IMMUNOLOGY

HISTORY OF IMMUNOLOGY
 430 BC- Thucydides recorded that individual who had previous contracted the disease recovered
 1000 AD- Chinese practiced a form of immunization
 15th century- powdered smallpox crust were inserted with a pin into the skin

SIGNIFICANT MILESTONES IN IMMUNOLOGY

LOUIS PASTEUR- Father of Immunology

DATE SCIENTIST DISCORVERY

1718 Lady Montagu Injecting material fromcrusts or fluids from small pox
blisters
1798 Edward Jenner Demonstrated cross immunity. Smallpox vaccination
1862 Haeckel phagocytosis
1880-1881 Pasteur live, attenated chicken cholera and anthrax vaccines
1883-1905 Ellie Metchnikoff cellular theory of immunity through phagocytosis
1885 Pasteur therapeutic vaccination,first report of live attenuated
vaccine for rabies
1890 Emil Von Behring, Kitsata humoral theory of immunity proposed
1891 Robert Koch demonstration of cutaneous (delayed-type)
hypersensitivity
1894 Jules Bordet discovered complement
1894-1895 Pfeiffer Buchner Complement mediated cytolysis
1896 Gruber and Durham 1iscovered agglutination reaction
1896 Ferdinand Widal devised an agglutination reaction for the diagnosis of
typhoid fever
1897 Robert Kaus discovered precipitins
1900 Paul Erlich antibody formation theory
1900 Karl Landsteiner Discovery of the ABO blood group
1901 Emil Von Behring Discovery of serum therapy or serum antitoxins
1902 Charles Richet and P. Portier Described anaphylaxis
1903 Maurice Arthus Described arthus reaction
1903 Von Pirquet and Schick Described serum sickness
1903 George Snell Worked out the genetics of the murine major
histocompatibility complex and generated the
congenic strains needed for its biologic analysis
1903 White and Douglas Demonstrated that acquired immunity resulted from
both humoral and cellular elements; they also
decribed opsonization
1903 Almoth Wright Demonstrated immune response involving both
cellular and humoral immunity
1905 Robert Koch Demonstrated cellular immunity in TB
1906 Von Pirquet and Schick Described the relationship of immunity and
hypersensitivity
1906 Jean Dausset An early pioneer in the study of MHC and HLA

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 1911 Neil Jerne Developed hemolytic plaque assay and several
important immunological theories including an early
version of clonal selction
1920 Prausnitz and Kustner Described the immunologic basis of some allergy
1920 Jules Bordet Received nobel prize for his pioneering works on
complement
1920 Baruj Benacerraf Discovered immune response genes and collaborated
in the first demonstration of MHC restriction
1920-1985 Rodney Porter Worked out the polypeptide structure of the antibody
molecule laying out the groundwork of its analysis in
protein sequencing
1921 Albert Calmette and Camille First successful vaccine against tuberculosis
Guerin
1924-1926 Heidelberg Introduced precipitin analysis
1928 Alexander Flemming Discovered penicillin
1929 Gerald Edelman Discoveries about the structure of Ig including first
complete sequence of Ab molecule
1932 Gerhard Domagk Developed Prontosil an agent against Streptococcus
1938 Marrack Hypotheis of antigen-antibody binding
1944 Medawar Described immunologic processes in transplantation
1949 Salk and Sabin Development of polio vaccine
1951 Reed Vaccine against yellow fever
1952 Bruton Described hypogammaglobulinemia
1953 Grabar and Williams Introduced immunoelectrophoresis
1957 Alic Isaacs and Jean Discovered interferon
Lindemann
1957 Frank Mcfarlane Burnet Introduced clonal selection theory
1972 Gerald Edelman and Rodney Structure of the antibodies
Porter
1975 Kohler and Milstein Monoclonal antibodies production using hybridoma
technology
1977 Rosalyn Yalow Radioimmunassay
1986 Mossmann Th1 versus Th2 model of T helper function
1978-1987 Susumo Tonegawa Discovery of principles underlying generation of
antibodies with different specificities (antibody
diversity)
1980 Luc Montagier Isolated a retrovirus from a non-immune deficient
homosexual man with lymphadenopathy and called
the virus lymphadenopathy associated virus (LAV)
1980 Robert Gallo Renamed retrovirus as Human Immunodeficiency
virus
1980 George Snell, Jean Dausset MHC
and Barul Benaceraf
1984 Niels Jerne Immunoregulation
1984 Discovery of T-cell receptor genes
1987 Schwartz Discovered the Major Histocompatibility complex’s
importance in antigen presenatation

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 1991 Edward Thomas and Joseph Transplantation
Murray
1996 Peter Doherty and Rolf Described the process of how cytotoxic T cell
Zinkernagel recognize virally infected cells
2005 Frazer Development of Human Papilloma Vaccine
2008 Francoise Barre- Sinoussi, Luc HIV
Montagnier

IMMUNOLOGY- study of the molecules, cells, organs and systems responsible for the -
recognition
_______________ and ______________
disposal of foreign material.

THE IMMUNE SYSTEM

 PRIMARY LYMPHOID TISSUE
o THYMUS- primary or central lymphoid tissue responsible for processes of lymphocytes
into the T type of cell. This ductless glandlike structure is located beneath the sternum
o BONE MARROW- spongy material inside bones that contains hematopoietic tissues and
is the main source of hematopoietic stem cells, which develop into erythrocytes,
granulocytes, monocytes, platelets and lymphocytes

 SECONDARY LYMPHOID TISSUE

o LYMPH NODES- act as a _______________
Lymphoid filter in the _________________
Lymphatic system
o SPLEEN- act as a __________________
Lymphoid filter within the ____________________
Blood vascular tree
o GUT- ASSOCIATED LYMPHOID TISSUE- it includes lymphoid tissue in the intestines
and the liver.
- Important for the development of tolerance to ingested
antigens
o THORACIC DUCT- thoracic duct lymph is a rich source of ________________. mature T cells
o BRONCHUS-ASSOCIATED LYMPHOID TISSUE- includes tissue in the
______________________
lower respiratory tract and hilar lymph nodes
o SKIN-ASSOCIATED LYMPHOID TISSUE- antigens introduced to skin are presented
by _________________________,
epidermal langerhans cells which are bone marrow- derived accessory cells
o BLOOD- circulating blood has enough mature T cells to produce a graft-versus-host
reaction important lymphoid organ and immunologi effector tissue
most frequently sampled lymphoid tissue: Blood
CELLS OF THE IMMUNE SYSTEM
 LYMPHOCYTES- specialized cells that ______________
capture and _______________
display microbial
antigen and effector cells that eliminate microbes

PRINCIPAL FUNCTION OF LYMPHOCYTES

1. Specific ____________
recognition of antigens
2. _____________
Capture of antigens for display to lymphocytes
3. _____________
Elimination of antigens

ROLE OF IMMUNE SYSTEM

1. Defending the body against infections
2. Recognizing and responding to foreign antigens
3. Defending the body against the development of tumor
Immunocompromised- may underlying condition
Immunosuppressed- medically induced

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COMPOSITION OF IMMUNE SYSTEM
 HUMORAL IMMUNITY- _______________________________________________________
Soluble substances: antibodies, salivary enzymes, lysozymes

 CELLULAR IMMUNITY- _______________________________________________________

Cells: EBCs and APCs

BODY DEFENSES: RESISTANCE TO MICROBIAL DISEASE

 INNATE/NON-ADAPTIVE/NON-SPCIFIC-
___________________________________________
natural means, present at birth, early stages of defense
 ACQUIRED/ADAPTIVE/SPECIFIC-
_________________________________________________
occurs following encounter, adapts depending on the type of antigen

COMPARISON OF TYPES OF ACQUIRED IMMUNITY

TYPE MODE OF ANTIBODY DURATION EXAMPLE
ACQUISITION PRODUCED OF IMMUNE
BY HOST RESPONSE
natural infection yes long natural disease process
ACTIVE
Antigen artificial vaccination yes long COVID vaccine
tetanus toxoid
transfer in vivo; antibodies transmitted
natural colostrum no short
PASSIVE from mother
Antibody artificial vaccination; infusion anti- HBS, anti- tetanus
of serum/plasma no short HBiG; Rhogam/ RhIg

toxoid- modified subject by heating

INNATE IMMUNITY
 FIRST LINE OF DEFENSE
 Body fluids, specialized cells, fluids and resident bacteria which allow the respiratory,
digestive, urogenital, integumentary, and other systems to defend the body against
microbial infection

I. ANATOMICAL or PHYSICAL BARRIERS

II. RESIDENT FLORA

Skin- S. epidermidis
Oral cavity- Viridans streptococci
Intestines-Bacteroides sp. (90%), 10% e. coli
Vagina- Lactobacillus acidophilus

III. HEREDITARY OR GENETIC INFLUENCES

Interspecies differences
Racial or individual differences
Blood phenotypes
Inherited HLAs

 SECOND LINE OF DEFENSE

 Activated once microorganisms penetrate the 1 st line of defense. It includes phagocytic
cells, complement and acute inflammatory reaction.

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 CELLULAR COMPONENTS

I. MAST CELLS AND BASOPHILS

 Less than 1% of the WBC population
 Contain histamine, heparin and eosinophil chemotactic factor-A
 Cell involved in Type I hypersensitivity
 Release inflammatory mediator such as ________________________
Histamine
 Mast cell resemble basophils but contain ACP, ALP and protease
II. EOSINOPHIL
 1 to 3% of circulating WBC
 Capable of phagocytosis but lack digestive enzyme
 Increases during allergic reactions and parasitic disease
 Neutralize basophil and mast cell products and killing certain parasites
 Primary granules
degradation product of eosinophils: Charcot Leyden crytals- evident in type 1
 ACP and Arylsulfatase hypersensitivity such as ASTHMA
 Secondary granules what type of infection can u see the highest increase in eosinophil: T. spiralis
 MBP, eosinophilic cationic protein, eosinophilic peroxidase, eosinophil derived
neurotoxin, phospholipase, histaminase, aminopeptidase and ribonuclease
III. PHAGOCYTES
 Examples are the neutrophils and monocyte-macrophages
 Functions- phagocytosis and antigen presentation
 Process involved in phagocytosis
i. INITIATION
ii. CHEMOTAXIS
 Process by which cells tend to move in a certain direction under the stimulation
of chemical substances Chemoattractants/ tour guides of phagocytes: C5a and C8a
 Cells are guided to the site of injury by __________________________
CHEMOTACTIC AGENTS/ CHEMOATTRACTANTS
 Chemotactic response can be positive or negative
 Without chemotaxis, cell motion is _____________________________
RANDOM
 Ameboid movement of cells such as monocytes and polymorphonuclear
neutrophils to a site of inflammation is called ______________________
DIAPEDESIS
 _________________________-
Job's syndrome impaired chemotaxis of WBC
 __________________________-
Lazy leukocyte syndrome impaired random movement and chemotaxis of
WBC
iii. RECOGNITION
 Direct recognition via Primitive Pattern Recognition Receptors (PPRR)
o WBCs have PPRR
o PPRR van recognize primitive patterns also known as PAMPS/
____________________________________________________
Pathogen Associated Molecular Patterns

 Indirect recognition via opsonization

o Use of opsonins to coat pathogen
o Opsonins include- _____________________________________
C3b, CRP, Immunoglobulins
 OPSONIZATION processes
1. Antibody attached to the surface of bacteria bins FC phagocyte
receptor
2. Complement C3b attaches to the surface of the bacterium and
loosely binds to pahocyte C3b receptor
3. Both antibody and C3b attaches to the surface of the bacterium
and bound tightly to the phagocyte
opsonins are like sprinkles to coat pathogen

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 iv. ENGULFMENT or INGESTION
 Once the phagocytic cell has arrived at the site of injury, the bacteria can be
engulfed through active membrane invagination.
 Formation of phagosome
 Fusion of phagosome and lysosome to form __________________________ PHAGOLYSOSOME
v. DIGESTION or DESTRUCTION Phagolysosome contains digestive enzymes of the phagocytes

 Phagocyte granules contain degradatory enzymes such as

Chronic Granulomatous disease 1. Primary or azurophilic, granules containing enzymes. most important enzymes
- impaired oxidative burst because if MPO is damages, oxidative burst
kinain lang cell pero di na digest Example- _______________________________________________
Myeloperoxidase, lysozymes is impaired
2. Secondary, or specific, granules containing substances
Example- _______________________________________________
Lactoferrin
3. Tertiary granules containing substances
Example- ________________________________________________
Caspases

IV. NEUTROPHILS
 Associated with __________ ACUTE types of infection
 50-70% of WBC population; Most PREDOMINANT
 Marginating pool- ________ 50% walls of blood vessels; di nag cicirculate
 Circulating pool- _________ 50%
V. MONOCYTE-MACROPHAGES
 Associated with __________ CHRONIC types of infection
 Largest cell in the peripheral blood largest cell in bone marrow: megakaryocyte

 Macrophages are __________________________

TISSUE MONOCYTES
 MACROPHAGES ACCORDING TO THEIR TISSUE LOCATION
 Alveolar macrophages Lungs
 Kupffer cells Liver
 Microglial cells Brain
 Mesangial cells Kidney
 Histiocytes Connective tissue
 Lymph node macrophage other name: most potent and efficient APC---- has 5 protrusions---can present multiple
antigens--DENDRITIC CELLS
 Splenic macrophage Littoral cell
 Placental macrophage Hauffbauer cell
 Skin macrophage Langerhans cell
 Receptors found on macrophages and neutrophils
 FcγR IgG
 CR1 Receptor for C3B
 CRP-Receptor
VI. DENDRITIC CELLS
 Cell covered with long membranous extensions
 Phagocytize antigen and present it to T-helper cells
 _________________ MOST phagocytic cell in the tissues

 DENDRITIC CELLS NOMENCLATURE

 Skin and mucous membranes-______________________________________
Langerhans cell
 Heart, lungs, liver, kidneys and gastrointestinal tract-_____________________ Interstitial dendritic cells
 Secondary lymphoid tissues and thymus-_______________________________
Interdigitating dendritic cells
VII. NATURAL KILLER CELLS Kills virally infected cells and tumor cells even without sensitization
 5-15% of total lymphocyte population
 Positive for- ______________________________
CD16 and CD56 NK cells
no B cell and T cell markers
 ______THIRD population of lymphocytes null lymphocyte/ large granular lymphocyte
part of innate immunity

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VIII. ANTIGEN-PRESENTING CELLS Dendritic cells, macrophage, B cells
 Collective term used to describe cells that present antigens either to the CD4 or CD8
positive cells though MHCs or HLA

HUMORAL COMPONENTS

I. COMPLEMENT C1- C9
 Proteins which is considered to be the __________________ MAJOR HUMORAL component of natural
immunity Major humoral component of ADAPTIVE immunity: Antibodies

II. LYSOZYMES
III. INTERFERON -cytokines produced by T cell and other cell lines that inhibit viral synthesis or act as immune regulator
; or simply interfere viral replication
Innate  __________-
ALPHA treat Hepatitis C, Kaposi’s sarcoma, leukemia and lymphomas
- Produced by null lymphocytes, anti-viral and activates NK cells
Innate  __________-
BETA treatment of Multiple Sclerosis
- Derived from dsRNA-induced fibroblasts
Adaptive  __________- derived from lymphocytes (T cells), responsible for immunoregulation
GAMMA
IV. TUMOR NECROSIS FACTOR
 A cytokine that can destroy tumor cells
 First isolated in tumor cells
 Against gram negative organisms; from LPS-activated phagocyte
V. BETA-LYSINS
 Heat-stable cationic substance released by platelets
 Bactericidal for gram-positives
VI. INTERLEUKINS
 Cytokine or chemical messenger produced by leukocytes that affect the inflammatory
process through an increase in soluble factors or cells
 Some interleukines produced by macrophages and monocytes
 IL-1= induces fever; proinflammatory
 IL-6= induces production of CRP
 IL-8= potent stimulator of neutrophils in chemotaxis
 IL-12= enhances NK cell function
 Other types of Interleukins
 IL-2= initiate proliferation or clonal expansion of activated T cells
 IL-3= principally promotes the growth of early hematopoietic cell lines
 IL-4= growth factor for early activation of resting B cells that influences the
synthesis of some immunoglobulins
 IL-5= not active in early lymphoid cells
 IL-7= stimulates early B cell progenitor cells
 IL-9= __________
POTENT lymphocyte growth factor
 Il-10= inhibits cytokine synthesis in various cells
 IL-11= regulator of hematopoietic stroma that stimulates the production of
____________
megakaryocyte and __________________________.
myeloid progenitors
 IL-13= similar to that of IL-4
 IL-14= B cell growth factor
 IL-15= biologically similar to IL-2
 IL-16= acts as a T cell ______________
CHEMOATTRACTANT and participates in the regulation of
many cytokines
 IL-17= increased production due to histamine and serotonin
 IL-18= synergist with IL-12 in some of their effects, especially in the induction
of IFN-γ production and inhibition of angiogenesis

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  IL-19= similar to that of IL-10; it regulates the functions of macrophages and
suppresses the activities of T helper cells
 IL-20= plays an important role in skin inflammations
 IL-21= regulates hematopoiesis and immune response and influences the
development of lymphocytes
 IL-22= similar to IL-10 but does not prevent the production of proinflammatory
cytokines through monocytes
 IL-23= cytokine that shares some in vivo functions with IL-12, including the
activation of STAT-4
 IL-25= secreted bone-marrow stroma-derived growth factor; also called SF-20
VII. INFLAMMATION
 Tissue reaction to injury caused by physical or chemical agents, including
microorganisms.
 Involves the cellular and vascular responses by the tissues of the body in response to
injury
 CARDINAL SIGNS OF INFLAMMATION
o ______________-
CALOR heat production Increased blood flow, presence of blood contents, transfer of internal
heat,
o ______________-
TUMOR swelling
increased capillary permeability causing extravasation of blood fluid
o ______________-
RUBOR redness capillary and arteriolar dilatation with increased rate of blood flow
capillary and arteriolar dilatation with increased rate
of blood flow towards the site of injury
o ______________- pain
DOLOR towards the site of injury
Overcrowding-- naiipit rin nerve endings, nagkaroon pressure, nerve
o ______________-
FUNCTIO LAESA loss of function endings are sensitive to pain
damaged normal cells
 Primary response in acute inflammation is ______________________________
LOCALIZED VASODILATION

 ACUTE PHASE REACTANTS

 Group of glycoproteins associated with non-specific inflammation of body
tissues
 Innate body defense and is a nonspecific indicator of an inflammatory process
 MOST POTENT acute phase protein- ____________________________
C- REACTIVE PROTEIN
ESR- obsolete o APRs with 1000x increase- _____________________________
CRP and SERUM AMYLOID A
o APRs with 2x or 5x increase- ____________________________
ALPHA-A- ANTITRYPSIN and FIBRINOGEN
o APRs with 2x increase- _________________________________
CERULOPLASMIN and C3
 Main biological sign of inflammation- ____________________________
increased ESR
 _______________
ALBUMIN is a negative acute phase reactant
NEG- lahat mag iincrease except albumin
 Final stages of inflammatory process include __________ RESOLUTION and __________ REPAIR
 End product of inflammatory response
o complete repair and total restoration of function
o formation of an abscess with some loss of function
o granuloma formation

ADAPTIVE IMMUNITY

CHARACTERISTIC OF TWO TYPES OF ADAPTIVE IMMUNITY

HUMORAL-MEDIATED CELL-MEDIATED IMMUNITY
IMMUNITY
MECHANISM antibody mediated cell mediated

CELL TYPE B cells T cells

MODE OF ACTION presence of antibodies in serum direct cell to cell contact or soluble products secreted by cells
(cytokines)
PURPOSE primary defense against bacterial infection defense against viral and fungal infections, intracellular organisms
tumor antigens and graft rejections

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 THIRD LINE OF DEFENSE
o It is a more recently evolved mechanism that allows the body to __________, RECOGNIZE
REMEMBER RESPOND
___________, and ____________ to a ___________ SPECIFIC stimulus, an antigen.
o Can result in the elimination of microorganisms and recovery from disease and the host
often acquires a _______________________.
SPECIFIC IMMUNOLOGIC MEMORY

 COMPONENTS OF ADAPTIVE IMMUNITY

o Cellular- _________________________________________________
B AND T LYMPHOCYTES
o Humoral- ________________________________________________
CYTOKINES (secreted by products of T cells) AND ANTIBODIES

 TYPES OF LYMPHOCYTE
o T- CELLS
 Cell responsible for cellular immune response and involved in the regulation of
antibody reactions
 60-80% of total lymphocytes
 _____________________
CELL-Mediated immunity can be transferred to a normal individual with
lymphoid cells from a hypersensitive individual
 T CELL MATURATION
1. _________________________-
DOUBLE NEGATIVE STAGE no CD4 and CD8
2. _________________________-
DOUBLE POSITIVE STAGE CD4 and CD8 positive
3. _________________________-
MATURE T CELLS CD4 or CD8 positive
_______
2/3 of the T lymphocytes are CD4
_______
1/3 of the T lymphocytes are CD8
Th1= _________________________
against INTRACELLULAR antigens
Th2= _________________________
against EXTRACELLULAR antigens
4. _________________________-
ACTIVATED T CELLS/ memory T cell CD25 positive
 Some cytokines produced by T cells
1. _______-
IL-2 T cell growth factor
2. _______-
IL-3 multicolony stimulating factor
3. _______-
IL-5 activates eosinophils
4. _______-
IL-12 increases cytolytic activity of NK cells
 T cell Markers
1. _______-
CD2 receptor that reacts with sheep red blood cells to form
rossettes
2. _______-
CD3 mature T cell marker; part of T cell antigen receptor
3. _______-
CD4 helper/ inducer T cells
4. _______-CD8 cytotoxic and suppressor T cells
5. Normal ratio of CD4:CB8 _______ 2:1
6. Ratio of CD4:CD8 in HIV infection _______ REVERSED
o B CELLS
 Lymphocyte subset type that secretes antibody; the humoral element of adaptive
immunity
 Humoral immunity can be transferred to a normal individual with a serum from a
hypersensitive individual
 20-30% of total lymphocytes
 B CELL MATURATION
1. ______________-
Pro- B cell CD19, CD45R, CD24
2. ______________-
Pre- B cell CD19, CD45R, CD24, mu chains in cytoplasm
3. ______________-
Immature B cell CD19, CD45R, CD24, IgM molecules on the surface,
monomer
CD21, CD40, MHC Class II
Peyer's Patch- for the development of pre b cell

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 4. ______________-
Mature B cell CD19, CD45R, CD24, IgM and IgD molecules on the
surface, CD21, CD40, MHC Class II
5. ______________-
Activated B cell added marker CD25
6. ______________-
Plasma cell produces antibodies, non-dviding
7. MEMORY CELLS are long lived T or B cells that have been stimulated
Hallmark of B cell- IgM and IgD by a specific and recall prior antigen exposure

COMPARISON OF T and B CELLS

T CELLS B CELLS
Developed in thymus developed in the bone marrow

60-80% 20-30%

Identified by rosette formation with sheep RBCs through CD2 Identified by surface immunoglobulins
End products of activation are cytokines End products of activation are antibodies
Antigens include CD2, CD3, CD4, CD8 Antigens include CD19, CD20, CD21, CD40, and MHC Class II

Located in the paracortex of lymph nodes Located in the cortex of lymph nodes

 LYMPHOCYTE ACTIVATORS
o Antigens monoclonal activators
o Superantigens oligoclonal activators
o Mitogens polyclonal activators
lectin- substances that are extracted from  Mitogens that activate B cells _________________
LIPOPOLYSACCHARIDE
plants/animals
 Mitogens that activate T cells________________________________
ConA and PHA lectin ( from extracts of jack beans)

 Mitogens that activate both T and B cell_____________________________

POKEWEED mitogen ( from legumes)
concavalin A and Phytohemaglutinin lectin
SURFACE MARKERS ON T CELLS AND B CELLS
CELLS ASSOCIATED COMMENTS
CD2 T cells Sheep RBC receptor
CD3 T cells Part of the T-cell antigen receptor
CD4 Helper T cells Co-receptor for MHC class II;
receptor for HIV
CD5 Mature T cells CD marker for CLL
CD7 T cells
CD8 Cytotoxic/ Suppressor T cell Co-receptor for MHC class I
CD10 B and T cell precursor Marker for pre-B CALLA; Burkitts
and follicular lymphoma
CD16 NK cells Marker of natural killer cell
CD19 B cells
CD20 B cells
CD21 B cells Receptor for C3d and EBV
CD22 B cells CD marker of HAIRY CELL
LEUKEMIA
CD23 B cells Regulation of IgE synthesis; CD
marker for CLL

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 CD25 Activated T and B cells Receptor for IL-2
CD34 Stem cell Hematopoietic stem cell marker
CD45 WBCs CD marker of all white blood cells
CD56 NK cells Marker of natural killer cell and
abnormal plasma cell

ANTIGENS
 A foreign substance that can stimulate the production of antibodies
ANTIGENICITY
 Ability of an antigen to stimulate an immune response
EPITOPES
 A single antigenic _______________.
DETERMINANT It is functionally the portion of an antigen that combines
with an antibody
FORMS OF ANTIGENS
 ______________-
IMMUNOGENS are antigens capable of stimulating a host’s immune system
 ______________-
HAPTENS very small molecule that can bind to a larger carrier molecule and behave as an
antigen
 AUTOLOGOUS ANTIGEN also known as autoantigen; refers to the host’s own antigen
______________-
 ______________-
ALLOANTIGENS antigens from other members of the host’s species
 ______________- an antigen that induces an antibody production and reacts specifically with it
HOMOLOGOUS ANTIGEN

HETEROLOGOUS
______________-
ANTIGENSan antigen that reacts with an antibody it did not induce, causing cross-reaction
 ______________-
IMMUNIZATION a process of exposing the body to specific antigens to stimulate immunity
Sequelaes strep reaction-- Heterologous
PSAGN- post streptococcal acute glumerulonephritis
PHYSICAL NATURE OF ANTIGENS ARF- Acute rheumatic heart fever
 FOREIGNESS The more foreign, the more antigenic
oIs the degree to which antigenic determinants are recognized as nonself by an
individual’s immune system.
 CHEMICAL COMPOSITION
o ____________________˃______________________˃_______________________
PROTEINS POLYSACCHARIDES LIPIDS AND NUCLEIC ACID
 MOLECULAR WEIGHT
o Bigger molecules are immunogenic
o ˃10,000 daltons
o More than 10,000 da but less than 60,000 da are _______________________
Weakly immunogenic
 DEGRADABILITY
o Antigens must not be easily disintegrated or destroyed by host’s immune system
 STRUCTURAL STABILITY
o Antigen structure should be stable
 COMPLEXITY
o The more complex an antigen, the greater is its effectiveness
o Complex proteins are better antigens than large repeating polymer of substances
 GENETIC COMPOSITION
o HLA A9- high response to tetanus toxoid
o HLA A5- low response to tetanus toxoid
 ROUTE and DOSAGE
o Intravenous and intraperitoneal are effective
o Intradermal is more effective than subcutaneous or intramuscular
o Higher dose of exposure= greater immune response

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ADJUVANTS -precipitate substances leading
 Is a substance, distinct from antigen, that enhances T cell activation by; to increase in size and prolongs
persistence
o Promoting accumulation of APCs at the site of antigen exposure -activates the macrophages enhancing
phagocytosis
o Enhancing the expression of co-stimulators and cytokines by APCs -stimulates the local inflammatory process
o Present in antigen preparations of vaccine
o Includes- ________________________________________________________
alum precipitate, squalene, MF-56, Freunds complete adjuvant
ANTIGEN RECOGNITION
 By cells of innate immunity
o _________________________
Direct recognition

o _________________________
Indirect recognition

 By cells adaptive immunity

o _________________________
Via B cells
o _________________________
Via T cells
MAJOR HISTOCOMPATIBILITY COMPLEX
 Highly polymorphic genes encoding the proteins that regulate immune response
 Genes found in the ___________________________
short arm of Chromosome 6

 Codes for the production of proteins known as _______ HLA

HUMAN LEUKOCYTE ANTIGENS

 MHC gene products
 Known as HLA since they were first found in WBCs
 Often used as a molecular basis for T cells to discriminate self from non-self
 SIGNIFICANCE
o Can be associated with diseases
 Addison’s disease- __________
HLA- B8

 Ankylosing Spondylitis- ______HLA-27

 Rheumatoid Arthritis-_______
HLA-DR4
 Reiter’s syndrome- ________
HLA-B27
 Graves’ disease- ________
HLA-B8
 SLE, Autoimmune Thyroid disease, Dermatitis Herpetiformis- _______
HLA-DR3

 Yersinia arthritis- ________

HLA-B27
 Salmonella arthritis- _________
HLA-B27
 Sjogren’s syndrome- _________
HLA-Dw3
 Adult rheumatoid arthritis- ________
HLA-Dw4
 Autoimmune thyroiditis-________
HLA-Bw35
 Anterior uveitis- ________
HLA-B27
 Myasthenia gravis- ________
HLA-B8
 Multiple sclerosis- ________
HLA-B7

o For PATERNITY TESTING

o For anthropologic marker
o An important factor for the success of organ transplantation
o Involved in antigen presentation
 CLASSES OF MHC MOLECULES OR HLA
CLASS I can be found on APC CLASS II cannot be found on CLASS III
most nucleated cells
specific only for APC's
Loci HLA-A, HLA-B, HLA-C HLA-DN, DO, DP, DQ, DR Complement components

Distribution most nucleated cells APC's serum

Function present antigen to CD4 NO ROLE FOR ANTIGEN

present antigen to CD8
PRESENTATION

 FAB
HISTOCOMPATIBILITY TESTING
 HLA typing
o Test by which the HLA antigens or genes in a candidate or donor is identified
o Test can either be HLA _______________
PHENOTYPING or HLA ______________
GENOTYPING
 HLA PHENOTYPING
o Principle- ________________________________________________
COMPLEMENT DEPENDENT CYTOTOXICITY
o Panels of antisera that define the individual are incubated with _______________
LYMPHOCYTES from
the individual to be HLA typed
o ______________________
PURIFIED T CELLS are used for ____________________
HLA CLASS 1 TYPING

o ______________________ are used for ____________________

PURIFIED B CELLS HLA CLASS II TYPING
o After incubation, __________________
COMPLEMENT is added
o End result- _______________
CELL LYSIS
o Vital dye is then added to distinguish live from dead cell microscopically
 HLA GENOTYPING
o Detects specific HLA genes
o Makes use of molecular-based test such as PCR
 HLA antibody screening and identification
o Antibodies to HLA can be detected in candidates and recipients of solid organ
transplantation
o METHODS
 CDC method- panels of lymphocytes with defined HLA phenotypes are
incubated with the patients’ serum
1. Binding is detected by the addition of complement and a vital dye to
assess cell death microscopically
 ELISA- uses purified HLA antigens bound to wells of microtiter plate
1. Patient serum is added
2. Bound antibody is detected using enzyme-labeled AHG reagent
3. Color change is detected spectrophotometrically
 FLOW CYTOMETRY- single analysis is the __________________PREFERRED method for
crossmatching and antibody identification
1. Antibody in the patient serum is incubated with latex bead coated with
HLA antigen
2. Bound antibody is detected using ___________________________
FITC- LABELED AHG REAGENT

TRANSPLANTATION TERMS
TERM DEFINITION

AUTOGRAFT Graft transferred from same individual

SYNGRAFT Graft transplanted between different but identical recipient and donor

ALLOGRAFT Graft between genetically different recipient and donor of the same species relationship between mother and baby

XENOGRAFT Graft between individuals of different species

1st organ that is successfully transplanted using xenograft: Pig's heart

IMMUNOGENICITY OF ORGANS IN TRANSPLANTATION

 Most immunogenic-_______________________
bone marrow, skin, islets of Langerhans, heart, kidney, liver, bone

 Least immunogenic-_______________________
cornea
Avascular and di madaling puntahan ng immune cells

 FAB
 CATEGORIES AND CHARACTERISTICS OF GRAFT REJECTION
Type Time of Tissue damage Predominant mechanism
Hyperacute w/in minutes Humoral
Accelerated 2-5 days Cell-mediated
Acute 7-21 days Cell-mediated
Chronic ˃3 mos Cell-mediated
 Graft - vs- Host disease- immunologic reaction of engrafted cells against host
G vs H- immunocompromised; di na kaya ng host magreject
H vs G- immunocompetent
ANTIBODIES
 CHARACTERISTICS
o May be present on the surface of B cells or secreted by ________________________
PLASMA CELLS
o Antibody determinants are known as _____________________;
PARATOPES they are the binding sites
in the antibody
o Can be separated from other proteins during electrophoresis
o Found in the _________________
GAMMA REGION during electrophoresis
o _____________________
SVEDBERG UNIT of Ig indicates sedimentation rate using analytical centrifuge
o Antibodies according to
 Temperature
Agglutinating antibody (can be seen
 COLD ANTIBODIES- IgM (Reactive at 4C and/or Room temp) 31.Phases in crossmatching
Immediate spin / saline phase
by the naked eye since it is pentamer)- IgM  WARM ANTIBODIES-IgG (Reactive at 37C) 2. 37 C phase
Precipitating antibody- IgG 3. AHG phase - antibody that is directed
IgG- more clinically significant to another antibody
 Occurrence
 NATURAL ANTIBODIES- ABO antibodies
 IMMUNE ANTIBODIES- Rh blood group
 Species which produce them
 AUTOANTIBODIES
 ISOANTIBODIES/ALLOANTIBODIES
 HETEROPHILE ANTIBODIES
 Reaction with antigen
 Agglutinins - antibody involved in agglutination
 Precipitins -antibody involved in precipitation
 Agglutinoids -agglutinating antibodies that are modified by heat
 Hemmagglutinins -antibodies involved in hemagglutination
 Lysins - antibodies capable of cell lysis
 Allergic antibodies antibodies involved with allergens
 Opsonins- ex: IgG
 Neutralizing abs, antitoxins, complement fixing abs, blocking/ inhibitory
abs

 FAB
  In-vitro behavior

IgM COMPLETE IgG INCOMPLETE

SYNONYMS Bivalent; saline acting Univalent; blocking;
coagglutinating
RESPONSE TO Thermolabile Thermostable
TEMPERATURE
ABILITY TO CROSS Cannot cross Can cross
PLACENTA
OCCURRENCE Early in immunization Late in immunization
REACTION Saline acting Albumin acting

 FUNCTIONS
o Neutralize toxic substances
o Facilitate phagocytosis and kill microbes
o Combine with antigens on cellular surfaces; binding would then initiate phagocytosis by
WBCs or lysis by complement action
o Immunoglobulins are considered to be humoral branch of immune response
 PROPERTIES OF ANTIBODY
o ____________
PROTEIN in nature
o With high molecular weight
o Present on different bodily secretions

 STRUCTURE OF IMMUNOGLOBULIN
o MONOMER- basic structural unit of an antibody
 ‘’Y’’ shaped molecule that consist of four polypeptide chains
o Has a pair of ______________
HEAVY CHAIN and a pair of ____________
LIGHT CHAIN
Most predominantly distributed light chain: Kappa
o Light chains are divided into two types- ______________________________
KAPPA and LAMBDA Ratio of Kappa to Lambda: 2:1
o Heavy chains are divided into five types- ________________________________
GAMMA, ALPHA, MU, DELTA, EPSILON

o Each heavy and light chain has

 Variable region located in the ________________________
AMINO TERMINAL END
 Constant region located in the ________________________
CARBOXYL TERMINAL END
o _____________-
HINGE REGION flexible part of the antibody located in the heavy chains made up of proline
o _____________-
DISULFIDE BONDS unity among four peptide chains
 Two types of Disulfide bonds
1. Interchain- connects light chain to heavy chain
2. Intrachain- connects different regions of immunoglobulin
o Each chain has two regions
o DOMAINS- are globular regions on the polypeptide chain
stabilized by intrachain disulfide bonds
 Domains on the Heavy chain- __________________ VH, CH1, CH2, CH3

 Domains on the Light chain- __________ VL, CL

o POLYMER- immunoglobulin consisting of more than a one basic monomer Ex: IgM, IgA
o J-CHAIN- polypeptide chain in which normally holds polymeric Ig
o SECRETORY COMPONENT- a substance attached to polymeric Ig found on secretions IgA
 PARTS OF AN IMMUNOGLOBULIN MONOMER
o Antigen-binding site involves- _____________________________________
1 light chain and 1/2 heavy chain
o Hinge region is between- ________________________________
CH1 and CH2
o Complement binding site- ____________________________________
IgG- CH2; IgM- CH3 IgA- can also activate complement using alternative pathway
o WBC binding site- __________________
Fc receptor

 FAB
  When subjected to enzyme digestion, formation of fragments will ensue;
o Fragment antigen- binding
o Fragment crystallizable
 PEPSIN- hydrolyzes antibody into ___ 2 fragments
 PAPAIN- hydrolyzes antibody into ___ 3 fragments

 IMMUNOGLOBULIN VARIABILITY
o ______________-
ISOTYPE refers to the heavy chain constants region structure associated with
different classes and subclasses
 ______________-
Subclasses are differences in constant region (of heavy chains) present
within an individual
o ______________-
ALLOTYPE refers to the unique, minor variations within the constant region of
gamma and alpha chains, as well as the kappa lambda chains present in some individuals
but not with others
- Are differences in the constant region between
individuals
o ______________-
IDIOTYPES variations in the variable regions that give individual antibody
molecules their specificity to an antigen

CHARACTERISTIC OF IMMUNOGLOBULIN CLASSES

IgG IgM IgA IgD IgE
MOLECULAR WEIGHT 150,000 900,000 160,000 180,000 190,000
SEDIMENTATION COEFFICIENT 7S 19S 7S 7S 8S
HEAVY CHAIN SUBCLASSES 4 0 2 0 0
PERCENT OF TOTAL Ig 70-75 10 10-15 <1 0.002
SERUM CONCENTRATION (mg/dL) 800-1600 120-150 70-350 1-3 0.005
SERUM HALF-LIFE (days) 23 6 5 1-3 2-3
ELECTROPHORETIC MIGRATION γ2- α1 γ1-β12 γ2-β2 γ1 γ1
COMPLEMENT FIXATION YES YES NO NO NO
CROSSES PLACENTA YES NO NO NO NO
IgA can be considered as a complement fixing antibody
if it uses alternative pathway
 TYPES OF ANTIBODIES
o ________
IgG
 Most predominant antibody in the total Ig pool (75-80%); life span- 1-3 weeks
 Has 4 major subclasses
 IgG1Most effective in crossing the placenta 1>4>3 chronological order in effectivity to cross
placenta; no IgG2 because it cannot cross placenta
 IgG2 cannot cross the placenta
 IgG3 most efficient in complement fixation 3>1>2 order of effectivity upon fixing complement; no
 IgG4 do not fix complement IgG4 because it cannot fix complement

 Easily distributed in the different fluid compartments with detectable amounts in

CSF and urine
 Appear later than IgM in primary response but they form the major antibody of
the secondary immune response
 Maternal IgG is actively and selectively transferred across the placenta to the
fetus and imparts passive protection to the newborn for 6-9 months

 FAB
  ROLES- activation of the classical complement pathway, opsonization, mediator
of antibody-dependent cell-mediated cytotoxicity, neutralization of viruses
o IgM
_________
 The largest of the immunoglobulin molecule, 5-10% of the total Ig pool
 Star-shaped in the free state, crab-like once activated
 Earliest antibody to appear in the primary immune response but does not persist
for long kasi pinapalitan na ng IgG
also a useful indicator of intrauterine infection
 Does not cross placenta
 A powerful agglutinator of particulate antigen
 Has 2 subclasses
 Pentameric found in the plasma, it contains j chain
 Monomeric found in the surface of B cells
o IgA
_________
 Represents 15-20% of the total circulatory Ig pool
 Predominant immunoglobulin in secretions such as saliva, tears, colostrum, milk
and intestinal fluids
 Has 2 subclasses
 ________________
MONOMERIC IgA most commonly found in blood cannot activate complement system
 ________________
DIMERIC IgA found in secretions; contains __________________
secretory component
which enables IgA to be transported into the mucosal surface can activate complement system
 Activation of B cells leading to IgA production occurs primarily in the
mucosa-associated lymphoid tissue (MALT)
o IgD
_________
 Heat labile immunoglobulin, found in very low concentrations in plasma,
accounting for less than 1% of the total Ig pool.
 Primarily a cell membrane Ig found on the surface of B lymphocytes in
association with IgM
o IgE
_________
 Previously known as _______________________________
REAGINIC ANTIBODY
 Binds to mast cells, eosinophils and basophils
 For immune response to helminthic infections-when parasite-IgE complex binds
to the FcεR on eosinophils, the eosinophils release major basic protein which is
cytotoxic to helminthes
 Responsible for allergies and anaphylactic shock

 THEORIES OF ANTIBODY PRODUCTION

o EHRLICH’S SIDE CHAIN THEORY
 Certain cells have had specific surface receptors for antigen that were present
before contact with antigen occurred. Once antigen was introduced, it would
select the cell with proper receptors. Combination would take place, receptors
will break off and enter circulation as an antibody molecule. New receptors will
be formed in place of those broken off and this process could be repeated
o THE TEMPLATE THEORY
 Antibody-producing cells are capable of synthesizing a generalized type of
antibody, and when contact with an antigen occurs, the antigen 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
o SELECTIVE THEORY
Watch recorded lec for more detailed explanation

 FAB
 
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 cell rather than from antigen
o CLONAL SELECTION THEORY
 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 ANTIBODY
o Purified antibodies cloned from a single cell
o Uses HYBRIDOMA TECHNOLOGY
o ______________-
HYBRIDOMA cells that have the ability to reproduce indefinitely and synthesize
adv polyclonal antibody
antibody combination of B cell and a myeloma cell-- -multiple ways to attack certain antigen
o Steps kasi kayang mag produce indefinitely -useful in agglutination and immunoprecipitation based methods

1. Immunization of mouse with the antigen of interest Disadv:

-low in concentration
2. Removal of spleen from the mouse -can lead to common producing cross reactions reducing its
specificity and sensitivity because of multiple ways to attack
3. Removal of RBCs from the spleen cells
4. Fusion of spleen cells and myeloma cells
- to remove unwanted
5. Cell mixture is placed on a medium with _______________________________
HYPOXANTHINE-AMINOPTERIN-THYMIDINE combination
6. Identify the hybridoma cell that produces the antibody of interest B cell and B cell
Myeloma and Myeloma
o USES OF MONOCLONAL ANTIBODIES B cell and Myeloma-- this is the
 Identifying and quantifying hormones target combination

 Typing tissue and blood

 Identifying infectious agents
 Identifying clusters of differentiation
 For the classification of leukemias, lymphomas and follow-up therapy
 Identifying tumor antigens and autoantibodies
 Delivering immunotherapy
o ANTIGEN-ANTIBODY INTERACTIONS
 _____________-
AFFINITY is the initial force of attraction that exists between a single Fab
site on an antibody molecule and a single epitope or determinant site on the
corresponding antigen
 _____________-
AVIDITY strength with which a multivalent antibody binds to a
multivalent antigen
 _____________-the
SENSITIVITY frequency of positive results obtained in testing a population
of individuals who are positive for antibody
 _____________-
SPECIFICITY the proportion of negative test results obtained in the population
of individuals who actually lack the antibody in question
 CROSS-
_____________-
REACTIVITY a condition in which some of the determinants of an antigen are
shared by similar antigenic determinants on the surface of apparently unrelated
molecules and a proportion of these antigens interact with the other kind of
antigen Ex: cowpox and small pox
 Antigen-antibody reactions are ________________
REVERSIBLE
o PRIMARY INTERACTIONS
 Hydrophobic interactions such as ____________________________
Van der waals force
 Hydrophilic interactions such as _____________________________
ionic bonds and hydrogen bonds

 DOES NOT include _________________________________

o SECONDARY INTERACTIONS
 Are those that occur between antigens and antibodies when the antigen is
multivalent
 ___________________-
PRECIPITATION
is the term for the aggregation of soluble test antigens.

 FAB
 -It is the combination of soluble antigen with soluble
antibody to produce a visible insoluble complex
 ___________________-
AGGLUTINATION process whereby PARTICULATE antigens aggregate to
form a larger complex in the presence of specific antibody
 Stages of agglutination
o Sensitization/coating
o Lattice formation
o FACTORS THAT AFFECT ANTIGEN-ANTIBODY BINDING
 Antigen- antibody ratio
 ___________________-
ZONE OF EQUIVALENCE point at which the most antibody is precipitated
by the least amount of antigen Ratio should be 2:1 ; 2 antibody,1antigen
 ___________________-
PROZONE antibody in excess, insufficient reactive sites on
antigen for lattice formation
 ___________________-
POSTZONE antigen in excess, insufficient reactive sites on
antibody for lattice formation
 OPTIMUN SERUM TO CELL RATIO in BB- ________ 2:1
 pH
 Optimum pH for antigen-antibody binding-_____________ 6.5-7.5
 Antibody in blood banking that is enhanced by acidic pH- __________ Anti M and Anti P1
 Centrifugation/rotations
 Enhances physical contact between antigen and antibody
 Addition of colloids and enhancement media
 Enhancement media such as 22% albumin, LISS or PEG
o Action of LISS- _______________________________
decrease zeta potential -negative charge of RBC
o Action of PEG-_______________________________
remove water to increase binding of antigen and antibody
 Length of incubation
 A certain amount of contact time must occur for antibody to bind the
antigen
o 22% albumin incubation time- ________ 30-60 mins
o LISS incubation time- _____________ 10-15 mins
 Number of antigens
 The more antigens present on the cell surface, the faster the LATTICE
FORMATION will occur
 Location of antigens
 Binding occurs faster if the antigens are accessible to antibody’s reach
 Rouleaux formation vs True agglutination
 Can be differentiated with the addition of ________ NSS
 True agglutination- ______ INTACT

 Rouleaux formation- __________ DISPERSED

 Type of antibody according to temperature of reacting media
 IgM- ____________________
ROOM TEMPERATURE
 IgG- _____________________
37 C
THE COMPLEMENT SYSTEM
 Is a heat-labile series of 18 plasma proteins, many of which are enzymes or proteinases
 Complement system proteins are named with a capital C followed by a number, a small letter
after the number indicates that the protein is a smaller protein "b"- larger fragment except
C2a is the larger fragment
 Synthesized in the liverexcept C1- that is produce from the intestinal
epithelial cells; Factor D-produce from adipose tissues
 It is the major fraction of the ______________
BETA REGION during serum protein electrophoresis

 FAB
 FUNCTIONS
o Opsonins c3b
o Anaphylatoxins all with letter "a"
o Chemotactic agents c5a and c8a
o Clearance of immune complex and cell lysis MAC- C5-C9
 Complement plays a role in CYTOLYTIC DESTRUCTION
 Complement activity is destroyed by heating sera _____
56 C for ________
30 mins if elapsed for more than 4 hrs=
 ____________ are the only Ig that react with complement
IgG and IgM heat inactivate again at 56 C for 10 mins

o Some antibodies can activate complement, some does not

 ___________
IgM most potent antibody in terms of complement activation
 ___________
IgG3 is the most active IgG in complement activation
 ___________ is the IgG that does not activate the complement
IgG4
o During activation, complement proteins initially bind the ____________
CH2/CH3 of antibodies
COMPLEMENT PATHWAYS IgG IgM

 CLASSICAL PATHWAY
o Antibody directed
 ALTERNATIVE PATHWAYOld name: Properdin pathway; generally abs independent however it can be initiated by IgA
o Bacterial cell wall, fungal cell wall, yeast, viruses, virally infected cells, tumor cell
lines,parasite
 LECTIN PATHWAY/MANNOSE BINDING LECTIN
o Bacteria, yeast, viruses, parasites containing MANNOSE
STAGES OF COMPLEMENT ACTIVATION
 Recognition C1, C3b, MBL
 Activation C2, C4 and C3
 MAC C5- C9

 FAB
CLASSICAL PATHWAY ALTERNATIVE PATHWAY MB LECTIN PATHWAY

Antigen-Antibody Activating surfaces MB lectin binding

complex to pathogen
surfaces

C3 CONVERTASE

C5 CONVERTASE

TERMINAL PATHWAY

 FAB
COMPONENTS OF COMPLEMENT PROTEINS
 __________-
C1q binds to Fc portion of IgM and IgG molecules
 __________- activates C1s
C1r

 __________-
C1s cleaves C4 and C2
 __________-
C4 part of the C3 convertase
 __________-
C2 binds to C4b to form C3 convertase
 __________-
C3 central pivot point and key intermediate in all pathway
 __________- initiates MAC
C5

 __________-
C6 binds to C5b in MAC
 __________-
C7 binds to C5b6 in MAC
 __________-
C8 starts pore formation in membrane
 __________-
C9 polymerizes to cause cell lysis
 __________-
Factor B binds C3b to form C3 convertase C3bBb
 __________-
Factor D cleaves factor B
 __________-
Properdin stabilizes the C3bBb complex
 __________-
MBL binds to mannose; homologous to C1q
 __________-
MASP-2 homologous to C1s; cleaves C4 and C2
 C3 convertase- ___________________
C4b2a

 C5 convertase- ___________________
C4b2a3b
REGULATORY PROTEINS
 _____________-
C1INH inactivates C1 by dissociating C1r and C1s; Inactivates MASP 2
 _____________-
FACTOR 1 cleaves C3b and C4b
 _____________-
FACTOR H cofactor to Factor I to inactivate C3b; prevents binding of B to C3b
 _____________-
C4-BP acts as cofactor with Factor I to inactivate C4b
 S-CHON/
_____________-
VITRONECTIN prevents attachment of C5b67 complex to cell membrane
 _____________-
DAF decay- accelerating factor; (CD55); capable of dissociating C3 convertase
DEFICIENCIES OF COMPLEMENT COMPONENTS
 _____________-
C1 (q,r,s) SLE-like syndrome
 _____________-
C2 and C4 immune complex disorder; SLE-like syndrome
 _____________-
C5 to C8 Neisseria infections
 _____________-
C3 severe recurrent infections, glomerulonephritis
 _____________- hereditary angioneurotic edema
C1 INH

 _____________-
DAF and HRF PNH/ Marchiafava-Micheli
 _____________- Recurrent bacterial infections
FACTOR H and I
 _____________-
C2 deficiency MOST COMMON complement protein deficiency
 _____________-
C3 deficiency MOST SEVERE type of complement protein deficiency

SEROLOGY
SEROLOGICAL TEST
 Are test that uses antigen and antibody interactions as tools for diagnosis
 ____________-
ANALYTES are the substances being assayed in immunoassay
 Antigen and antibody interactions are used to detect and measure the analyte of interest
 Serological tests interpretation
o Qualitative- presence or absence of antigen or antibody
o Semi- quantitative- increase in antibody titer

 FAB
  _________
TITER - measurement of serological reactions increase in titer 4-fold/4x--- clinically significant
- The concentration or strength of an antibody expressed
as the highest dilution of the serum that produces
agglutination
 DILUTION formula- ____________________
sample volume/ total volume Total volume= sample volume plus diluent volume
 Diluent- _____________
0.85% NSS NSS

TYPES OF SEROLOGICAL TEST

 Agglutination
 Precipitation
 Neutralization/inhibition
 Complement fixation
 Labeled immunoassay
 Nucleic acid test

IMMUNOLOGIC REACTIONS
1. Primary Ag-Ab reaction
 Non visible reaction, first union of Ag-Ab
 ELISA, RIA, IFA
2. Secondary Ag-Ab reaction
 Visible reaction
 Precipitation, agglutination, neutralization, complement fixation
3. Tertiary Ag-Ab reaction
 In-vivo Ag-Ab reaction
 Phagocytosis, opsonization, chemotaxis

AGGLUTINATION
 Types of agglutination reactions
o DIRECT AGGLUTINATION- agglutination of __________________
NATURAL ANTIGENS
- Carrier can be RBC’s or bacteria
Example
 FEBRILE AGGLUTININ TEST
 _________________-
WIDAL TEST Detects Salmonella ANTIBODY
 _________________-
WEIL FELIX Detects Rickettsia ANTIBODY
 ABO CELL TYPING

o INDIRECT or PASSIVE AGGLUTINATION- agglutination of ____________________

ARTIFICAL ANTIGEN
- Carriers are coated with antigen that is NOT normally
found on their surfaces Carriers: latex particle, polystyrene, silicate
- Detects ANTIBODY and bentonite
Example
 ASO latex agglutination test detect ASO antibody
Reagent: Streptolysin O in latex particle directed for Streptococcus pyogenes

o REVERSE PASSIVE AGGLUTINATION- uses _______________________________

ARTIFICIAL ANTIBODY
- Carriers are coated with antibodies that are NOT
normally found on their surfaces
- Detects ANTIGEN
Example
 CRP Latex agglutination test

 FAB
 o AGGLUTINATION INHIBITION- blocking principle
 Positive
_____- absence of agglutination
 Negative
_____- presence of agglutination
 Has TWO stages
o 1st- neutralization of antigen upon addition of soluble reagent
antibodies
o 2nd- indicator phase; addition of antigen-coated particles to bind
 Often used when antigen is to be detected on biological fluids
 EXAMPLE- HCG for pregnancy testing watch recorded lec for more detailed explanation

o AHG-MEDIATED AGGLUTINATION REACTION

 A.k.a Antiglobulin test or COOMB’S TEST
 Uses anti-human globulin
 AHG serves as bridge to connect two non-agglutinating antibody
Example- DAT in vivo test of sensitization
 Specimen- RBCs
o Hemolytic transfusion reaction
o Hemolytic disease of the fetus and newborn
o Autoimmune hemolytic anemia
Example- IAT
 Specimen- Serum
o Antibody detection
o Antibody titration
o RBC phenotyping

o VIRAL HEMAGGLUTINATION
 Used in viruses that can be quantified based on their ability to spontaneously
agglutinate red blood cells, such as influenza virus

o HEMAGGLUTINATION INHIBITION
 Similar to agglutination inhibition except that antigen-coated RBC are added
instead of antigen- coated latex particles
 HBsAg detection

o Sources of error
FALSE POSITIVE
Overcentrifugation
Contaminated glassware, slides or reagents
Autoagglutination
Saline stored in glass bottles
Presence of cross-reactivity
Presence of rheumatoid factor
Presence of heterophile antibody
Delay in reading a slide test
FALSE NEGATIVE
Under centrifugation
Inadequate washing of cells, especially in
antiglobulin
Reagents not active
Delays in testing procedures
Incorrect incubation temperature
Insufficient incubation time
Pro zone phenomenon and post zone
phenomenon in AHG test
Failure to add antiglobulin reagent

 FAB
 o COMPLEMENT FIXATION TEST
 Positive result- NO hemolysis
 Negative result- hemolysis
 STEPS
i. Add reagent antigen to serum
ii. Add complement
iii. Add sensitized RBC
iv. Observe for hemolysis/absence of hemolysis
watch recorded lec for more detailed explanation

PRECIPITATION
 Aggregation of soluble test antigens with soluble antibodies
 TYPES OF PRECIPITATION REACTION
o PRECIPITATION TECHNIQUE BY LIGHT MEASUREMENT
 Nephelometry- measurement of _________________________
LIGHT SCATTERED
- For quantitation of _________________
immunoglobulins
 Turbidimetry- measurement of _________________________
LIGHT BLOCKED
- Measures cloudiness of solution
o GEL PRECIPITATION
 Single immunodiffusion
 Only the antigens diffuse; the antibody is incorporated in the gel or vise
versa
 TYPES
o Single linear immunodiffusion- presence of precipitin band
o Single radial immunodiffusion -radial precipitin

Types of radial Immunodiffusion

__________________________-
FAHEY- MCKELVEY kinetic method
o Measurement after ________
18 hrs
o Diameter of precipitation ring is proportionate to log of antigen concentration
__________________________-
MANCINI end point method
o Measurement after ______
24 hrs for IgG to ______
50-70 hrs for IgM

Double immunodiffusion/ Ouchterlony double diffusion

 Both antigen and antibody are free to move passively toward each other
 Antigen is placed on the outer wells; antibodies on the inner well
 Results
o Identity- ____________________________
smooth arc
o Non-identity- crossed/intersecting
_________________________
lines
watch recorded lec

o Partial identity- ________________________

single spur, pointing towards the simpler antigen
o ELECTROPHORESIS
 General steps in electrophoresis
 Electrophoresis/separation
 Staining to visualize bands; amido black, ponceau S, coomasi brilliant blue
 Densitometry to quantify bands; to determine concentration of antigen antibody complex
 Electrophoretic techniques intended for separation
 Examples
o Zone electrophoresis 5 bands- albumin, a1, a2, B, y
o High resolution electrophoresis 12 bands
 ONE- STAGE ELECTROPHORESIS
 Counterimmunoelectrophoresis

 FAB

 TWO-STAGE ELECTROPHORESIS
Immunofixation electrophoresis
Electrophoresis plus immunodiffusion
Abs is overlain in the gel
positive result- precipitin bands
Application in Bence jones protein present in
px urine with multiple myeloma
WATCH RECORDED LEC
o Antigen towards the _________
ANODE
o Antibody towards the _________
CATHODE
o Positive result- _____________
PRECIPITIN LINE
 Rocket electrophoresis
o Also known as immunoelectrophoresis or voltage-facilitated
single immunodiffusion or one-dimension
electroimmunodiffusion or LAURELL ELECTROPHORESIS
o Antibody is incorporated in the gel; only the antigen diffuses
with the aid of electric current
o Positive result- formation of precipitin rockets
Cross immunoelectrophoresis
 Crossed immunoelectrophoresis 1ST: ELECTROPHORESIS
 Immunofixation electrophoresis 2ND: ELECTROPHORESIS/IMMUNODIFFUSION
2 Electrophoresis: Abs is incorporated in the gel
 Classic immunoelectrophoresis Positive result- precipitin rockets

 IMMUNOFIXATION/RESSLER’S TECHINIQUE
Classic immunoelectrophoresis
Electrophoresis plus immunodiffusion
 A cellulose acetate strip impregnated with antiserum is placed over the
Abs is placed in the trough positive result is separate proteins after serum, urine or CSF are electrophoresed.
precipitin arc
 Diffusion of antiserum into the gel occurs rapidly, resulting in
precipitation
 Cellulose acetate strip are then removed and the precipitin bands are
stained
NEUTRALIZATION
 Mixture of an antigen and antibody is injected to a test animal
 Positive neutralization- _______________________
ANIMAL LIVE

 Negative neutralization- ______________________

ANIMAL DIE

 Examples
o Toxin neutralization
 ASO titration
 Animal protection test
 Schick and Dick test
o Virus neutralization
 Pock and plaque reduction
 Metabolic inhibition test
 Tissue culture technique
 In vivo and in ovo test
FLOCCULATION
 A specific type of precipitation that occurs over a narrow range of Ag concentration
 Aggregation of _________________________
COLLOIDAL PARTICLES in a serological reaction
 Example- VDRL, RPR

LABELED IMMUNOASSAY
 Involves antigen and antibody interaction, with either reactant labeled with a marker so that the
amount of binding may be monitored
 Terminologies
o ____________-
LIGAND any substrate that will complex to another substance; substance to be
measured
o _______________-
LIGAND ASSAY one reactant is labeled so that the amount of binding can be measured
o ____________-
LABELS act as marker to detect antigen-antibody reaction
 Characteristics of a good label
 Must not alter the reactivity of the molecule

 FAB
  Should remain stable for the shelf life of the reagent
 Classification of labeled immunoassays
 Enzyme immunoassay Enzyme
 Fluorescence immunoassay Fluorochrome dyes
 Radioimmunoassay Radioisotopes
 Chemiluminescence Chemiluminol
 Electrochemiluminescence Current plus luminol

o LABELD SPECIES
___________________- may be an antigen or antibody to which the label is attached
o ___________________-
SOLID PHASE media such as the well or microplate, to which an antigen or
antibody may be attached to
 Two types of Immunoassay
o __________________-
HETEROGENOUS requires a step to physically separate from unbound analyte
- Involve a solid phase
- Competitive or non-competitive
o __________________-
HOMOGENOUS no separation step is necessary because enzyme activity
diminishes when binding of antibody and antigen occurs
- Liquid phase only
- Faster and easier to automate
 Separation step
o Purpose- provides a simple way to separate bound and free reactants
o Methods used
 Physical means
 Decanting, centrifugation, or filtration
 This is followed by a washing step to remove any remaining unbound
analyte
 Solid-phase vehicle
 Polystyrene test tubes, microtiter plates, glass or polystyrene beads,
magnetic beads and cellulose membranes
 Antigen or antibody is attached by physical adsorption
 When specific binding takes place, complexes remain attached to the
solid phase
 Detection step
EIA- spectrophotometer
o Dependent on the label used RIA- gamma solid scintillation counters
FIA- fluorometers, fluorescent microscope, flow cytometer or spectrofluorometers
CHLIA- spectrophotometer
ENZYME IMMUNOASSAY
 It uses enzyme as ligand and addition of chromogenic substrate produces a colorimetric reaction
 Can be used either for antigen or antibody detection
 Advantages- cheap and readily available, have a long shelf life, are easily adapted to automation,
and measurements can be done using inexpensive equipment; no disposal problems and health
hazards
 Enzymes used
o Acetylcholinesterase Electrophorus electicus
o Alkaline phosphatase E. coli
o Beta-galactosidase E.coli
o Glucose oxidase A. niger
o G6PD Leuconostoc messenteroides
o Lysozymes Egg white
o Malate dehydrogenase Pig heart
o Peroxidase Horseraddish

Recall question:Malunggay- san nanggaling enzyme for immunoassay in PH

 FAB
  FORMS
o NON-COMPETITIVE
- Direct
- Detects- ANTIGEN
- Labeled specie- ANTIBODY DIRECTED TO ANTIGEN
- Solid phase- NONE
- Concentration- DIRECT
- Indirect
- Detects- ANTIBODIES
- Labeled specie- ANTI-IMMUNOGLOBULIN
- Solid phase- ANTIGEN
- Concentration- DIRECT
- Capture/sandwich
- Detects- ANTIGEN
- Labeled specie- ANTIBODIES DIRECTED TO THE ANTIGEN
- Solid phase- ANTIBODIES
- Concentration- DIRECT
o COMPETITIVE
- Detects- ANTIGEN
- Labeled specie- ANTIGEN
- Solid phase- ANTIBODIES
- Concentration- INVERSE
o IMMUNOENZYMETRIC
- Detects- ANTIGEN
- Labeled specie- ANTIBODIES
- Solid phase- UNATTACHED ANTIGENS
- Concentration- DIRECT
FLUORESCENCE IMMUNOASSAY
 Method of localizing antigen by the use of fluorescence
 Fluorescent probes
o FITC- GREEN
o Phycocyanin- RED
o Texas red- RED
o Tetramethyl rhodamine- RED ORANGE
 FORMS
o Single layer immunofluorescence assay
Similar to direct EIA; fluorochrome label

o Double layer immunofluorescence assay

similar to indirect EIA; fluorochrome label

o Immunofluorescence antigen sandwich

similar to sandwich EIA; fluorochrome label

o Immunofluorescence- Complement fixation

 FAB
 -
Similar to indirect except that the globulin conjugate is directed against the
species supplying the complement
o Immunofluorescence inhibition
- Blocking specific Ag-Ab reaction by initial exposure to a different aliquot of
homologous antibody
o Fluorescence polarization immunoassay
- Non classical, two stage, competitive immunoassay
- Detection of illegal and therapeutic drugs
RADIOIMMUNOASSAYS
 First type of immunoassay developed
 Radioisotope labels used- 131 I, 236 I, 3H
 Disadvantages
- Health hazard, difficult and expensive maintenance of license and compliance with
federal regulations, disposal problems, short shelf life, expensive equipment
 Forms of RIA
- Non-competitive (indirect and capture), competitive, immunoradiometric
 Applications
- RIST Radioimmunosorbent test
- RAST Radioallergosorbent test- allergen specific
NUCLEIC ACID TESTS
 Probe amplification methods
- Cleavase/invader technology
 Signal amplification methods
- Branched DNA
- Hybrid capture assays
 Whole genome amplification
 Target amplification
- Polymerase chain reaction
 Reverse-transcriptase polymerase chain reaction
 Nested polymerase chain reaction
 Multiplex polymerase chain reaction
 Digital polymerase chain reaction
 End-point quantitative polymerase chain reaction
 Rapid-cycle polymerase chain reaction
- Transcription based amplification
- Loop mediated amplification
- Helicase dependent amplification
- Strand-displacement amplification
Polymerase chain reaction
- DNA target amplification procedure
- Amplifies tiny quantities of nucleic acid up to detectable levels
- Specimen DNA is isolated from the sample, containing the target sequence
- Reagents-
________________________________________________________________________
MASTER MIX, WATER, BUFFER, NUCLEOTIDES, FORF and REVERSE PRIMER, TAQ POLYMERASE
universal media, maintains pH, building blocks of DNA, starting pt, reverse pt, thermos aquaticus- a heat stable bacteria that thrives in hot
________________________________________________________________________
springs
- STEPS
 Denaturation
Heating of the sample at 95 C
Purpose- separate the dsDNA into ssDNA, denatures the DNA, breaking H bonds that holds the strand together
 Annealing
Cooled at 52 C, primers bind to the complementary sequence on each DNA strands

 FAB
  Extension or Elongation or Synthesis
takes place at 72 C
Heat stable DNA polymerase enzyme binds to each primer to synthesize new strand of dsDNA

Molecular blotting techniques

 Southern blot DNA
 Northern blot RNA
 Eastern blot LIPID
 Western blot PROTEIN

DISEASES OF THE IMMUNE SYSTEM

 HYPERSENSITIVITY
o Normal but exaggerated or uncontrolled immune response to an antigen that can produce
inflammation, cell destruction, or tissue injury

TYPE I TYPE II TYPE III TYPE IV

ANAPHYLACTIC CYTOTOXIC IMMUNE COMPLEX CELL- MEDIATED
Immune IgG and IgM IgG and IgM T cells
IgE, Basophil
mediator
Antigen allergens sensitized antigens
cell bound antigens soluble ag to ins complex
involved
Immune release of inf, mediators cell lysis due to antibody deposition of Ag-Ab complex release cytokines
from mast cells and on walls of organs
mechanism basophils
and complement

Example transfusion reactions,

anaphylaxis, hay fever, serum sickness, arthus tuberculin test;
AIHA, HDFN
food allergies, asthma reaction, SLE contact dermatitis

AUTOIMMUNITY
 Body’s immune system mounts an immune response against the host’s antigens
 Autoimmune disease- damage to tissues or organs due to the presence of
___________________________
AUTOANTIBODIES
 Molecular mimicry- similarity between an infectious agent and a self- antigen that causes
antibody formation in response to the infectious agent to cross-react with self
o Poliovirus VP2- acetylcholine receptors
o Measles VP3- Myelin basic protein
o Papilloma virus E2- insulin receptors
 Autoantibodies and associated autoimmune diseases
o Systemic lupus erythematosus-_________________________________________
Anti-dsDNA, Anti-sm
o Rheumatoid arthritis-_________________________________________________
Rheumatoid factor
o Diabetes mellitus type 1- ______________________________________________
Anti- beta cells, Anti- insulin
o Pernicious anemia- ___________________________________________________
Anti-parietal cell
o Autoimmune hemolytic anemia- ________________________________________
cold and warm autoantibodies
o Hashimoto’s thyroiditis- _______________________________________________
Anti-microsomal, Anti-thyroglobulin
o Grave’s disease- _____________________________________________________
Anti-TSH receptor antibody
o Goodpasture syndrome- ______________________________________________
Anti-glomerular basement membrane
o Wegener’s granulomatis/granulomatosis-_________________________________
Anti-neutrophilic cytoplasmic antibodies
o CREST- _____________________________________________________________
Anti-centromere antibodies
o Addison’s disease- ____________________________________________________
Adrenal glands antibodies
o Multiple sclerosis-____________________________________________________
Anti- myelin sheath antibodies
o Myasthenia gravis- ___________________________________________________
Anti-acetylcholine receptor antibodies
o Primary biliary cirrhosis- _______________________________________________
Anti-mitochondrial antibodies

 FAB
 o Chronic active hepatitis- _______________________________________________
Anti-smooth muscle antibodies
o Idiopathic or immune thrombocytopenic purpura- __________________________
Platelet antibodies
 Tests related to autoimmune disorders
o Rheumatoid arthritis
 Latex tests are more sensitive but sheep cell agglutination test is more specific
 RF latex slide test
 Reagent- latex coated with human gamma globulin
 Specimen- serum dilutions
 Positive result- agglutination
 RF latex tube test
 Reagent- latex with adsorbed gamma globulin
 Specimen- serum dilutions
 Positive result- agglutination
o Systemic lupus erythematosus
 Characterized by ___________________________________________
BUTTERFLY RASH, LE CELL, LUPUS INHIBITOR/ANTICOAGULANT
 Substrate used to detect anti-dsDNA- ___________________________
CRITHIDIA LUCILIAE

 Test- anti-nuclear antibodies by immunofluorescence

 Patterns
o Homogenous diffuse- anti-DNA, anti-RNP
o Peripheral outline- anti-dsDNA
o Speckled anti-smith, anti-RNP
o Nucleolar anti-RNA precursors
 FORMS
1. Discoid
o Limited to the skin and is identified by biopsy of the rash that
may appear on the face, neck and scalp
2. Systemic
o More severe and can affect the skin, joints and almost all body
system
3. Drug-induced
o Antihypertensive- hydralazine hydrochloride
o Anticonvulsants- phenytoin
o Antiarrhythmic- procainamide hydrochloride
4. Neonatal
o From passage of maternal antibodies, specifically anti-Ro/SS-A
or anti-La/SS-B that can affect the skin, heart and blood of the
fetus and newborns
IMMUNODEFICIENCY
 A dysfunction in the body defense mechanisms that cause a failure to detect foreign antigens and
produce antibodies against these foreign substances
 Classified as
o Primary immunodeficiency- results from developmental defects; inherited
o Secondary immunodeficiency- caused by factors such as chronic disorders, nutritional
defects, aging, drug therapy, lifestyle, stress, viruses; acquired
B cell disorders
 X-linked (Bruton’s) agammaglobulinemia- B cells fail to mature and to secrete immunoglobulins
- Inherited form, transmitted to males through the X
chromosome
 Selective IgA deficiency- most common of the immunodeficiency disorders

 FAB
T cell disorders
 DiGeorge’s syndrome- immunodeficiency disease resulting from failure of the parathyroid nd
thymus glands to develop before birth
 Purine nucleoside phosphorylase/ PNP deficiency- defect in purine metabolism, decreased in T
cells due to accumulation of toxic purine metabolite deoxyguanosine triphosphate

Combined T and B cell disorders

 Severe combined immunodeficiency
 Wiskott Aldrich syndrome
 Ataxia- Telangiectasia

Phagocytic disorder
 Lazy leukocyte syndrome- defect in chemotaxis and random WBC movement
 Job syndrome- defect in chemotaxis
 Chronic granulomatous disease- defect in the respiratory/ oxidative burst
 Chediak Higashi syndrome- defect in the lysosomal granules
 Leukocyte adhesion deficiency-failure of the neutrophils to leave the vasculature and migrate to the site of infection
due to defective adhesion receptors

TUMOR IMMUNOLOGY
 Neoplasia- uncontrolled growth of normal cells
 ___________________-
CARCINOMA epithelial tissue of origin
 ___________________-
SARCOMA connective tissue of origin
 ___________________- occurs when the malignant cells travel through the body, causing new
METASTASIS
foci of malignancy until body function is so disrupted that death eventually occurs

FUNCTIONS OF TUMOR MARKERS

i. Localization of metastasis
ii. Disease monitoring and therapy selection
iii. Tumor screening and diagnosis
iv. Histopathologic evaluation and staging
TUMOR ANTIGENS/MARKERS
ONCOFETAL TUMOR ANTIGENS
Carcinoembryonic antigen/CEA COLORECTAL CA
Alpha-fetoprotein HEPATOCELLULAR CA

CARBOHYDRATE ANTIGEN/CA
CA125 OVARIAN CA
CA15-3 BREAST CA
CA19-9 PANCREAS AND GASTRIC CA
CA72-4 GASTRIC CA
ENZYMES
Prostate specific antigen/ PSA PROSTATE CA

Alkaline phosphatase BONE AND LIVER CA

HORMONES
Beta-HCG TESTICULAR CA
Calcitonin MEDULLARY THYROID CA
Gastrin GASTRIC AND PANCREATIC CA

CYTOKERATIN 19 FRAGMENTS LUNG CA

 FAB
 SUSCEPTIBILITY GENES AND ONCOGENES
BRCA-1 and BRCA-2 mutations BREAST CA
HER2/neu BREAST CA

SEROLOGICAL TEST FOR SYPHILIS

 History
o _____________________
CHRISTOPHER COLUMBUS together with his sailors brought the disease from America to
Europe
o Causative agent T.pallidum is not recovered in the blood, serum or plasma stored at
_____4C for more than ______ 48 hrs
Wasserman ag- diphosphatidyl
o ToRCHeS- vertically-transmitted infections Glycerol/cardiolipin

 Other Treponema specie

o T. pallidum subspecies pertenue- _____________ YAWS
o T. pallidum subspecies endemicum- ____________________ ENDEMIC SYPHILIS/ BEJEL
o T. carateum- ___________________
PINTA
o T. cuniculi- _____________________
RABBIT SYPHILIS
 STAGES OF SYPHILIS
not demonstrated serologically; spirochetes is viewed under the
o Primary syphilis- characterized by ________________________
HARD CHANCRE
microscope (DFM, PCM, FM); check for cork screw motility
o Secondary syphilis- disseminated infection, with the appearance of multiple, widespread
lesions on mucus membranes or on skin-spreads on the blood, can be demonstrated through serological test (RPR and VDRL);
condylomata lata
o Latent syphilis- asymptomaticbest stage to diagnose, generally after 2nd yr of infection
o Tertiary/late syphilis- manifests __________________,
DISSEMINATED GUMMAS, CARDIOVASCULAR SYPHILIS or NEUROSYPHILIS
Diagnosis:10 yrs after infection;
____________________________ Gummas- systemic or multiple organ involvement test diagnostic for neurosyphilis is "VDRL"
or _______________________
 Direct demonstration of Treponemes
o Darkfield Microscopy- _____________________________
PRIMARY STAGE; CORKSCREW MOTILITY
o Levaditi’s silver impregnation method
o Direct fluorescent- antibody staining for T. pallidum
 Non- Treponemal tests for syphilis
o Includes
 VDRL and RPR
 Wassermann complement fixation test
 Reagin screen test
 Unheated serum reagin
 Toluidine red unheated serum test
 Automated reagin test
 Kahn flocculation test
 Plasmacrit
o Characteristics
 Detects anti-cardiolipin antibodies
 Non-specific; often used as screening test
 Flocculation is the reaction between reagin and cardiolipin
 _______________
FLOCCULATION is a physical process of contact and adhesions wherein the
aggregates form larger-size clusters called flocs/flakes being excluded from the
suspension
o VDRL
 Principle- qualitative/quantitative slide flocculation test
 Used in both serum and CSF specimen
 Requires serum inactivation
 Inactivation is done by heating serum for __________________ 56 C FOR 30 MINS
within
________________
4HRS

 FAB
  If more than 4 hrs has elapsed, serum must be reinactivated by heating at
56 C FOR 10 MINS
______________________
 Reagents
 Alcoholic solution of cardiolipin-cholesterol-lecithin
o ______________________-
0.03% CARDIOLIPIN phospholipid isolated from beef
heart; responsible for reactivity
o ______________________-
0.09% CHOLESTEROL serves as center for absorption of
tissue lipids to increase the antigen’s size
o ______________________-
0.21% LECITHIN for standard reactivity

 HAMILTON SYRINGE gauge for antigen suspension

o For qualitative test, 18g= 60±2 drops of Ag susp/mL
o For quantitative test, 19g= 75±2 drops of Ag susp/mL
23g= 100±2 drops of saline /mL
 Serum-antigen mixture is placed in rotator for _______________,
4 MINS AT 180 RPM on
room temperature
 Results must be read under LPO microscope
 Reporting of result
o ______________=
REACTIVE medium to large clumps
o ______________=
WEAKLY REACTIVE small clumps

o ______________=
NON-REACTIVE no clumping

o RPR
 Antigen
 Cardiolipin, lecithin cholesterol
 EDTA, Na2HPO4, thimerosal, charcoal, choline chloride
 Serum-antigen mixture is rotated for _______________
8 MINS AT 100 RPM

 Gauge number of needles for antigen suspension

- 20g needle= 60±2 drops/mL
 Advantages
 No heat inactivation Because of choline chloride
 Antigen suspension is more stable, can be stored
 Charcoal makes the reaction visible and can be read macroscopically
 Can be automated, more sensitive and rapid than VDRL
 Treponemal test
o Characteristics
 Confirmatory test, more specific, antibody used are directed towards T.
pallidum specie
o FTA-abs(Fluorescent Treponema pallidum Antibody-Absorbed test)
 Specificity of test enhanced by pre-absorption of serum with Reiter
treponemes
 Principle- Indirect fluorescent immunoassay
 Antigen- Nichol’s strain dried fixed on slide
 Label- FITC-AHG
 Sorbent- Reiter treponemes

 FAB
 o TPI (Treponema pallidum Immobilization test)
 Most specific test for syphilis, STANDARD TEST
 Test of reference, first test developed for the detection of treponemal
antibody
 Principle- antibody against T. pallidum plus complement will immobilize
live treponemes
 Specimen- serum
 Reagent- suspension of motile T. pallidum from experimental rabbits, guinea
pig complement
 Interpretation of result
 Positive- _________________________
>50% IMMOBILIZED

 Negative-_________________________
<20% IMMOBILIZED

 Doubtful- _________________________
20-50% IMMOBILIZED

o AGGLUTINATION TESTS
 TPHA (Treponema pallidum Hemagglutination)
 Confirmatory test for syphilis, indirect hemagglutination test
 Use tanned sheep red cells coated with antigen from the Nichol’s strain

 MHA-TP (Microhemagglutination-Treponema pallidum)

 Similar to TPHA except that it is a microtechnique

 HATTS (Hemagglutination treponemal test for syphilis)

 Automated conversion for TPHA
 Use glutaraldehyde-stabilized turkey RBCs

 TP-PA (Treponema pallidum Particle Agglutination)

 Use gelatin particles (instead of RBCs) sensitized with T. pallidum antigens
 An indirect agglutination test

SEROLOGICAL TESTS FOR HIV

- Related genes- _________________________________
POL, ENV, GAG (polymerase, envelope, group specific antigen)
- ____________________________-
P24 ANTIGEN and VIRAL RNA first marker to be positive in HIV
- ____________________________-
P24 and P55 antibodies to Gag protein
- ____________________________-
GP41, GP120, AND GP160 antibodies to the envelope proteins
- Screening test- _________________
ELISA
- Confirmatory test- __________________________
WESTERN BLOT

 Bands- ______________________
p24, gp41, and gp120/160
- Ratio of CD4:CD8 in HIV- ________________
1:2

- _______________-
<200/ul CD4 count indicative of AIDS

SEROLOGICAL TEST FOR HEPATITIS B

- Hepatitis virus transmitted via fecal-oral route- ___________
A and E
- Mode of transmission of Hepatitis B and C- _______________________________
VENEREAL, PERINATAL, PARENTERAL

 FAB
  Infectious Hepatitis- _____________
Hepatitis A

 Serum Hepatitis- _____________

Hepatitis B
- Hepatitis B vaccine
 Formalin killed
 Recombinant vaccine made from _________________
Saccharomyces cereviciae
- Most common cause of post transfusion Hepatitis- ________________
NON-A NON-B HEPATITIS
- Quantitative HCV RNA test used is to measure- _________________
HCV VIRAL LOAD
- Surrogate testing for HCV uses measurement of _____________________
ALT and ANTI-HBC
- Markers of Hepatitis B infection
 ________________-
HBsAg Australia antigen, first marker to appear, indicative of active
Hepatitis B infection
 ________________-
HBcAg located in the nuclei of hepatocytes, not part of Hepatitis B profile
test
 ________________-
HBeAg active Hepatitis B with high degree of infectivity
 ________________- first antibody to appear; current or recent acute Hepatitis B; marker
Anti- HBc
used in detecting infection in cases where HBsAg is undetectable
 ________________-
Anti-HBc predominantly IgG, current or past Hepatitis B infection; high titers
in carriers
 ________________-
Anti-HBe good prognostic; sign of recovery
 ________________-
Anti-HBs marker of immunity to further infection, produced after
immunization with Hepatitis B vaccine; _________________
>/=10mlU/mL of serum = protective titers; not
produced during chronic HBV infection
- Tests for HBsAg detection
 First generation
 Ouchterlony
 Second generation
 Complement fixation, rheophoresis, counterelectrophoresis
 Third generation
 RIA, RPHA, ELISA, RPA

SEROLOGICAL TEST FOR INFECTIOUS MONONUCLEOSIS

- Caused by _________
EBV which infects ___________.
B CELLS
- Characterized by _________________________________
VARIANT/ REACTIVE/ ATYPICAL T LYMPHOCYTES
- Associated with _____________________
BURKITT'S LYMPHOMA and _________________
ANTI-i
- Tests
o Paul-Bunnell screening/ general test; principle-hemagglutination sheep RBC and guinea pig
o Davidsohn differential test traditional/ classical test- confirmatory test
o Monospot rapid test- uses HORSE RBC

SEROLOGY OF STREPTOCOCCAL INFECTION

- Causes suppurative conditions, non-suppurative conditions and toxigenic conditions
- _______________________________________-
ARF and PSAGN sequela of S.pyogenes infection
- Streptococcal tests employing extracellular products
o ASO test
 Streptolysin O- immunogenic, oxygen-labile hemolysin
 ASO can be detected using latex agglutination test or by tube test
 Latex agglutination test
 Based on _________________________________
 Tube test is based on _______________________________
 Dilution of patients’ serum is incubated with streptolysin O reagent

 FAB
  RBCs are added and tubes are observed for the presence or absence of
hemolysis
 Inhibition of hemolysis is related to the presence of ASO in patient
serum
 Reported in titer
 Reporting of result
o _______________-
TODD UNITS when the streptolysin reagent standard is
used
o _______________-
IU UNITS when WHO international standard is used
 Normal= ____________
<200 TODD UNITS
 Controls
o _______________=
HEMOLYSIS SLO control
o _______________=
NO HEMOLYSIS RBC control
o Other test for extracellular products
 Streptozyme
 Anti-DNAse B
 Anti-streptococcal hyaluronidase test
 Anti-streptokinase test
 Anti-NADase

o Streptococcal test employing cellular antigen from Streptococcal wall

 Anti-M protein antibodies
- Positive test correlates with convalescent state
 Anti-A carbohydrate antibodies
- Positive test differentiates rheumatic from non-rheumatic valvular
disease

SEROLOGICAL TEST FOR Salmonella

- Carriers of Salmonella typhi- _________________________________
DETECTED USING BILDE FORM GALL BLADDER
- Cook who spread the febrile condition- ________________________
MARY MALON
- Test- ________________
WIDAL TEST
o Detects antibodies to Salmonella
- Antigens used
O Somatic antigen LPS (inc in G- bacteria)
H Flagellar antigen CHON
K or Vi capsular/ envelope antigen Polysaccharide/sugar

SEROLOGICAL TESTS FOR Helicobacter pylori

- Associated with peptic ulcer
o Tests that require endoscopy
 H. pylori culture
 Histological exam of gastric biopsy
 Urease biopsy test
o Tests that do not require endoscopy
 Urea breath testing
 Enzyme immunoassays for bacterial antigens in the feces
 Molecular tests for H. pylori
 Antibody test

 FAB
SEROLOGICAL TESTS FOR Rickettsia
- Causes fever and typhus
- Tests- _________________________
WEIL-FELIX
o A non-specific test for the detection of antibodies against Rickettsial diseases
Rickettsial Disease OX- 19 OX-2 OX-K
Scrub R. tsutsugamushi - - ++++
RMSF R. ricketsii ++++ + -
Q fever Coxiella - - -
Rickettsial fever R. akari - - -

SEROLOGICAL TEST FOR Mycoplasma pneumoniae

- Mycoplasma pneumoniae causes- __________________________________
PRIMARY ATYPICAL/ WALKING PNEUMONIA
- Associated with- __________________________
COLD AGGLUTININS

SEROLOGICAL TESTS for MALARIA

- Caused by Plasmodium species of parasite
THICK and THIN SMEAR
- Definitive test- __________________
- Immnochromatographic assays
o OptiMAL assay
 Detects ___________
pLDH
 Can distinguish between species
o MalaQuick standby Malaria test
 Detect P. falciparum’s ___________________
HRP-2

SEROLOGICAL TEST for Histoplasmosis

- Two precipitin bands of diagnostic significance
o ____________________-
H LINE most specific but can be found up to 2 years after recovery
o ____________________-
M LINE found in patients with active infection, but can also be found in
patients with past infection and patient who had a recent skin test
SEROLOGICAL TEST for Influenza virus
- Influenza types can be determined using
o _______________________-
HEMAGGLUTININ required for entry into the cell; H1 to H8
o _______________________-
NEURAMINIDASE facilitates release of virus from the cell; N1 to N11
- Mutations in influenza
o _______________________-
ANTIGENIC DRIFTS minor mutations that occur at the very site of the virus as it
replicates; pre-existing antibodies cross react and can render protection
o _______________________-
ANTIGENIC SHIFTS major antigenic change in influenza A, resulting in HA and
HA; occurs about every 10 years; sudden in onset and can cause serious epidemics
Other serological tests for other infectious agents
i. MTB- Tuberculin test, vollmers patch
ii. C. trachomatis- Frei test
iii. C. diptheriae- Schicks test
iv. S. pyogenes- Dicks test
v. Brucella- Brucellergin test
vi. T. spiralis- Bachman/trichinella skin test
vii. B. anthracis- Ascoli test
viii. Toxoplasmosis, Echinococcosis- Toxoplasmins/cassonis skin/ intradermal test
ix. M. leprae- lepromin, mitsuda test, fernandez test
x. S. pneumoniae-Francis test

 FAB