IMMUNOLOGY-SEROLOGY SEMINAR
SEROLOGY
Medical science that deals with blood sera,
their components and immunologic
properties and reactions
Serologic Tests/Procedures
o Blood tests or any laboratory
procedure carried out on blood
serum
o Detect the presence of antibodies
against microorganism
o Designed to demonstrate ______
ANTIGEN VS. IMMUNOGEN
Antigen vs. Immunogens
o Antigen foreign substances that
stimulates antibody formation but
may NOT ALWAYS be able to evoke
an immune response
o Immunogen macromolecules
capable of triggering an adaptive
immune response inducing (1)
antibodies or (2) sensitized T-cells
o Epitope portion of
the antigen that is
recognized by the
antibody antigenic
determinant
o Hapten
Non-immunogenic by itself
Needs to be complexed with
larger molecules to be able
to
stimulate
immune
response
Only has
limited
epitopes
Attached to a carrier
molecule to become an
Immunogen
Hapten + Carrier = Complete
antigen
Types of antigens
1. Autoantigen endogenous antigen
recognized as non-self (i.e.
Autoimmune diseases)
2. Alloantigen antigen existing in
allelic forms in a species (i.e. Blood
group antigens)
3. Heteroantigen antigen originating
from a species foreign to the
antibody producer
4. Heterophile Antigen antigens that
cross react to antigens of similar
nature
Prerequisites for immunogenicity
1. Molecular size
Minimum molecular size =
10,000 daltons
Except: (1) Nylon, (2)
Polyacrylamide and
(3) Teflon
Preferred = 100,000 daltons
2. Chemical composition and
complexity
More complex composition,
better immunogenicity
Proteins more complex due
to amino acid sequence
Polysaccharides sugars
are less complex due to
repetitive composition
Best bound to
something else, more
immunogenic
Glycolipids AB
blood group
Glycoproteins Rh
blood group
Lipids
Nucleic acids not as good
of an Immunogen because
the double helix stops it from
being accessed by
antibodies
3. Foreignness degree to measure as
non-self
4. Ability to be processed and
presented with MHC molecules
MHC I all nucleated cells
MHC II antigen presenting
cells
ANTIBODIES
Also known as: immunoglobulins
Specific proteins that combines with an
antigen
Composed of 4 polypeptide chains
o 2 lights chains
o 2 heavy chains
Considered as a MONOMER but has two
binding sites
o Fab antigen binding site where
you find the paratope that binds with
the antigen
o Fc site for complement fixation
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 Immunoglobulin classes o Avidity
Most in number Represents the sum of all
Does not cross attractive forces between an
the placenta antigen and an antibody
(IgG2) Multivalent
IgG Takes into consideration all
Capable of
complement the binding sites of an
activation (IgG3) antibody
Monomeric TYPES OF IMMUNOLOGIC REACTIONS
Present in body Primary Reactions
IgA o Specific recognition and combination
secretions
Largest of antigen and a corresponding
immunoglobulin antibody
Pentameric (2 o More sensitive than secondary
binding sites reactions
per Fab) = 10 o Reaction is invisible unless there is
IgM binding sites the addition of a label
Good agglutinin o Examples:
because of its ELISA
large size Immunofluorescence
Radioimmunoassay
Cannot cross
Chemilluminescence
the placenta
Secondary Reactions
Surface markers of B-
IgD o Conformation of amino acid chain
cells
due to interchain hydrogen bonding
Reagin antibody for
IgE o Occurs after primary reactions
allergies
o Cross linking leading to formation of
ANTIGEN-ANTIBODY REACTIONS immune complexes
Immune reactions involving the formation of o Visible reactions
Antigen-antibody complex Agglutination and
Involves the interaction of the epitope and precipitation
Fab region Complement fixation
Result of NON-COVALENT and Neutralization
REVERSIBLE intermolecular interactions Tertiary Reactions
Reaction is not too strong o Occur as biologic reactions
Four types of intermolecular forces o Involves folding of polypeptide
1. Ionic or electrostatic oppositely chains through hydrophobic and
charged particles hydrogen bonds
2. Hydrogen bonds Antigen and o Examples:
antibody are in close proximity; Phagocytosis
attraction between polar molecules Opsonization
3. Van der Waals interaction between Chemotaxis
the electron clouds of oscillating Immune adherence
dipoles Cellular degradation
4. Hydrophobic bonds occurs IMMUNOASSAYS
between non-polar molecules
Quick and accurate tests that can be used
Reaction characteristics
on-site and in the laboratory to detect
o Invisible and precise specific molecules and/or measure the
o Engagement comparable to a lock
concentration of a molecule in a solution
and key
through the use of an antibody or
o Kinetics of antigen-antibody reaction
immunoglobulin
complies with law of action mass Rely on the inherent ability of an antibody to
Affinity vs. Avidity
bind to the specific structure of a molecule
o Affinity initial force of attraction that
referred to as analyte and which
exists between an epitope and Fab chemically in many cases a protein
usually monovalent
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 An antibody molecule recognizes and binds
to an antigen
Binding is related to:
1. Concentration of each reactant
2. Specificity of antibody for antigen
3. Affinity and avidity for pair
4. Environmental conditions
Types of Immunoassay Formats
1. Homogenous
Consists only of a liquid
phase
Allowing to make an assay-
measurement by a simple
mix and read procedure
No washing or separation
step
EMIT Enzyme Multiplied
Immunoassay Technique
2. Heterogenous
Involves the use of a solid
phase
Requires the use of a
microwell or beads to
discriminate the free from the
bound components
ELISA and RIA
PRIMARY IMMUNOLOGIC REACTIONS
Since the antigen-antibody reaction is not
visible, labels are employed because it is
too small
Labeled Immunoassays
o Labels improve the analytic
sensitivity of the test more
sensitive
o Addition of labels to antigen-
antibody interactions allows
quantitative measurement of target
analyte becomes quantitative
3 categories of Indicator Labels
o Radioactive Isotopes
With unstable nuclei and emit
radiation spontaneously
125I and 131I most
commonly used isotopes in
the clinical laboratory
Use scintillation counter
Application:
Radioimmunoassays
o Fluorochromes
Molecules that absorb light
and become transiently
excited excited molecule
becomes stable and is
emitted as visible light
(fluorescence)
FITC Fluorescein
Isothiocyanate
Application:
Immunofluorescence
Direct antigen
Indirect antibody
o Enzymes biological catalysts
Acts upon the substrate
added in the reaction leading
to a chance that can be
measured
Enzymatic activity can be a:
Colored product
Chemiluminescence
Examples:
Horseradish
peroxidase
ALP
Application: ELISA
Classification of Indicator Labeled
Immunoassays
1. Competitive
Principle two analytes;
labeled and unlabeled
(usually antigens) can
compete for the constant and
limited biding sites on the
solid phase
Can be carried out in an
antigen-capture or antibody-
capture format
The measured signal is
inversely proportional to the
concentrations of analyte in
the sample
Higher concentration,
less unlabeled
samples
Examples:
RIA
EMIT drugs of
abuse/therapeutic;
body fluids are used
(urine)
FPIA (Fluorescence
Polarization
Immunoassay)
Hormones, drugs of
abuse/therapeutic
2. Non-competitive
Detection of either antigen or
antibody in a biological
sample
Antigen and antibody are
permitted to interact without
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 deliberate introduction of
competing molecules
Examples:
ELISA (Direct,
indirect, sandwich)
o Non-competitive: Direct Detection
Method uses a labeled
primary antibody that reacts
directly with the antigen
Antigen can be immobilized
on the assay plate or in a
capture assay format
Application:
immunohistochemistry
o Non-competitive: Indirect Detection
Method is the most popular
format for ELISA
It uses a labeled secondary
antibody for detection has
specificity for the primary
antibody
More sensitive due to
amplified reaction
Antigen attached to the well
and then incubated (with
albumin) to block non-
specific binding sites
Add sample: antibody and
then add secondary antibody
that as a label
Secondary antibody:
Has anti-IgG
Binds to Fc
portion of
complex
o Non-competitive: Sandwich Assay
Sensitive, robust and most
powerful assay format
Analyte to be measured is
bound between 2 antibodies
Antibody sources:
Capture mouse IgG
Detection rabbit IgG
For sandwich assays, it is
beneficial to use secondary
antibodies that have been
crossed-adsorbed remove
any antibodies that have
affinity for capture antibody
Two types:
Direct 1 antibody
Indirect 2 antibodies
Capture antibody has to be in
the correct orientation
uses Protein A
Add albumin to block non-
specific sites
Components of a Labeled Immunoassay
1. Labelled and unlabeled analyte
2. Receptor molecule
3. Standard or calibrator solutions
4. A means to separate the free from
the bound components
5. A mean to measure the labelled
product
Separation Methods for Heterogenous
Immunoassays
o Adsorption onto particles
Uses particles to trap small
antigens (labelled or not)
Mixture of _______ is most
commonly used
o Precipitation
Occurs when environment is
altered, affecting solubility of
protein
Ammonium sulfate, ethanol,
PEG
o Use of solid phase
Used to immobilize reagent
antibody or antigen and
separate free from bound-
labeled reactant after
washing
Glass beads, polyacrylamide
beads, paper
SECONDARY IMMUNOLOGIC
REACTIONS
Precipitation
o Involves the interaction of antigen
with antibody resulting in a visible
precipitate
Antigen is soluble
multivalent
Antibody is bivalent (IgG)
o Phase change present from liquid
to solid
2 Phases:
Initial
Formation of lattice
work
o Zone of equivalence does not
have to be equal; just needs to be
proportional
o Antigen involved must be SOLUBLE
o Precipitation Procedures
Beta Ramon procedure
Antibody varying
Antigen fixed
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 Alpha Dean-Webb
procedure
Antibody fixed
Antigen varying
Agglutination
o Antigen involved is
insoluble/particulate
o Formation of
agglutinates/aggregates
o 2 mechanisms:
Sensitization
Lattice formation
o Detects antibody (IgM)
multivalent
TYPES OF PRECIPITATION REACTIONS
Precipitation in Liquid
1. Test tube method
Measures turbidity
Quantitated by
nephelometry/turbidimetry
2. Slide Method Use of microscope
3. Capillary Method Dense
granulation in the interphase
4. Ring test Precipitin ring formation
Antigen is carefully layered
over the antiserum, without
mixing so that an interphase
is formed diffusion of each
reagent occurs into the other
Homologous system
precipitation will occur at the
point in the tube where the
proper ratio if antigen to
antibody is reached
Example:
Ascholi test
anthrax
CRP
Precipitation in Semi-solid Medium (1%
Agarose Gel)
o Agarose gel has no charge
Antibody (+)
Antigen (-)
o Single Immunodiffusion, Single
Dimension
AKA: Ouidin Test
Patient sample with
ANTIGEN agarose agar
impregnated with ANTIBODY
(+) Results precipitin ring;
the higher diameter, the
more complexes formed
o Double Diffusion, Single Dimension
AKA: Oakley-Fulthorpe Test
Both antigen and antibody
are moving but at the same
direction
Antibody incorporated in
agar, poured into a tubed,
allowed to harden
Plain agar layer of agar
without antibody, poured
above antibody agar and
allowed to solidify
Antigen placed about plan
agar
(+) Result: Precipitin band
appears in the plain agar
o Single diffusion, double dimension
(Radial immunodiffusion)
Uses a plate containing agar
WITH ANTIBODY
ANTIGEN is placed on the
wells
Diameter or precipitation line
directly proportional to the
concentration of the
TARGET ANTIGEN
Methods:
Mancini end point
method (24-72
hours); square of the
diameter is directly
proportional to the
concentration of
antigen
Fahey-McKelvey
kinetic method (18
hours); diameter is
proportional to the log
of the concentration
of the antigen
o Double Diffusion, Double Dimension
Ouchterlony/double angular
immunodiffusion
Diffusion of both antigen and
antibody
Diffusion of 3 reagents
placed in wells
2 antigens
1 antibody
Used to find out relationship
between antigens
Qualitative
Complicated diffusion
patterns
Immunoelectrophoresis (Grabar and
Williams)
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 o Double diffusion technique o Examples:
incorporates voltage current to Western Blot
enhance results Southern Blot
o Serum source of antigen Northern Blot
o Trough where to put the known o Procedure:
antibody antiserum Aliquots of the patients
o Double diffusion occurs at right serum placed in each well
angles (tracks 1-6)
o Expected results: Precipitin line/arc Perform electrophoresis
develops separation of proteins
o Procedure: Apply antibody solution to
Antigens migrate under an electrophoretic migration
electric current tracks 2-6
Reagent antibody is added in Track 1 treated with
the trough diffusion through a fixative; serves as a
gel reference for the
Formation of precipitin arc patients complete
Counter immunoelectrophoresis protein profile
o Counter current Incubate, add deproteination
immunoelectrophoresis solution to removed unfixed
o Voltage facilitated double proteins stain proteins
immunodiffusion Destain to remove non-
o Double immunodiffusion (one specific staining
dimension) assay Types of Agglutination Reactions
With current Direct Agglutination
At pH 8.6 to the medium o Detection of febrile agglutination
o Apply antigen to one side, antibody o Antigen is found naturally on the red
to the other placed in opposite cell surface
wells o Application:
Antigen negative Blood typing
Antibody positive Febrile agglutination tests -->
o Rapid but not sensitive Widal test
Rocket electrophoresis Paul Bunnel
o Laurell single diffusion technique hemagglutination
o Single diffusion, one dimension Heterophile antigen
Quantitative on 2% RBCs
o Antiserum is incorporated into the Passive Agglutination
gel and the unknown antigen is o Antigen is attached/coated to a
placed in the wells and carrier particle latex particle
electrophoresed o Application: RF Latex Test
o The height of the rocket (conical) o Carrier particles:
measured from the well to the apex RBC
directly proportional to the Latex
amount of antigen Bentonite
Immunofixation electrophoresis Charcoal
o First described by Alper and Reverse Passive Agglutination
Johnson o Antibody are attached to a carrier
o 2 Stage Technique: particle
Electrophoresis separate o Antigen is detected
proteins o Application: Detection of CRP
Immunoprecipitation similar Agglutination Inhibition
to immunoelectrophoresis o Homologous antigen inhibits
except that antiserum is agglutination of antigen coated
applied directly, rather than a particle
trough o 2 Step Procedure
o Agarose or cellulose acetate is used
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 1st step soluble antigen in Best source Rabbit
patient sample + known antisera
antibody reagent o Indicator cells Sensitized sheep
2nd step particulate antigen RBCs coated with hemolysin
is added o (+) No lysis; (-) Presence of lysis
o (+) Result no agglutination NEUTRALIZATION
Antiglobulin Techniques Antigenic activity is
o AKA Coombs Test stopped/nullified/neutralized by its
o Primarily employed in blood banking neutralizing antibody
o Coombs Reagent Target: to detect toxins, viral agents or
Added to bridge the gap antibodies to the toxins or viral agents
between the cells Types of neutralization tests
Composition: anti-IgG o Toxin Neutralization
(monospecific) Schick Test
Binds to Fc portion to act as Dick Test
a bridge linking the gap ASO Titration Test
o Sensitization attachment of o Virus Neutralization
antibody (IgG) to RBC FLOCCULATION
o Types: AKA: Natural clumping
Direct Coombs Involves aggregation of colloidal particles
Indirect Coombs Observed as fleecy mass when a
Coagglutination suspension of antigen and antibody is
o Term given to systems using agitated
bacteria as the inert particles to Midway reaction between agglutination and
which antibody is attached precipitation
o Staphylococcus aureus cowan strain Cardiolipin non-cellular particulate
most frequently used antigens
o Protein A surface protein; naturally Examples:
adsorbs the Fc portion of antibody o VDRL
o Active sites face outward and are o RPR
capable of reacting with specific
antigen
o Exhibits greater stability than latex
particles
o More refractory to changes in ionic
strength
COMPLEMENT FIXATION
Principle: Antigen combines with an
antibody to form a complex activates
complement to attach complement is
FIXED by sticking to the Fc portion of the
the immune complex no more free
complement
Two systems involved for complement
fixation
o Test system bacteriolytic system
o Indicator system hemolytic system
Components:
o Antigen known target antigen
reagent
Beef heart extract
Bacterial antigen
o Complement guinea pig serum
o Antibody Hemolysin or
amboreceptor
Sensitize indicator cells
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