2. Basic Immunologic
Procedures
Terry Kotrla, MS, MT(ASCP)BB
Fall 2005
Introduction
 Detection of antigen/antibody reactions
difficult
 Sensitization is the binding of a specific
antibody to its’ specific antigen
 Cannot be visualized
 Multitude of laboratory methods have been
developed to make this visible
Three Distinct Phases of
Antigen/Antibody Reactions
 Primary Phenomenon – Sensitization
 Secondary Phenomenon – Lattice formation
 Tertiary Phenomenon – Detected by affect on
tissues or cells.
Primary phenomenon
 Sensitization – binding of antibody to antigen – not
visible
Secondary Phenomenon
 Lattice Formation
 The Fab portion of the Ig molecule attaches to antigens on 2
adjacent cells-visible results in agglutination
 If both antigen and antibody are SOLUBLE reaction will become
visible over time, ie, precipitation
 http://www.cehs.siu.edu/fix/medmicro/agabx.htm
Tertiary Phenomenon
 Reaction not visible, detected by affect of
reaction on tissues or cells.
 http://www.cellsalive.com/mac.htm
Phagocytosis
Secondary Phenomena Most
Frequently Utilized
 Precipitation – soluble antibody reacts with
soluble antigen
 Agglutination – particulate antigens bound
together by antibody
 Complement Fixation – antibody binding to
antigen triggers activation of complement
Antigen-Antibody Binding
 Affinity
 Avidity
 Law of Mass Action
Affinity
 Antibody affinity is the strength of the reaction
between a single antigenic determinant and a
single combining site on the antibody.
 It is the sum of the attractive and repulsive
forces operating between the antigenic
determinant and the combining site .
 Affinity is the equilibrium constant that
describes the Ag-Ab reaction as illustrated in
Figure 3. Most antibodies have a high affinity
for their antigens.
Affinity
Avidity
 Avidity is a measure of the overall strength of binding
of an antigen with many antigenic determinants and
multivalent antibodies.
 Affinity refers to the strength of binding between a
single antigenic determinant and an individual
antibody combining site whereas avidity refers to the
overall strength of binding between multivalent
antigens and antibodies.
 Avidity is influenced by both the valence of the
antibody and the valence of the antigen.
 Avidity is more than the sum of the individual
affinities.
Avidity
Law of Mass Action
 Governs the reversibility of the antigen-antibody
reaction.
 Reversible reaction, visible reaction occurs when the
rate of binding exceeds the rate of dissociation.
Precipitation Curve
 Prozone – antibody excess, many antibodies
coat all antigen sites- results in false negative
 Postzone – antigen excess, antibody coats
antigen but cannot get lattice formation,
results in false negative
 Zone of Equivalence – antigen and antibody
present in optimal proportions to bind and
give visible reaction
Precipitation Curve
Precipitation Curve
Measurement of Precipitation by Light
 Antigen-antibody complexes, when formed at
a high rate, will precipitate out of a solution
resulting in a turbid or cloudy appearance.
 Turbidimetry measures the turbidity or
cloudiness of a solution by measuring amount
of light directly passing through a solution.
 Nephelometry indirect measurement,
measures amount of light scattered by the
antigen-antibody complexes.
Precipitation/Flocculation
 When soluble antibody binds
to soluble antigen
(sensitization) there will
come a point where lattice
formation will occur resulting
in precipitation occurring
resulting in a visible reaction
 These immune complexes
have fallen out of solution.
The Ab at the bottom in the
illustration at right is still in
the soluble phase.
Turbidimetry
 Measures turbidity or cloudiness of a solution by measuring the
amount of light PASSING THROUGH the solution.
 Soluble antigen and antibody join and once they join in sufficient
amounts precipitate, results in cloudiness.
 The more cloudy the solution, the less light can pass through.
Nephelometry
 Measures SCATTERED light bouncing off antigen-
antibody complexes.
Nephelometry
Passive Immunodiffusion
 Reactions in gels
 Migrate towards each other and where they
meet in optimal proportions form a
precipitate.
 http://perso.wanadoo.fr/svt.ronsard/svt.ronsar
d/travaux/exper/albumine/anim1.gif
Four Methodologies
 Single diffusion, single dimension
 Single diffusion, double dimension
 Double diffusion, single dimension
 Double diffusion, double dimension
Ouidin
Single Diffusion, Single Dimension
Oudin Precipitation
 Solution of antibody is carefully layered on top of a solution of
antigen, such that there is no mixing between the two.
 With time at the interface where the two layers meet, antigenantibody complexes form a visible precipitate. The other two
tubes are negative controls, containing only antibody or only
antigen plus an irrelevant protein in the second layer.
Radial Immunodiffusion
Standard Curve
RADIAL IMMUNODIFFUSION
Precipitin Rings
A
B
Standards
Standard Curve
C
a
b
Samples
c
Ouchterlony Gel Diffusion
 Holes punched in agar.
 Known antibody or antigen added to center
well.
 Known sample added to outer well.
 Unknown sample added to outer well next to
unknown sample.
 Wait for bands to form.
Ouchterlony Immunodiffusion
Ouchterlony - Identity
 The precipitation appears as a continuous line in the
form of an angle between those two wells and the C
well. There are no spurs at the angle and this type of
reaction is termed a band of identity.
Ouchterlony – Partial Identity
 FIGURE 2:
If a solution with antigens X and Y is placed in well 1, a solution with
antigen X only is placed in well 2, and antiserum containing antibodies
specific for both X and Y is placed in well 3, a reaction similar to that
appearing in Fig. 2 will occur. Notice that there is a spur reaction
towards the XY well. This indicates that the two antigenic materials in
wells 1 and 2 are related, but that the material in well 1 possesses an
antigenic specificity not possessed by the material in well 2. Such a
reaction with spur formation indicates partial identity
Ouchterlony – Non-Identity
 If the material in wells 1 and 2 do not possess
common antigens and the antiserum in well 3
possesses specificities for both materials, the
reaction will appear as two crossed lines as in Fig. 3
Ouchterlony-Interpret
 Determine which interpretation fits for samples 1, 2
and 3.
Electrophoretic Techniques
 Immunodiffusion can be combined with electrical
current to speed things up.
Rocket Immunoelectrophoresis
 Antigen is electrophoresed into gel containing
antibody. The distance from the starting well to
the front of the rocket shaped arc is related to
antigen concentration.
Rocket Electrophoresis
Immunoelectrophoresis
Immunoelectrophoresis
 Two-dimensional immunoelectrophoresis. Antigens are
separated on the basis of electrophoretic mobility. The
second separation is run at right angles to the first which
drives the antigens into the antiserum-containing gel to
form precipitin peaks; the area under the peak is related to
the concentration of antigen.
Immunoelectrophoresis-Antivenom

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Each antibody molecule can bind two separate sites on an antigen
molecule (venom toxin), consequently antibodies have the ability to cross
link many antigen molecules simultaneously. This cross-linking causes
the antibody antigen-complex to become insoluble and precipitate out
from the solution.
The immunoelectrophoresis technique makes use of this capability of the
antibodies to form giant insoluble complexes with their respective
antigens. The antigen-antibody precipitate which forms can be
visualised by specific staining techniques, or quantified by various means.
Immunofixation Electrophoresis
 Immunofixation Electrophoresis (IFE) combines zone
electrophoresis with immunoprecipitation.
 This technique may be used to identify and
characterise serum proteins.
 In IFE, proteins of sample are first separated by
electrophoresis on a support (agarose) according to
their charge and after that the medium is overlaid
with monospesific antiserá reactive with specific
protein - antigen.
 If the antigen is present a characteristic
immunoprecipitin band will be formed.
Immunofixation Electrophoresis
Immunofixation Electrophoresis
Enhancement of Agglutination
 Additive to neutralize charge
 Viscosity
 Treatment with enzymes
 Agitation and centrifugation
 Temperature
 pH
Direct Agglutination
 Antigen found naturally on particle.
 Blood Grouping is an example, antigen on
cell
ABO Blood Grouping
Passive Agglutination
 Employs particles that are coated with antigens, ie ,
RBCs, polystyrene latex, bentonite or charcoal.
Reverse Passive Agglutination
 Antibody attached to carrier particle instead of
antigen.
 Serologic Typing of Shigella: Positive Test
Agglutination Inhibition
 Based on competition between particulate
and soluble antigens for limited antibody
combining sites.
 Patient sample added to reagent antibody
specific for antigen being tested, if antigen is
present it binds to reagent antibody.
 Reagent particles (latex or RBCs) coated with
the same antigen are added, if antigen was
present in the sample all reagent antibody
binds to it so no antibody is present to react
with antigens coating the particles
Agglutination Inhibition
 In row A wells 1-8 are positive.
Coagglutination
 Name given to systems using bacteria as the inert
particles to which antibody is attached.
Labeled Immunoassays
 Some antigen/antibody reactions not detected by
precipitation or agglutination.
 Measured indirectly using a labeled reactant.
 Referred to as receptor-ligand assays.
 Ligand is the substance to be measured and is
defined as a molecule that binds to


nother molecule of a complementary configuration,
usually it binds to the
substance the test is trying to detect.
 The receptor is what binds the specific target
molecule.
Labeled Immunoassays
 Ligand – substance to be measured.
 Receptor – binds the specific target molecule
 Sandwich technique
 Next slide - example
“Sandwich” Technique ELISA
Labels
 Used to detect reaction which has occurred.
 Radioactive, fluorescent, chemiluminescent
and enzymes.
 Competitive or non-competitive
 Heterogenous or homogeneous
Standards or Calibrators
 Substance of exact known concentration.
 Usually run for each new lot number
 Based on results create standard curve.
 Standard curve used to “read” results or built
into machine to provide results.
Competitive Binding
 Add known labeled antigen
 Add unknown antigen
 Will compete with each other for sites on
bound antibody molecule.
 Must wash off unreacted substances.
 Type of label on known antigen will determine
method of detection.
Competitive Binding
Radioimmunoassay

http://www.castleviewuk.com/Frameless/Forensics/Presumptive_Tests/Drugs/ria/ria.htm
Radioimmunassay (RIA)
 Competitive binding
 Uses Iodine 125 (I
125)
as label
 Radioactive label competes with patient for
sites
 High radioactivity, small amount of patient
substance
 Low radioactivity high amount of patient
substance
 Textbook page 159 figure 11-2
Radioimmunassay
Radioimmunoassay Competitive
 A plasma (or urine) sample that contains ADH is mixed with the
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antibodies.
A known amount of "standard" ADH that has been tagged with
radioactivity is added to the antibody mixture.
The "standard" ADH is something that has already been measured and
that can be used to compare the unknown quantity of natural ADH.
A radioactive tag is added to the standard ADH so that it can be
distinguished from the natural ADH that is in the plasma.
As the antibodies, the standard radioactive ADH, and the plasma are
mixed, one main requirement must be met: there must be too little
antibody to bind completely to both the radioactive hormone and
the natural hormone in the fluid to be assayed.
Therefore, the natural hormone in the plasma and the radioactive
standard hormone compete for the binding sites on the antibody (next
slide)
Radioimmunoassay Competitive
Immunoradiometric Assay (IRMA)
 Labeled antibody plus patient antigen
 Solid phase antigen added to bind excess
antibody
 Spin down to separate
 Labeled antibody/antigen remain in solution.
 Measure radioactivity
 Textbook page 160 fig 11-3
Enzyme Immunoassay
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Advantages
Reason for choosing enzyme as label
Enzymes used
Two classifications:


Heterogenous – separation of reactants must
be done
Homogeneous – no separation step
 Two types:
 Competitive – known and unknown compete
 Noncompetitive – only unknown reacts
Enzyme Immunoassay

http://www.castleviewuk.com/Frameless/Forensics/Presumptive_Tests/Drugs/elisa/elisa.htm
 http://tinyurl.com/ckdaz
Heterogenous Enzyme Assays
 Competitive ELISA
 Noncompetetive ELISA
 Immunoenzymometric Assay
 Sandwich or capture assays
Competitive ELISA
 Unknown antigen competes with labeled known
antigen
 Enzyme produces color reaction
Noncompetitive ELISA
 Referred to as “Indirect” ELISA
 Antigen bound to solid phase
 Add patient, antibody will bind if present
 Add known labeled antibody
 Measure enzyme label
 Textbook page 161 figure 11-4
 Disadvantage more manipulation of test
Immunoenzymometric Assay
 Noncompetitive ELISA
 Detects unknown antigen by means of excess
labeled antibody
 Textbook page 162 figure 11-5
Sandwich or Capture Assays
 Used for antigens with multiple epitopes, ie,
HCG
 Antibody to one epitope fluid, antibody to
second epitope fixed.
 Enzyme label used to detect reaction
 Textbook page 163 figure 11-6
Homogeneous Enzyme Assay
 Labeled reagent antigen and patient sample
added.
 Compete for bound antibody
 Antibody binds to patient antigen causing
inactivation of enzyme
 Competitive Assay
 Enzyme activity proportional to concentration
of patient antigen
Fluorescence Immunoassay
Markers
 Fluorophores or fluorochromes
 Ability to absorb energy and emit light
 Two most commonly used:

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Fluorescein – green
Tetramethylrhodamine – red
Fluorescence
Fluorescence
 Antibodies and bacteria are fixed on a glass-plate.
 The surplus i.e. non-bounded antibodies are washed out,
antibody-bacteria-complexes ("sandwiches") remain.
 The "sandwich" becomes visible by adding fluorescent anti
bovine immunoglobulin which can be seen as green light in the
fluorescence microscope.
Fluorescent Immunoassay
 Direct immunofluorescence

Tagged antibody added to unknown antigen
fixed to slide
 Indirect immunofluorescence
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Patient plus known fixed antigen
Add tagged anti-antibody
Fluorescence
Positive
Heterogeneous Fluorescent
Immunoassays
Homogenous Assays
Fluorescence Polarization
Immunoassay

http://www.castleviewuk.com/Frameless/Forensics/Presumptive_Tests/Drugs/fpia/fpia.htm
References
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http://web.indstate.edu/thcme/PSP/labtests/precip.htm
http://www.gla.ac.uk/departments/immunology/education/nursing/lectures/antibo
dy.htm
http://www.cellsalive.com/mac.htm
http://jeeves.mmg.uci.edu/immunology/Assays/Assays.htm
http://www.medschool.lsuhsc.edu/microbiology/DMIP/dmex03.htm
http://www.tulipgroup.com/Common/html/TurbidTech.pdf
http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm
http://www.mercodia.se/global/mainpage.asp?page_id=41 ELISA
http://www.clinprointl.com/technical.htm ELISA
 http://www.nsbri.org/HumanPhysSpace/focus4/sf-hormonal.html
 http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm
molecular diagnostics
References (Continued)
 http://www.liv.ac.uk/~agmclen/Medpracs/practical_5/theory_5.html
 http://www.fao.org/docrep/W0049E/w0049e06.htm