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Instant Review In

IMMUNOLOGY AND
SEROLOGY

Immunology and
Serology

Comprehensive review
Overview of Immune System
Cells of Immune system
Lymphoid system
Antigens
Antibodies
Complement System
MHC
Cytokines
Disorders of Immune System

Immunology

The study of a hosts reaction when foreign


substances are introduced into the body.
It includes:
Physiological function
Disorders of the Immune system
Methods in laboratory practices ( Serology)

Historical Background

Edward Jenner (1700s) observed the


relationship between exposure to
cowpox and immunity to small pox.
Developed the first method of
Vaccination.
Louis Pasteur improve the efficiency and
affectivity of vaccination by using
attenuated vaccines.

Divisions of Immunity

Natural Immunity

Natural/innate immunity the ability of


the individual to resist infection by
means of normally present body
functions. This includes the External and
Internal defenses.
External defense ( 1st line of Defense)
Barriers/Skin
Mechanical Movements
Normal microbial flora

Natural Immunity
Internal defenses ( 2nd line) Cellular immunity.
Immune cells ( Phagocytosis) ( Cyto-toxic)
WBCs
Platelets

Acute Phase reactants


CRP
Complement system
Ceruloplasmin
Fibrinogen
Alpha1 anti-trypsin

Inflammatory reactions

Immune cells

Cellular Defense

Neutrophils (50-70%) circulating.


Diaedesis Move toward the walls of blood

vessels.
Selectins Sticky projections for adherance.
Chemotaxis allow the cells to migrate to
the site of infection.
Promote phagocytosis ( enhanced by
complement proteins)

Cellular Defense

Eosinophils (1-3%)
Increase during parasitic infections and allergic

reactions. ( neutralize basophils)

Basophils ( < 4%)


Bind to IgE and promote allergy.
Contains dense blue granules ( secrete

histamines)

Mast cells Similar to Eosinophils but are


found in connective tissue.
Contains ACP and ALP
Life span is 9-18 months
Bind to IgE and promote allergy.

Cellular Defense

Monocytes 4-10% ( contains peroxidase)


Phagocytic cell, becomes macrophage and

stays in the infected sites/tissues.

Macrophage Specialized Phagocytic cells.


Kupffer cells Liver

Mesangial Cell Kidney


Microgial cells CNS
Histiocytes Connective tissues
Alveolar Lungs
Functions: Phagocytic, APC and anti-tumor.

Cellular Defense

Dentric cells resembles nerve dendrites.


Function as APC ( Most potent phagocytes in

tissues)
Found in skin (Langerhans)
Organs Interstitial dentric cells
Also found in Thymus.

Toll-like receptors ( TLRs)


Bind to cell walls of bacteria
TLR-2 gram +
TLR 4 gram
TLR -1 Mycobacteria

Cellular Defense

T cells develop in the thymus


T-Cytotoxic Antibody dependent

cytotoxicity.
Natural killer
T helper

B cells develop in the bone marrow.


Plasma cells Produce antibodies
B Memory cells

T helper cells

CD4 cells

secrete cytokines that promote the


proliferation and differentiation of T
cells, B cells and macrophages, and
that recruit and activate inflammatory
leukocytes.
TH1
TH2

Subsets of T helper cells

TH1 (inflammatory helper cells)


Produce interleukin-2 (IL-2)
interferon-gamma (IFNg)
Tumor necrosis factor-beta (TNFb)
Cytokines that promote cellular immune responses.

TH2 (helper cells)


Produce IL-4, IL-5, IL-6, IL-10 and
TGFb - protein that controls proliferation, cellular

differentiation.
Promote antibody synthesis by B cells.

T cytotoxic

Lyse cells that produce foreign


antigens, e.g. tumor cells, virusinfected cells, and foreign tissue
grafts.

Tc cells are identified by the presence


of the CD8 marker.

Natural killer (NK) cells

Natural killer (NK) cells NK cells are lymphocytes that do not


express either sIg or TCR = T cell antigen receptor (TCR

All NK cells express CD16, a receptor for a portion of the


IgG antibody molecule called the Fc fragment.

CD16 allows the NK cell to kill target cells coated with IgG.

NK cells are also important in eliminating tumor cells and


virus-infected cells.

B cells

B Cells B lymphocytes acquired their


name because in birds they were first
shown to mature in an organ called
the Bursa of Fabricius.
Surface immunoglobulin (sIg) is the B
cell's receptor for antigen and its
definitive marker.
Becomes plasma cells ad secrete
antibodies.

Cluster Differentiation CD Markers

Phagocytosis

Steps
Physical contact
Formation of Phagosome.
Fusion of cytoplasmic granules
Digestion and release of debris.

Acute Phase Reactants

Cytokines produce by immune cells to regulate


the function of immune system.
CRP Acute phase reactants prouced by the liver
to promote opsonization.
Serum Ameyloid A Removes chol from
macrophage at the site of injury.
Complement protiens Cell lysis
Fibrinogen Coagulation
Ceruloplasmin Copper transport (Fe to Ferritin)
Haptoglobin Capture free Hgb from plasma

Inflammation
The overall reaction of the body towards
injury or invasion by agents.
Cellular
Dilation of blood vessels
Increase blood flow
Increase vasopermeability
Increase chemotaxis
Increase coagulation

Humoral Secretion of histamines

Adaptive immune
system

Composed of:
T and B cells
Apcs
Antibodies
Complement system

Innate Vs Adaptive
Innate / natural immunity

Adaptive/ acquired immunity

Readily available, present at birth

Later developed

Non-specific

Very specific

No memory

Memory

Composed of Structural
components

Humoral secretions ( antibodies)

Lymphoid Organs

The lymphoid organs are those organs in which maturation,


differentiation and proliferation of lymphocytes take place.

The primary, or central, lymphoid organs are those in which


T and B lymphocytes begin expressing their antigen
receptors.

The secondary lymphoid organs are those in which


lymphocytes are induced to proliferate and differentiate by
contact with antigen.

LYMPHOID ORGANS

The THYMUS and BONE MARROW are primary lymphoid


organs.

They are the sites of maturation for T and B cells,


respectively.

Cellular and humoral immune responses occur in the


secondary (peripheral) lymphoid organs and tissues; effector
and memory cells are generated here.

Secondary lymphoid organs can be classified according to


the body regions which they defend.

The spleen responds predominantly to blood-borne


antigens.
Lymph nodes mount immune responses to antigens
circulating in the lymph, absorbed either through the skin
(superficial nodes) or from internal viscera (deep lymph
nodes).
Tonsils, Peyer's patches and other mucosa-associated
lymphoid tissue (MALT, boxes) respond to antigens which
have penetrated the mucosal barriers.
Note that bone marrow is both a primary and secondary
lymphoid organ. Recently, it has become accepted that the
liver is also a hematopoietic organ, giving rise to all
leukocyte lineages.

Primary (central) lymphoid organs

There are two major primary lymphoid organs, one in


which T cells develop (thymus), and the other in which B
cells develop (bone marrow). The structure of the thymus
is presented below.

Congenital absence of a thymus results in an immediate


and drastic reduction in T cells that produces a potentially
lethal wasting disease (DiGeorge syndrome).

Patients who survive the immediate neonatal period are


prone to developing recurrent or chronic infections with
viral, bacterial, fungal or protozoal agents.

Secondary lymphoid organs

The secondary lymphoid organs have two major functions:


Trap and concentrate foreign substances,
They are the main sites of production of antibodies and
antigen-specific T cells.

The major secondary lymphoid organs include:


Spleen - which is responsive to blood-borne antigens
The lymph nodes - which protect the body from antigens that
come from skin or internal surfaces via the lymphatic system.
The mucosa-associated lymphoid tissue (MALT)
scattered along mucosal linings, which protects the body from
antigens entering the body directly through mucosal surfaces.

The spleen

The Spleen is the major organ in which antibodies are


synthesized and released into circulation.

The spleen is composed of two types of tissue-the red pulp


and the white pulp.
The red pulp contains plasma cells, resident macrophages,
erythrocytes, platelets, granulocytes and lymphocytes.

It is the site where aged platelets and erythrocytes are


destroyed (hemocatheresis).

Spleen

Lymph Node

Lymph Node Clusters of lymph nodes (ovoid structures


usually less than 1 cm in diameter) are strategically placed
in the neck, axillae, groin, mediastinum and abdominal
cavity, where they act to filter antigens from the interstitial
tissue fluid and the lymph during its passage from the
periphery to the thoracic duct.
Lymph nodes that protect the skin are termed somatic
nodes, while deep lymph nodes protecting the respiratory,
digestive and genitourinary tracts are termed visceral
nodes.

Lymph Node

LymphNode

Mucosa-associated lymphoid tissue

(MALT) The bulk of the body's lymphoid tissue (>50%) is


found associated with the mucosal system.

MALT is composed of gut-associated lymphoid tissues


(GALT) lining the intestinal tract, bronchus-associated
lymphoid tissue (BALT) lining the respiratory tract, and
lymphoid tissue lining the genitourinary tract. The major
effector mechanism at these sites is secretory
immunoglobulin A (sIgA) secreted directly onto the mucosal
epithelial surfaces.

Examples of MALT include the Peyer's patches lining the


small intestine, the tonsils and the appendix.
Diffuse accumulations of lymphoid tissue are seen in the
lamina propria of the intestinal wall.
The intestinal epithelium overlying the Peyer's patches is
specialized to allow the transport of antigens into the
lymphoid tissue. This particular function is carried out by
epithelial cells termed "M" cells, so called because they
have numerous microfolds on their luminal surface. M cells
absorb, transport, process and present antigens to
subepithelial lymphoid cells.

Antigens

An antigen is any substance that causes immune


system to produce antibodies.
Can be foreign substance from the environment such
as chemicals, bacteria, viruses, or pollen.
Can also be formed within the body, as with
bacterial toxins or tissue cells.
Prerequisite for Immunogenicity
Foreign
Chemically complex
High molecular weight

TERMINOLOGIES

Antigen (Ag) - Substance that triggers and immune


response.
Antigenecity / immunogenicity Effectiveness of
Antigen to activate cells of adaptive immunity.
Epitope / Antigenic determinants Specific regions of
Ag recognized by the Abs.
Recombinant proteins Synthetically manufactured
antigens.
Antibody (Ab) immunoglobulin specifically produced
by B cells in response to antigenic stimulation.

Antigen Structures

Epitopes can be located allover the antigen structure.


This antigenic determinants can activate B cells to
produce specific Abs for each epitopes.

Degree of Immunogenicity

Different biological substances have different immunogenicity.

Substance

Degree of
Immunogenicity

Composition and Structure

Proteins

+4

Complex macromolecules

Polysaccharides

+2

Repeating monosaccharides

Lipids

+1

Fats and triglycerides

Nucleic Acids

+1

DNA and RNA amino acids

The more complex structure the more antigenic.


Heavier and larger molecules are also highly antigenic.

Proteins
Complex macromolecules made up of amino acids.
Due to its high complexity, Proteins are the most immunogenic types

of antigens.

Polysaccharides
Macromolecules of repeating monosaccharide.
Commonly known as simple sugars.
Less immunogenic compared to proteins.
Can elicit immune response when link to proteins.

Lipids
Group of organic molecules that include fats and triglycerides.
Main component of cells.
Non-immunogenic unless coupled with proteins.

Nucleic Acids
DNA and RNA are not usually immunogenic unless coupled with

more complex and macro proteins.

Activators of Lymphocytes

3 TYPES OF MOLECULES THAT TRIGGER LYMPHOCYTE


ACTIVATION
Monoclonal activators - Antigens
Oligoclonal activators Superantigens (Typically derive from bacteria)
Polyclonal activators Mitogens ( Typically plant proteins)
ADAPTIVE RESPONSE TO ANTIGEN
T-dependent antigens
Will only activate B cells in the presence of T cells and the protein that

they secrete ( cytokines) ex. Proteins

T-independent antigens
Can be activate B cells in the absence of T cells. Ex. Polysaccharides.

Monoclonal activators
Substances that stimulate cells expressing antigen receptor specific for

an epitope.
Activation of T and B cells after contact with antigen can produce one
clone of cell population.

Immune-Antigen
Recognition

In order to produce immunity to certain substance, the immune system


must activate by recognition mechanisms.
Lymphocyte Activation
Monoclonal- (Monoclonal or Antigens)immune response to Ag are very
specific.
T-dependent Activate B cells in the presence of T cells and the proteins that

they secrete.
T-independent antigens- can only activate B cells in the absence of T cells
(polysaccharides)

Oligoclonal (superantigens) Derived from bacteria, Activate subsets of


T cells w/ particular common feature in the antigen receptor.
Polyclonal (mitogens) Typically plant proteins that bind to molecules
present in virtually all T cells and B cells.

Antigen Recognition
I. Antigen Recognition by Innate cellular immunity.
A. Direct Antigen Recognition
A1. Phagocytic activity
A2. NK attacks Virally infected cell

B. Indirect Antigen Recognition


B1. Phagocyte w/ Ag receptor attached to opsonin from
Ag.
B2. Eosinophil w/ IgE Fc receptor-IgE attached to
Helminth.
B3. NK w/ Fc receptor- attached to Fc of antibody from
virally infected cells.

Antigen recognition by innate


immunity.

Direct Antigen Recognition

Antigen
Natural killer
Phagocyte

Virally infected cell

Antigen recognition by innate


immunity.

Antigen recognition by innate


immunity.

Indirect recognition

Antigen recognition by innate immunity.

Indirect recognition

Antigen recognition by Adaptive


Immunity

B cell sIg attached to Antigen


Recognize antigens via receptors present on
their cell surface ( sIg)
T cells recognition requires
internalized/fragmented antigens (epitope)
from APCs
Cytotoxic T cell receptors attached to MHC
class I from virally infected cell.
Helper cell w/ receptor to MHC class II from
APC.

Antigen Recognition by Adaptive


immunity

Antigen Recognition by
Adaptive immunity

Antigen Recognition by Adaptive


immunity

Antibody

Serum factor in blood formed in response to antigen exposure..


They are made by the plasma cells that are derived from the B
cells of the immune system.
Immunoglobulins can be separated by Electrophoresis.
Antibodies are large Y-shaped proteins.

Antibodies consists of four


polypeptides.
Two heavy chains and two light
chains joined to form a "Y"
shaped molecule

Antibody Class

Basic Structure of
Antibodies

Bifuctional Properties

Antigen binding domain and the Constant region


determines the Abs biological activity.
Antigen Binding Sites Part of Antibody molecule
that binds to the antigen.
Ag binding site (Variable region) determines the Abs

specificity.

Antibodies can only target antigens that are found


outside the cells.
Constant region(Heavy chain) determines the
immunological fate of microbes.
Abs can produce 5 different constant regions.

Antibody fragments
Determination of Bifunctionality.

Ag binding site and biological activity site


was determined from fragmentation of Ab
structure.
Proteolytic enzymes,papain and pepsin
were used to fragment Ab into 3
fragments.
Fab 2 fragments ( Fragment antigen
binding)
Fc 1 fragment (Fragment crystallizable)

THE COMPLEMENT
SYSTEM

Overview

The complement system is part of the


innate immune system (vs adaptive)
It is named complement system because
it was first identified as a heat-labile
component of serum that
complemented antibodies in the
killing of bacteria
It is now known that it consists of over 30
proteins and contributes 3 g/L to overall
serum protein quantities

Classical Pathway

Begins with antibody binding to a cell surface and ends


with the lysis of the cell
The proteins in this pathway are named C1-C9 (the order
they were discovered and not the order of the reaction)
When complement is activated it is split into two parts
a smaller of the two
B larger part and usually the active part (except with factor 2)

Remember 3 Key Words


ACTIVATION
AMPLIFICATION
ATTACK

Classical Pathway

ACTIVATION
C1q portion of C1 attaches to the Fc portion of

an antibody
Only IgG and IgM can activate complement
Once activated C1s is eventually cleaved which
activates C4 and C2
C4b & C2a come together to form the C4b2a
which is the C3 convertase
C3 convertase activates C3 to C3a and C3b

Classical Pathway

ACTIVATION
C3a binds to receptors on basophils and

mast cells triggering them to release there


vasoactive compounds (enhances
vasodilation and vasopermeability)
C3a is called an anaphylatoxin
C3b serves as an opsonin which facilitates
immune complex clearance

Classical Pathway

AMPLIFICATION
Each C1s creates many C4b and C2b fragments
Each C4bC2a creates many C3b (activated C3)
Each C3b goes on to create many Membrane

Attack Complexes
Example

1 C1S makes 100 C4bC2b


100 C4bC2b makes 10,000 C3b
10,000 C3b makes 1,000,000 MAC

Classical Pathway

ATTACK
Most C3b serves an opsonin function
Some C3b binds to C4bC2a to form the C5

convertase C4bC2aC3b
C5 convertase cleaves C5 leading to the
formation of the Membrane attack Complex
(C5-C6-C7-C8-C9)
The MAC punches holes in cell walls
resulting in lysis

C5a is a:
C2
C4

C3
1. Potent anaphylatoxin

C1q 2.

Chemoattractant for
neutrophils

2a
4
2b
4b
a

C3a binds to receptors on


basophils and mast cells
triggering them to release
there vasoactive compounds
(enhances vasodilation and
vasopermeability) ANAPHYLATOXIN
C3a
C3b

C5

C5bC5a
C3-convertase
C5-Convertase

C7
C8
C9

Classical
Pathway

C6

Alternative Pathway

Requires no specific recognition of


antigen in order to cause activation

Alternative Pathway

ACTIVATION
Spontaneous conversion from C3 to C3b

occurs in body
Normally, C3b is very short lived and quickly
inactivated by proteins on the surface of the
bodys own cell walls
However, bacteria or other foreign material
may lack these surface proteins allowing
C3b to bind and stay active

Alternative Pathway

AMPLIFICATION
Factor B binds to C3b
Factor B is then cleaved by factor D into Ba

and Bb
C3bBb remains which acts as a C3
convertase (C3 C3a and C3b)
C3bBbC3b is formed which acts as a C5
convertase

Alternative Pathway

ATTACK
C5 is cleaved to C5a and C5b
C5b then starts the assembly of the

Membrane Attack Complex

C3a
C3
C3b
C3
C5

Anaphylatoxin

Alternative
Pathway
C3-Convertase
C5-Convertase
C7
C8
C9

C6

C3b
C3a
C5b C5aD

Bb
BBa

Summary - Activation

Complement can be activated by the


binding of antibody (Classical) or by the
adherance of C3b to foreign material
(Alternative)
The two pathways converge at the
formation of the C5 convertase (C4b2a3b
or C3bBbC3b)
The final common pathway is the formation
of the membrane attack complex

Summary - Function

Opsonization C3b
Chemotaxis C5a (attracts neutrophils)
Increases vasodilation & permeability
of capillary beds via mast cell and
basophil activation C3a & C5a
(Anaphylatoxins)
Cellular Lysis via the MAC

Major Histocompatibility complex

First called HLA Abs respond to circulating WBCs


MHC accounts how individual respond to a particular Ag.
Determines the survival of transplanted organ.
Found in all nucleated cells
Play a pivotal role in the development of cellular and humoral immunity.
Main function is to bring Ag to cells surface for recognition by T cells.
T cells are activated when Ag is bound to MHC.

Class I Highest in Lymphocytes and lowest in Hepatocytes. Neural

cells, muscle and sperm cells.


HLA-A and HLA-B important in transplantation.

Class II Found in APCs, include B cells, monocytes, macrophages

and dentric cells.


Involved in recognizing bacterial or exogenous antigens.

Cytokines

Soluble protiens that regulate immune system.


Produced by immune cells.
Effects of cytokines
Regulate growth
Differentiation
Gene expression WBCs

Secretion and effect can be:


Paracrine Target cells in proximity
Endocrine Systemic

Uncontrolled secretion of cytokines may cause


shock and even death.

Cytokines

TNF Tumor necrosis factor


Lysis of tumor cells, proteolytic cleavage
Secreted by monocytes, macro and activated T cells.
Increase of TNF during gram neg sepsis may casue DIC, shock and death.

( Present in Rheumatoid )

IL Interleukins are pro-inflammatory


IL1 educe fever, and increase Lymphocyte activity.
IL6- Acute phse reactants, differentiate B cells to plasma cells ( T, B mono
and macrophage)
Chemokines Promote migration and motility of WBC
Interferone anti-viral
Dentric cells, active in malignancies
Treatment for HCV

Increase NK activity and MHC1


CSF Colony stimulating factors
EPO erythropoeitin, G-CSF, granulocyte

Disorders of the Immune system

Autoimmunity
Hypersensitivity
Immunodefeciency

AUTOIMMUNITY

Autoimmunity

The pathologic condition arising from the


production f antibodies against autoantigens.
Antibodies and T cells mount an attack
towards own or self antigens that lead to
tissue injury.
Autoimmune disorders fall between type I or
II hypersensitivity.
Types of autoimmunity
Systemic Involving several organ system
Specific involving only one tissue or organ.

Factors influencing
Autoimmunity

Genetic
B-27 HLA is common to RH factor.
B-8 HLA is common to SLE and Graves

disease

Gender More common in Women


Environmental- Exposure to toxic
/carcinogenic and infectious agents.
Lifestyle Lack of exercise, smoking
and malnutrition.

Overview of Autoimmune
Disease

Systemic
Systemic lupus erythematosus
Rheumatoid arthritis
Scleroderma

Specific
Hashimotos thyroiditis
Graves disease
Myasthenia gravis
Type I D.M
Multiple sclerosis
Goodpastures syndrome
Rheumatic fever

List of Autoimmune Diseases

Pathogenesis of Autoimmune
Disease

The Sequestered Ag theory


Explains that during developmental stage of life, some tissues

are enclosed behind anatomical barriers thus cannot be


scanned by the immune system.
When these tissues are exposed to due to trauma,
deterioration or infection the immune system perceive this as a
foreign antigens.

The Clonal Theory


Explains that during fetal development, the fetus eradicates all

self-reacting lymphocyte clones called forbidden clones while


retaining only those clones that react to foreign antigens.
Autoimmune is triggered if these forbidden clones survive.

Pathogenesis of Autoimmune
Disease

The theory of Immune deficiency


Autoimmune is triggered by a mutations in receptor genes making them

reactive to self antigens, or a general breakdown in the normal suppression


of the immune response sets the scene for inappropriate immune response.

Inappropriate expression of MHC II markers


Cells that don't normally express them has been found to cause abnormal

immune reaction to self.


T cell activation may incorrectly turn on B cells that can react with self Ag.
(Bystander effect)

Molecular mimicry
Microbial antigens bear molecular epitopes similar to normal human cells.

Type I D. M and sclerosis are possible triggered by viral infection when the
virus alters cell receptors.

The Autoimmune regulator protein or AIRE.


This protein is believed to directs the transcription of many self antigens to

the thymus.

Systemic lupus
erythematosus (SLE)

Most sever form of autoimmune disorder


Also known a the butterfly-shaped rash across
the nose and cheeks.
Patients developed antibodies against his own
tissues and organs ( Kidneys, bone marrow,
skin, nerve, joints, heart and G.I tract.
Patients may also develop intracellular
antibodies ( Nucleus and Mitochondria)
SLE antibodies are usually deposited in the
basement of cells.

SLE Diagosis

Laboratory test for SLE


Anti-nuclear antibodies (ANA)
Methods
Fluorescence antibody
Radioimmunoassay

Butterfly face

Rheumatoid arthritis

Rheumatoid arthritis.
An autoimmune disease that damage the

eyes, skin and nervous system.


Autoantibodies form immune complex that
bind to the synovial membrane of the joints
and activate lymphocytes.
Presence of IgM Rheumatoid factor can be
detected in patients blood.

Rheumatoid arthritis

Endocrine Autoimmunity

Graves disease
Attachment of autoantibody to receptors on

the follicle cells that secrete Thyroxin.


Over secretion of thyroxin lead to
hyperthyroidism.

Hashimotos Thyroidism

Antibodies
destroy the
follicle cells and
inactivate
hormone
production.

HYPERSENSITIVITY

Hypersensitivity
Also known as allergy; a condition of altered
reactivity or exaggerated immune response that is
manifested by inflammation.
Types of Hypersensitivity :

Type 1 - Common allergy and anaphylaxis


Type 2 - IgG and IgM mediated cell damage
Type 3 Immune complex
Type 4 Delayed hypersensitivity

Overview of Hypersensitivity

Type I hypersensitivity

Results from excessive IgE production in response


to an exogenous antigen.
Forms of Type I
Atophy Localized allergy
Anaphylaxis Systemic and often fatal.

Portal of Entry skin, respiratory tract, G.I and GUT.


Activated by sensitizing dose of allergen and
expressed when a second provocative dose triggers
the allergic response.
Primary participant IgE, basophils, mast cells, and
agents of inflammatory response.

Hay fever or Allergic rhinitis

Hives

HDN

Review

Review

Review

Summary

HUMAN
IMMUNODEFICIENCY
VIRUS/ AIDS

Global Summary of the HIV/


AIDS epidemic December 2003

# of People living
with AIDS- 42M
# of People newly
infected as of 2002 is
5M
AIDS death as of
2002 is 3.1M

There is a low and slow


HIV transmission in the
Philippines

HIV Tracking systems

Passive Surveillance- AIDS registry


Active Surveillance
- HIV Sentinel Surveillance system
- Behavioral surveillance survey

Factors for Low HIV Transmission

Less number of sexual partners


Less exposure of males to Female sex
workers
MSM engage more in oral sex rather
than anal sex
Few injecting drug users
Delayed first sexual contact/ marriage

HISTORY

1981- PCP and other opportunistsic


infections in previously healthy
homosexuals in NY and SF
Intravenous drug users
Classic hemophilia patients

STLV-III African green monkeys


LAV Lymphadenopathy virus
ARV AIDS related virus
HTLV-III Human T-cell lymphotropic virus III

HUMAN IMMUNODEFICIENCY VIRUS

Retrovirus two single stranded RNA


and reverse transcriptase
Convert viral RNA to DNA
HIV-1 US and Europe
HIV-2 West Africa

TWO FACES OF AIDS

HIV INFECTION- entry of Human


Immunodeficiency Virus inside the body;
no signs or symptoms
AIDS Acquired Immunodeficiency
Syndrome; terminal stage; with signs
and symptoms; presence of
opportunistic infections

DIAGRAMMATIC
REPRESENTATION OF HIV

HIV
100 nm in diameter
Outer envelope glycoprotein 41, 120 and HLA
Core shell- protein24, 19
Cone shaped inner core RT, Int,P31, RNase

HIV GENOME

Structural genes
- gag p55>p18,p24m,p15
- pol RT(p66/51), RNase, Protease(p31), Int
- env gp160>gp120, 41
Regulatory genes
- tat p14 replication
- art/trs p20 expression of virus
- sor p23 viral infectivity
- 3orf latency period
- R p15

ENTRY OF HIV INTO THE


HOST

HOW IS HIV TRANSMITTED?

SEXUAL CONTACT
- vaginal
- anal
- oral

Infected blood and blood


products

Perinatal Transmission

Placental entry

During delivery

Breast feeding

You cant get HIV/ AIDS


from

Swimming pool

Mosquito bite

Toilet bowl

Casual contact

Kissing

Shaking hands

Sharing utensils

Hugging

PERSONS WITH HIV

PERSONS WITH AIDS

WHO ARE AT RISK?

Recipients of blood and blood products


Multiple sexual partners
Sexual partners of infected persons
Intravenous drug users
Men having sex with men

CLINICAL MANIFESTATIONS

PRIMARY STAGE
- asymptomatic, mild chronic lymphadenopathy
- antibodies- 2wks to 3 mos
INTERMEDIATE STAGE
- T cell deficiency, low CD4, inverted CD4/ CD8 ratio
- ARC- lymphadenopathy > 3 months
- fever more than 3 months
- >10%weight loss
- persisitent diarrhea
- fatigue
- night sweats
- Lab abnormalities CD4 < 400/ sq.m.
- CD4/ CD8 ratio < 1.0
- Leukopenia
- thrombocytopenia
- anemia
- positive HIV antibody test
- reduced balstogenesis
- elevated serum globulins
* 2 ARC + 2 Lab abnormalities = HIV infection

FINAL STAGE
- 2 to 10 years after initial infection
- severe CD4 depletion
- opportunistic infections and cancers
- AIDS
AGGRAVATORS:
- pre-existing medical condition
- HHV-6

LABORATORY DIAGNOSIS

HIV antigen active HIV infection


HIV antibody exposure to HIV

SCREENING TEST

ELISA: Enzyme Linked Immunosorbent Assaytest used to detect antibodies of HIV


HIV recombinant antigens are immobilized into
microtiter wells
Patients serum is incubated with it and washed
Antiglobulin antibodieswith HRP is added and
color formation occurs
The Screening test can only be performed by HIV
Proficient MedTechs in licensed HIV Testing
Laboratories

CONFIRMATORY TEST

WESTERN BLOT ASSAY: detects antibodies to the


major proteins that make up the virus.
HIV is disrupted and separated by MW into discrete
bands by electrophoresis onto polyacrylamide gels
Viral proteins are transferred to nitrocellulose strips and
cut.
Strips are incubated overnight with test serum, washed,
and incubated with AHG with enzymes or biotin.
Substrate is added and color develops
POSITIVE- gp41band appears
- Gp41/gp120/gp160 + p15,p18,p24,gp41,p55,p51,p66)

- Done only at SACCL Reference Laboratory in the


Philippines
- CD4: CD8= 2:1 ( HIV: 0.5:1)

HIV ANTIGEN TESTS

HIV ISOLATION before antibodies


develop
Monitor retroviral therapy
Ficoll Hypaque gradients used to collect

monocytes from peripheral blood samples

TESTS FOR DETECTING HIV


GENES

In situ hybridization
Filter hybridization
Southern Blot hybridization
DNA amplification

OPPORTUNISTIC DISEASES OF
AIDS

CANDIDIASIS Fungal infection


caused by Candida Albicans; one of the
most common opportunistic infections;
white patches on mouth, tongue, and
esophagus
CRYPTOCOCCOSIS affects the CNS

PROTOZOAN
INFECTIONS

Pneumocystis carinii Pneumoniainfection of the lungs


Toxoplasmosis Toxoplasma gondii
- CNS infection
Cyptosporidium enteritis GIT
- debilitating diarrhea

VIRAL INFECTIONS

Cytomegalovirus infection affects the


eyes, lungs and CNS
Herpes Simplex Virus infection
ulcerating skin lesions, mouthm genital
and anal area
Herpes Zoster infection cluster of
vesicles; dermatomal

BACTERIAL INFECTIONS

Mycobacterium tuberculosis lungs and


lymph nodes; common in the Philippines
Mycobacterium avium intracellularemost common systemic bacterial
infection; affects the bone marrow, liver
and lymph nodes

CANCER
KAPOSIS SARCOMA
Cancer of the small blood vessels
Most common cancer in AIDS
patients
Ulcerative patches or plaques in
the tongue which later become pale
violet, painless and non-tender

SIGNS AND SYMPTOMS


ASSOCIATED WITH HIV/AIDS

WEIGHT LOSS
PROLONGED FEVER
FALLING HAIR FUNGAL INFECTION OF THE SCALP
SUNKEN EYES- UNDERNOURISHMENT
NIGHT SWEATS TB
MENTAL DERANGEMENT CNS INFECTIONS
PERSISTENT COUGH
CHRONIC FATIGUE
MALAISE GENERAL FEELING OF ILLNESS OR DISCOMFORT
PERSISTENT DIARRHEA
PERSISTENT GENERALIZED LYMPHADENOPATHY

APPROACHES TO HIV/AIDS
MANAGEMENT

COUNSELLING
PREVENTION OF TRANSMISSION
SCREENING
EDUCATION
ANTIRETROVIRAL THERAPY
SYMPTOMATIC MANAGEMENT
ANTIMICROBIAL PROPHYLAXIS

CONSIDERATIONS OF
ANTIRETROVIRAL THERAPY

CLINICAL OBJECTIVES
Improve survival
Reduce impact of HIV disease
Improve quality of life
Increase in body weight

SURROGATE END POINTS


Reduce viral load
Improve immune function

ANTIRETROVIRAL DRUGS
(Highly Active Antiretroviral
Therapy <HAART>)

Fusion inhibitor
Nucleoside RT inhibitor- 3azido-3deoxythymidine/Azidothymidine (AZT/
Zidovudine)
Non-nucleoside RT inhibitor
Polymerase inhibitor
Alpha interferon- reduce viral budding

PREVENTION

ABSTINENCE
BE FAITHFUL
CONDOM USE
AVOID DRUG MISUSE
ENCOURAGE VOLUNTARY BLOOD
DONATION
INFECTED FEMALES PREGNANCY
ISSUES

ADVOCACY

MULTISECTORAL INVOLVEMENT
WORLD AIDS DAY CAMPAIGN
CANDLELIGHT MEMORIAL
CELEBRATION

LABORATORY METHODS

Serology(midterm)

PRECIPITATION AND
AGGLUTINATION
REACTIONS

Precipitation Reactions

Objectives
Describe how the laws of mass action

relates to Ag-Ab binding.


Distinguish precipitation and agglutination.
Define affinity and avidity and their influence
on Ag-Ab reactions.
Explain how zone of equivalence is related
to the lattice hypothesis.

Definition of terms
Affinity Initial force of attraction that exist

between a single Fab site on an antibody


molecule and a single epitope or
determinant site on the corresponding
antigen.
Avidity represents the sum of all the
attractive forces between an antigen and an
antibody.
Cross-reactivity The capability of Abs to
react to a similar Ag.

Precipitation Reaction
Involves combining soluble antigen with

soluble antibody to produce insoluble


complexes that are visible.
Rudolf kraus (1897) first observe
precipitation using pure culture of enteric
bacteria mixed with specific antibody.
Factors affecting precipitation
Type of antibody (Affinity and Avidity)
Relative concentration of Ag and Ab
Temperature

Law of Mass Action

Law of Mass Action states that free


reactants are in equilibrium with bound
reactants.
Equilibrium constant represents the difference

in the rates of the forward and reveres


reactions according to the following equation:
Binding strength increase the
K1

Ag+Ab AgAb
K2

tendency of AgAb to dissociate


decreases.

More precipitation reaction, the test


becomes more sensitive.

The Precipitation curve

Prozone Antibody excess can cause


antigens to combine with only one or two
antibody molecules causing to linkage.
Postzone occurs during small
aggregates are surrounded by excess
Antigen causing to lattice formation.
Zone of Equivalence the number of
multivalent sites of antigen and antibody
are approximately equal.

The Precipitation curve

Measuring precipitation

Light Scattering
Turbidimetry Detection device is placed in

direct line with the incident light, collecting


after it has passed through the solution. I
measure the reduction of light intensity due
to reflection, absorption or scattered.
Nephelometry Measures the light that is
scattered at a particular angle from the
incident beam as it passes through a
suspension.

Passive Immunodiffusion
Techniques

No electrical current is used to speed-up


the test.
Reactants are added to the gel and Ag-Ab
combination occurs by means of diffusion.
The rate of diffusion is affected by size of
particles, temperature, the gel viscosity
and the amount of hydration.
Radial Immunodiffusion
Ouchterlony Double Diffusion

Passive Immunodiffusion
Techniques

Radial Immunodiffusion
James Oudin used gels for precipitation

known a single diffusion.


Antibody is incorporated into agarose gel in
a test tube.
The antigen is layered on top and the
antigen moved down in to the gel.
Precipitation occurred and moved down the
tube in proportion to the amount of antigen
present the tube.

Radial Immunodiffusion

Passive Immunodiffusion
Techniques

Ouchterlony double diffusion


Classic immunological technique in which both

antigen and antibody diffuses independently


through a semi-solid medium in two dimensions
(Horizontally and vertically).
Wells are cut in gel, and reactants are added to
the wells. After 12 to 48 hrs incubation,
precipitin lines form where the moving front of
Ag meets the Ab.
The density of lines reflect the amount of
immune complex formed.

Ouchterlony double diffusion

Electrophoretic
Techniques

Separates molecules according to


difference in their electric charge when
they are placed in an electrical field.
Direct current is forced through the gel,
causing antigen, antibody or both to
migrate.
Rocket immunoelectrophoresis
Immunoelectrophoresis
Immunofixation Electrophoresis

Rocket immunoelectrophoresis

Developed by Laurell in 1960s.


One-dimension electroimmunodiffusion.
Antibody is cut in the gel and
electrophoresis is used to facilitate the
migration of the antigen into the agar.
The end result is a precipitin line that is
conical in shape resembling a rocket.
This method is used to quantitate
immunoglobulins at ph 8.6

Rocket immunoelectrophoresis

Immunoelectrophoresis

Developed by Grabar and Williams in 1953.


Double-diffusion technique that uses electrophoresis to
enhance result.
This method can be used to semi-quantitate a wide range of
antigens.
Serum is electrophoresed to separate out the main protien
fractions.
A trough is cut and the gel is incubated for 18 to 24 hrs.
Double diffusion occurs at right angles to the elctrophorertic
separation and precipitin lines developed where specific Ag
and Ab combination takes place.
The lines or arcs can be compared in shaped, intensity and
location to that of abnormal serum control to detect
abnormalities.

Immunoelectrophoresis

Immunofixation Electrophoresis

First described by Alper and Johnson.


Similar to Immunoelectrophoresis but
differs from the application of anti-serum
directly to the gels surface after
electrophoresis takes place.
This method is useful for Ag present in
CSF, serum and urine.

Sources of Errors

Applying the current in the wrong direction.


Sample run off
Unable to separate

In correct Ph of buffer and time.


Hinder proper separation

Inappropriate Ag-Ab concentration


No lattice formation.

Amount of current applied.


Incomplete separation

Summary

Immunoassays are developed to detect Ags and Abs.


Play a significant role in the diagnosis of diseases.
The assays principles are based on the principle of
precipitation which involves the binding of Abs to the
Ags.
The binding of Ab and Ags depends on several factors.
Affinity is the force of attraction and Avidity is as the
sum of all attractive forces occuring between multiple
binding sites.
For testing purposes, it is important to have an Ab with
high affinity and high avidity for the Ags in question.

Summary

Maximum precipitation reaction can be achieved if


a similar concentration of Abs combined with
similar numbers of Ags known as the ZONE OF
EQUIVALENCE.
When Ags are in excess it is called POST ZONE.
When Abs are excess, It is called PROZONE.
Precipitation can be detected by;
Turbidity measured as a reduction of light
intensity.
Nephelometry the amount of light scattered at a
particular angle.

Summary

Radial immunoassay, Ouchterlony


diffusion and electrophoresis uses
support medium as gel.
Electrophoresis utilizes electrical current
to hasten the precipitation reaction.
Precipitation is adaptable in manual or
automated method.

Agglutination Reaction

Course Outline
Historical Perspective
Agglutination Process
Types of Agglutination reaction
Applications in Serology
Advantages and Disadvantages

Objectives:
Explain the principles of Agglutination reaction.
Discuss the importance of agglutination reaction and

its application in Immunology and serology.


Interpret different agglutination reactions.

Before Serology?????

Transfusion of Dogs blood.

Blood transfusion of Sheeps blood.

Direct transfusion of human


blood to patient.

Primitive transfusion procedure.


ABO typing performed.

Agglutination Reaction

Agglutination

Visible aggregation of particulate antigen and


antibodies.
Antigen must be exposed to antibodies for binding.
Examples of Agglutinogens
RBC, Bacterial cells
Latex particles and cells.
First describe by Gruber and Durham in 1896, the
ability of antibodies to clump cells.
The finding becomes the basis for serology and
lead to the discovery of ABO blood group system.

Agglutination

Classification of Agglutination reactions


Direct agglutination
Passive agglutination
Reverse passive agglutination
Agglutination inhibition
Coagglutination

Steps in Agglutination
Sensitization Ag-Ab binding (attachment)
Lattice formation Sum interactions of Ag-Ab

via multiple antigenic sites.

Classification of Agglutination

Direct agglutination
Occurs when antigens are found naturally

on a particle.
ABO typing Hemagglutination
Widal test Typhoid fever

Classification of
Agglutination

Passive / indirect agglutination

Using particles that are coated with antigens not


normally found on their surfaces.
Examples of Particles
RBC, gelatin, salicates
Latex Widely used particles.
The methods is designed to detect IgM Abs.
Tests includes ASO, ANA, HIV and Hepatitis viruses
screening.
Advantage: Easy interpretation and stable reaction.
Disadvantage: Possibility of non-specific reaction.

Classification of
Agglutination

Reverse Passive Agglutination

Utilizing particles coated with Antibody.


Coated Abs must face outward so that the active binding
sites are exposed.
The method is designed to detect microbial antigens,
drugs, hormones and certain plasma proteins.
Examples:
Rapid identification of Streptococcus, Staphylococcus and
Neisseria species.
Monoclonal Antibodies are used to avoid cross reactivity.
Advantage: More reliable and easy to interpret
Disadvantage: Cross reactivity and possibility of false
positive reactions.

Classification of
Agglutination

Agglutination inhibition
Reactions are based on competition

between particulate and soluble antigens for


limited antibody binding sites.
The absence of agglutination is indication of
POSITIVE reaction.

Agglutination inhibition and


Hemagglutination inhibition

Agglutination inhibition - Haptens that are


complexed to proteins and attached to a
carrier particle.
Hemagglutination inhibition RBCs are
used as indicator particles.
Patient sample is made to react with
reagent antibody.
The addition of antigen coated particles.

Agglutination inhibition

Classification of
Agglutination

Coaggutination

Using bacteria as the inert particles to which antibody is


attached.
Staphylococcus aureus- most frequently used bacteria.
Protein A absorbs Fc portion of antibodies.
Antibodies are attached facing outward capable of
reacting to specific antigens.
Advantage: More stable than latex particles.
Disadvantage: Less sensitive for detecting small
quantities of antigens.
Test Examples: Detecting Neisseria gonorrhoeae, Vibrio
cholera and Haemophilus influenzae.

Agglutination
Enhancement

Controlling the factors that affects


agglutination reaction is one key to
enhance the reaction.
Low ionic strength saline
Temperature incubation and Ph
Agitation and centrifugation
Increasing Viscosity ( PEG, ALBUMIN)
Addition on Enzymes ( Bromelin, papain,

trypsin and ficin)


Enhancing agglutination by AHG.

Zeta Potential

Anti-human Globulin Test

Types of AHG
Direct AHG (DAT)
Demonstrate in vivo attachment of antibody or complement to individuals RBC.
Autoimmune haemolytic anemia
HDN, HTR and Drug induced haemolytic anemia.

Indirect AHG (IAT)


Demonstrate in vitro attachment of antibody.
Ab+Ag are made to react in vitro.
Washing is required to remove unbound proteins.
Agglutination is enhanced by the addition of AHG that forms linkage to Ab-Ag

complexes.
Crossmatching
Antibody identification
Weak D typing

End. Thank you for your time.

ELECTROPHORESIS

A technique in which molecules with a net charge are


separated when an electric field is applied to the system.
Separation of the molecules occurs only if these particles
have sufficiently different rates of movement in the
presence of an electrical field.
The electric field is applied to a solution via oppositely
charged electrodes that are termed CATHODE (-) and
ANODE (+).
Negatively charged particles will move toward the anode
and Positively charge will move towards the Cathode.

Applications

Separating proteins in biological samples.


Urine
Serum
CSF

Enhancing visualization
Stained
Precipitated
Transferred to another

Medium.

Non soluble support medium.

Agar and Agarose Polysaccharide mixture of agarose and


agaropectin.

Cellulose Acetate Prepared from cellulose treated with acetic


anhydride.

Polyacrylamide Polyacrylamide is a water-soluble polymer made


up of acrylamide subunits. Commonly used in Clinical Labs.
Note: Some medium causes endo-osmosis ( ions in the buffer bind to the

charge groups on the support medium.

Non soluble support medium.

Agarose

Cellulose

Polyacrylamide

Result

Factors Affecting the Migration

Strength of electrical field


Frictional force ( depending on the size of
proteins creating drag)
Depending of the viscosity of the buffer.
Support medium used.

Role of pH in
Electrophoresis

pH of solution can affect the charge on the protein in solution.


Isoelectric point (pI) When the + charges on the proteins, balance
the negative charges. (the protein becomes neutral)
When the pH is equal to PI, the molecule will not migrate.
Different proteins have different pIs.
The pH that is typically used for electrophoresis is the one that
results in an overall negative charge on most of proteins pH 8.0
The proteins will therefore migrate to the anode. The higher the pH
of support the less likely to interact with the support medium.

Role of Temperature

At higher temperatures, the mobility of proteins is increased,


but high temperatures cause proteins to unfold ( denature).

The denaturation of proteins increases frictional forces that


inhibit the migration.

At high temperatures, the buffer solution evaporates causing


an increased osmolality thereby changing the ionic strength
of the buffer.

Summary

Electrophoresis is a technique in which charged molecules are


separated when an electrical field is applied to the system.
The electrical field is applied to a solution via oppositely charge
electrodes that are termed cathode and anode.
Particles that are negatively charged will move toward the anode, while
those that are positively charged move toward the cathode.
Factors that affects the migration includes: Ph of buffer, charge of
molecule, size and shape and strength of electric field.
Support medium includes: Agarose, Cellulose acetate and
Polyacrylamide.
Applications of Electrophoresis includes: Detection of Viral proteins
(HIV) identification and quantification of proteins.

ELISA

( Enzyme Linked
Immunoassay)

A rapid test used for detecting and quantifying


antibodies or antigens and other materials.
In ELISA technology, the solid phase consists of
a 96-well polystyrene plate.
The function of the solid phase is to immobilize
either antigens or antibodies in the sample, as
they bind to the solid phase.
After incubation, the plates are washed to
remove any unbound material.
In some assays the conjugate is then added to
the plate and allowed to incubate.

The conjugate consists of either an antigen or


antibody that has been labelled with an enzyme.
Depending upon the assay format, the
immunologically reactive portion of the
conjugate binds with either the solid phase or
the sample.
The enzyme portion of the conjugate enables
detection. The plates are washed again and an
enzyme substrate (hydrogen peroxide and a
chromogen) is added and allowed to incubate.
Color develops in the presence of bound
enzyme and the optical density is read with an
ELISA plate reader.

ELISA Indirect

Indirect Format:
In the indirect format, the sample antibody is
sandwiched between the antigen coated on the plate
and an enzyme-labeled, anti-species globulin conjugate.
The addition of an enzyme substrate-chromogen
reagent causes color to develop.
This color is directly proportional to the amount of bound
sample antibody.
The more antibody present in the sample, the stronger
the color development in the test wells.
This format is suitable for determining total antibody
level in samples.

Blocking (Competitive) Format

In this format, the specific sample antibodies


compete with, or block, the enzyme-labeled,
specific antibody in the conjugate.
The addition of an enzyme substratechromogen reagent causes color to develop.
This color is inversely proportional to the
amount of bound sample antibody.
The more antibodies present in the sample,
the less color development in the test wells.

Antigen-Capture (Direct) Format:

In the antigen-capture format, the antigen in


the sample is sandwiched between antibodies
coated on the plate and an enzyme-labeled
conjugate.
The antibody conjugate can be either
monoclonal or polyclonal.
The addition of an enzyme substratechromogen reagent causes color to develop.
This color is directly proportional to the amount
of the target antigen present in the sample.

Materials

An ELISA is a set of standardized reagents


and microwell plates manufactured for a
specific test.
Coated Plates The 96-well plates are made
of polystyrene and coated with either
inactivated antigen or antibody.
This coating is the binding site for the
antibodies or antigens in the sample.
Unbound antibodies or antigens in the
sample are washed away after incubation.

Sample Diluent Most assays require a specific


dilution of the sample.
Samples are added to the sample diluent and
mixed prior to putting them onto the coated
plates.
Controls The positive control is a solution that
contains antibody or antigen.
The negative control is a solution without
antibody or antigen.
The controls help to normalize or standardize
each plate.
Controls are also used to validate the assay and
to calculate sample results.

Conjugate ELISA conjugates are enzyme-labeled antibodies or


antigens that react specifically to plate-bound sample analytes.
Unbound conjugate is washed away after incubation and before the
addition of substrate.

The optical density of the colorimetric substrate is directly


proportional to the quantity of bound enzyme present.

Substrate For peroxidase conjugates, the substrate is a mixture of


hydrogen peroxide and a chromogen that reacts with the enzyme
portion of the conjugate to produce color.

Wash Concentrate The wash concentrate is a buffered solution


containing detergent used to wash away unbound materials from the
plates.

Stop Solution The stop solution stops the enzyme-substrate reaction


and, thereby, the color development.

Materials

ELISA

( Enzyme Linked
Immunoassay)

Reading of Plates

Calculations

Reference

https://www.idexx.com/pdf/en_us/livesto
ck-poultry/elisa-technical-guide.pdf