and chemokines
that enhance the immune response, or activation of
the complement system, resulting in enhanced
recognition by the immune system.
● If the innate immunity is unsuccessful in eliminating
the parasite, the parasite may be eliminated
through activation of the adaptive immune
responses. This results in either a humoral or a
cell-mediated response to the parasite

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 WEEK 17: IMMUNOLOGY AND SEROLOGY OF PARASITE AND FUNGI

FUNGAL IMMUNOLOGY
● heterogeneous group of eukaryotic organisms that
are ubiquitous in the environment.
● Fungi can either be considered as parasites,
deriving their nutrition from living matter, or more
commonly as saprophytes, living off of dead and
decaying matter.

Immune Responses to Fungi

● The immune defenses to fungal agents range from
protective mechanisms that include innate immunity
present early in the infection to specific adaptive
mechanisms that are induced later.
● The first line of innate defense includes skin and
the mucous membranes of the respiratory,
gastrointestinal, and genitourinary tracts that
provide physical barriers that separate the host
from the environment.
● Fungi possess very few factors that allow them to
overcome those physical barriers; because of the
nutrients and environmental conditions needed for
many fungi, those mechanisms have not evolved.
● If the fungi penetrate the physical barriers, there are
a variety of innate mechanisms for recognizing the
organism.
● Innate immune cells express various
pattern-recognition receptors (PRRs) that recognize
specific structures and molecules present on
bacteria and fungi.
● These structures and molecules of the organism,
called pathogen-associated molecular patterns
(PAMPs), are conserved among microbial species.
● PRRs on the innate immune cells (phagocytes,
macrophages, and dendritic cells [DCs]) initiate the
immune response by sensing and recognizing the
presence of PAMPs present on the bacteria, fungi,
or viruses.

2 | Cyrille nicole bathan| BSMLS3-YA-1