Antigen & Antibody Interactions

ANTIGEN - ANTIBODY INTERACTIONS
Principles & Applications
A Presentation By
G. Prashanth Kumar
Department of Microbiology & Parasitology,
International Medical & Technological University,
Dar-es-Salaam, Tanzania.

INTRODUCTION
• The diagnosis of an infectious disease by culture
and biochemical techniques not possible for all
microorganisms certain factors can hinder.
• These factors include the inability to cultivate an
organism on artificial media, such as with
Treponema pallidum, the agent of syphilis, or the
fragility of an organism and its subsequent failure
to survive transport to the laboratory such as
with respiratory syncytial virus and varicella –
zoster virus.
• Fastidious nature and long incubation period.

INTRODUCTION
• Avoiding antibiotic treatment before sample
collection, If you partial treatment can impede
the diagnosis.
• In these cases, detecting a specific product of the
infectious agent in clinical specimen is very
important because this product would not be
present the specimen in the absence of the
agent.
• The direct detection of microorganisms in patient
specimen by using immunochemical methods.
• The Immunochemical methods use antigen and
antibodies as tools to detect microorganisms.

DEFINATIONS
• Antigens are foreign substances, usually high-
molecular weight proteins or carbohydrates, that
elicit the production of other protein called
antibodies, in human or animal host.
• Antibodies attach to the antigens and aid the host
in removing the infectious agent.
• Antigens may be part of the physical structure of
the pathogen, such as the bacterial cell wall, or
they may be a chemical produced and released
by the pathogen, such as an enzyme or
toxin(epitope).
• Antigens and antibodies, by definition, combine
with each other specifically and in an observable
manner.

USES OF ANTIGEN AND ANTIBODY
REACTIONS
• Helps antibody mediated immunity in
infection, and tissue injury in some
hypersensitivity and autoimmune diseases.
• Helps diagnosis of Infections.
• In epidemiological surveys
• Detections and quantization of antigens and
antibodies.
• Antigen-antibody reactions in vitro-
serological reactions.

SEROLOGY
• The branch of laboratory medicine that
studies blood serum for evidence of infection
and other parameters by evaluating antigen-
antibody reactions in vitro
• Serology is the scientific study of blood serum.
In practice, the term usually refers to the
diagnostic identification of antibodies in the
serum
• We can detect antigens too

SEROLOGICAL TESTS
• Antigen and antibody reactions in vitro are
known as serological tests. It can be studied in 3
stages
• 1st Antigen and antibody react with visible
effects, obeys the laws of physical dynamics. But
this reaction is reversible.
• The reaction is effected by weaker intermolecular
forces – Vander Waal’s forces, Ionic bonds,
Hydrogen bonding not by covalent bonding.
• It can be detected by Radioactive isotopes,
fluorescent dyes

SEROLOGICAL TESTS
• 2nd Reaction can occur as Precipitation,
Agglutination, Lysis and killing of live antigens,
Neutralization of toxins, Fixation of
complement, Immobilization of motile
microbes, Enhancement of Phagocytosis.
• 3rd reactions include humoral immunity
against infectious diseases as well as clinical
allergy and other immunological diseases.

SEROLOGICAL TESTS
• Types of serological tests/ antigen antibody
reactions:
• 1. Precipitation Reaction
• 2. Agglutination Reaction
• 3. Complement fixation tests(CFT)
• 4. Neutralization Tests
• 5. Immunofluorescence testing
• 6. Enzyme Immunoassays
• 7. Radioimmunoassay(RIA)
• 8. Chemiluminescence Immunoassay(CLIA)
• 9. Immunoelectroblot Techniques
• 10. Immunochromatographic Tests.

GENERAL FEATURES
• Specific reaction – combines with specific antigen
• Entire molecule reacts not fragments
• No denaturation of antigen or antibody
• Combination occurs as surface antigens to surface
of antibodies
• Combination is firm but reversible depends on
affinity and avidity
• Both antigens and antibodies participate
• Combine in varying proportions bivalent and
multivalent

GENERAL FEATURES
• Affinity: Antibody affinity is the strength of the
reaction between a single antigenic determinant
and a single combining site on the antibody.
• Avidity: It is a measure of the overall strength of
binding of an antigen with many antigenic
determinants and multivalent antibodies.
• Dilution: Estimating the antibody by determining
the greatest degree to which the serum may be
diluted without losing the power to given an
observable effect in a mixture with specific
antigen

GENERAL FEATURES
• Sensitivity
• –Analytical Sensitivity – ability of a test to detect very
small amounts of a substance
• –Clinical Sensitivity – ability of test to give positive
result if patient has the disease (no false negative
results)
• Specificity
• •Analytical Specificity – ability of test to detect
substance without interference from cross-reacting
substances
• •Clinical Specificity – ability of test to give negative
result if patient does not have disease (no false positive
results)

MEASUREMENT
• Many methods are available for the
measurement of antigen and antibodies i.e.
Mass Nitrogen (microgram) or As Units or As
titre.
• Titre: The highest dilution of the serum that
shows an observable reaction with the antigen
in the particular test.

PRECIPITATION - REACTIONS
• In precipitation when a soluble antigen combines
with its antibody in the presence of electrolytes
(NaCl) at a suitable temperature and pH, the
antigen and antibody complexes form a insoluble
precipitate.
• When instead of sedimenting, the precipitate
remains suspended as floccules this reaction is
known as flocculation.
• This reaction can take place in liquid medium,
gels, agar, agarose, polyacrylamide.

PRECIPITATION- REACTION
• The precipitation test may be carried out as a
qualitative or a quantitative test.
• It is very sensitive in the detection of antigens and as
little as 1 µg of protein can be detected.
• It therefore finds forensic application in the
identification of blood and seminal stains, and in
testing for food adulterants.
• Precipitation is relatively less sensitive for the
detection of antibodies.

Zone Phenomenon
• Precipitation occurs most rapidly and abundantly when
antigen and antibody are in optimal proportions or
equivalent ratio.
• These proportions are constant for all dilutions of the
same reagent.
• Zone of antibody excess:
 In this zone uncombined antibody shall be present.
 This is called zone of antibody excess or prozone.
 The prozone is of importance in clinical serology
since a serum sample having large amount of
antibody may give a false negative precipitation
result until several dilutions are tested.

Zone Phenomenon
• Zone of equivalence:
 In this zone both antigen and antibody are
completely precipitated and no uncombined antigen
or antibody is present.
 Maximum complement fixation also takes place in
this zone.
• Zone of antigen excess:
 In this zone all antibody has combined with antigen
and additional uncombined antigen is present.
 In this zone, precipitation is partially or completely
inhibited because soluble antigen antibody
complexes form in the presence of excess antigen.

PRECIPITATION- REACTION
Applications
• The following types of precipitation and flocculation tests
are in common use:
• Slide flocculation test
• Tube flocculation test
• Immunodiffusion: Following four combinations can
result:
 Single diffusion in one dimension.
 Double diffusion in one dimension.
 Single diffusion in two dimensions.
 Double diffusion in two dimensions
• Immunoelectrophoresis:

SLIDE TEST
• When a drop each of the antigen and the antiserum are
placed on a slide and mixed by shaking, floccules appear.
• APPLICATIONS: VDRL test for syphilis(slide flocculation).
TUBE TEST
• Serum dilution of toxin or toxoid is added to tubes
containing a fixed quantity of antitoxin
• The amount of toxin that flocculates optimally with one
unit of antitoxin is defined as Lf dose
• APPLICATIONS:
• Kahn test for Syphilis – a tube flocculation test.
• Toxoid Precipitation for Diphtheria.
• Quantitative tube flocculation test used in
standardarisation of toxin/toxoid.

PRECIPITATION - REACTION
NON REACTIVE REACTIVE

IMMUNODIFFUSION
• This term denotes precipitation in gel which
provides more sensitive and specific results.
• The reaction is in the form of bands of precipitation
and can be stained for better viewing as well as
preservation.
• If a large number of antigens are present, each
antigen-antibody reaction will give rise to a line of
precipitation.
• This technique also indicates identity, cross reaction
and non identity between different antigens.
• Various types of immunodiffusion tests are in
practice.
• These are based upon number of diffusions and
dimensions in which these take place.

Single diffusion in one Direction
(Oudin Procedure Ring test)
• This, the simplest type of precipitation test,
consists of layering the antigen solution over a
column of antiserum in a narrow tube.
• A precipitate forms at the junction of the two
liquids.
• Ring tests have only a few clinical applications
now.
• APPLICATIONS:
• Ascoli's thermo precipitin test for B. anthracis.
• Milk ring Test for Brucella.
• Lancefield technique for the grouping of
streptococci.

Double diffusion in one dimension
(Oakley- Fulthorpe procedure)
• Here, the antibody is incorporated in gel,
above which is placed a column of plain agar.
• The antigen is layered on top of this.
• The antigen and antibody move towards each
other through the intervening column of plain
agar and form a band of precipitate where
they meet at optimum proportion.

Single diffusion in two dimensions
(Radial immunodiffusion)
• Here the antiserum is incorporated in agar gel
poured on a flat surface (slide or Petri dish).
• The antigen is added to the wells cut on the
surface of the gel.
• It diffuses radially from the well and forms ring-
shaped bands of precipitation (halos)
concentrically around the well.
• The diameter of the halo gives an estimate of the
concentration of the antigen.
• APPLICATIONS:
• Estimation of the immunoglobulin classes in sera.
• IgG, IgM antibodies to influenza viruses.

Single diffusion in two dimensions
(Radial immunodiffusion)

Double diffusion in two dimensions
(Ouchterlony procedure)
• This is the immunodiffusion method most widely
employed and helps to compare different
antigens and antisera directly.
• Agar gel is poured on a slide and well are cut
using a template.
• The antiserum is placed in the central well and
different antigens in the surrounding wells.
• If two adjacent antigens are identical, the lines of
precipitate formed by them will fuse.
• If they are unrelated, the lines will cross each
other.

• Cross-reaction or partial identity is indicated by
spur formation.
• A special variety of double diffusion in two
dimensions is the Elek test for toxigenicity in
diphtheria bacilli.
• When diphtheria bacilli are streaked at right
angles to a filter paper strip carrying the antitoxin
implanted on a plate of suitable medium,
arrowhead-shaped lines of precipitation appear
on incubation, if the bacillus is toxigenic.

Incubate the plate for 24-48 hrs at 370C
P T N
Elek’s gel precipitation test

After incubation – line of precipitation can be observed
Where the toxin and antitoxin meet at optimum conc.
The lines of precipitation will indicate that
the test strain is toxigenic
P T N
Elek’s gel precipitation test

ELECTROIMMUNODIFFUSION
• The development of precipitin lines can be
speeded up by electrically driving the antigen and
antibody.
• Various methods have been described combining
electrophoresis with diffusion.
• Of these, one-dimensional double
electroimmunodjffusion (counterimmuno-
electrophoreis) and one-dimensional single
electroimmunodiffusion (rocket electrophoresis)
are used frequently in the clinical laboratory.

IMMUNOELECTROPHORESIS
• The resolving power of immunodiffusion was greatly
enhanced when Grabar and Williams devised the
technique of immunoelectrophoresis.
• This involves the electrophoretic separation of a
composite antigen (such as serum) into its constituent
protein, followed by immunodiffusion against its
antiserum, resulting in separate precipitin lines,
indicating reaction between each individual protein
with its antibody.
• This enables identification and approximate
quantitation of the various proteins present in the
serum.
• The technique is performed on agar or agarose gel on a
slide, with an antigen well and an antibody trough cut
on it.

IMMUNOELECTROPHORESIS
• The test serum is placed in the antigen well and
electrophoresed for about an hour.
• Antibody against human serum is then placed in the
trough and diffusion allowed to proceed for18-24
hours.
• The resulting precipitin lines can be photographed and
the slides dried, stained and preserved for record.
• Over 30 different proteins can be identified by this
method in human serum.
• This is useful for testing for normal and abnormal
proteins in serum and urine.

AGGLUTINATION REACTION
• When a particulate antigen is mixed with its antibody in
the presence of electrolytes at a suitable temperature
and pH, the particles are clumped or agglutinated.
• Agglutination is more sensitive than precipitation for the
detection of antibodies. The same principles govern
agglutination and precipitation.
• Agglutination occurs optimally when antigens and
antibodies react in equivalent proportions. The zone
phenomenon may be seen when either an antibody or an
antigen is in excess.
• 'Incomplete' or 'monovalent' antibodies do not cause
agglutination, though they combine with the antigen.
• They may act as 'blocking' antibodies, inhibiting
agglutination by the complete antibody added
subsequently.

APPLICATIONS
• SLIDE AGGLUTINATION: When a drop of the
appropriate antiserum is added to a smooth,
uniform suspension of a particulate antigen in a
drop of saline on a slide or tile, agglutination
takes place.
• A positive result is indicated by the clumping
together of the particles and the clearing of the
drop.
• The reaction is facilitated by mixing the antigen
and the antiserum with a loop or by gently
rocking the slide.
• Depending on the titre of the serum,
agglutination may occur instantly or within
seconds.

SLIDE AGGLUTINATION
• It is essential to have on the same slide a control
consisting of the antigen suspension in saline, without
the antiserum, to ensure that the antigen is not
autoagglutinable.
• Agglutination is usually visible to the naked eye but may
sometimes require confirmation under the microscope.
• APPLICATIONS:
• Blood Grouping for Blood Group antigens A, B, AB, O.
• Widal Test for Salmonella typhi, Salmonella paratyphi
A,B, and C.
• Bacterial Agglutination Test for Shigella, Salmonella and
vibrio

TUBE AGGLUTINATION
• This is a standard
quantitative method for the
measurement of antibodies.
• When a fixed volume of a
particulate antigen
suspension is added to an
equal volume of serial
dilutions of an antiserum in
test tubes, the agglutination
titre of the serum can be
estimated.
• Tube agglutination is
routinely used for the
serological diagnosis of:
• APPLICATIONS:
• Widal test for typhoid.
• Standard agglutination test
for Brucellosis.
• Weil-Felix Reaction for
serodiagnosis of typhus fever
• Indirect haemagglutination
test for Antibody detection in:
Amoebiasis.
Lymphatic filariasis.
Echinococcosis.
Toxoplasmosis.

PASSIVE AGGLUTINATION TEST
• The difference between the precipitation and
agglutination tests is the physical nature of the antigen.
• By attaching soluble antigens to the surface of carrier
particles, it is possible to convert precipitation tests
into agglutination tests, which are more convenient
and more sensitive for the detection of antibodies.
• Such tests are known as passive agglutination tests.
• The commonly used carrier particles are red cells, latex
particles or bentonite.
• Passive agglutination tests are very sensitive and yield
high titres, but may give false positive results.

Human or sheep erythrocytes:
• It will adsorb a variety of antigens.
• Polysaccharide antigens may be adsorbed by simple
mixing with the cells.
• APPLICATIONS:
• Passive hemagglutination test is the Rose- Waaler test.
• RA factor: In rheumatoid arthritis, an autoantibody
appears in the serum which acts as an antibody to
gammaglobulin.
• The RA factor is able to agglutinate red cells coated with
globulins.
• The antigen used for the test is a suspension of sheep
erythrocytes sensitised with a sub-agglutinating dose of
rabbit anti-sheep erythrocyte antibody (amboceptor).

POLYSTYRENE LATEX PARTCALS:
• Which can be manufactured as uniform spherical
particles, 0.8-1 m in diameter, can adsorb several
types of antigens.
• APPLICATIONS:
• Latex agglutination tests are widely employed in
the clinical laboratory for the detection of:
• ASO, CRP, RA factor, HCG, Cryptococcus
neoformans, Haemophilus influenzae,
Echinococcus granulosus and many other
antigens.

COMPLEMENT FIXATION TEST
• The complement fixation assay can be used to look for
the presence of i) specific antibody or ii) specific
antigen in a patient's serum.
• The test utilizes sheep red blood cells (SRBC), anti-SRBC
antibody and complement, along with specific antigen
or specific antibody
• If antibody (or antigen) is present in the patient's
serum, then the complement is completely utilized and
SRBC lysis is minimal.
• However, if the antibody (or antigen) is not present in
the patient's serum, then the complement binds anti-
SRBC antibody and lysis of the SRBCs ensues.
• APPLICATIONS: Treponema pallidum immobilization
Test for syphilis.

NEUTRALIZATION TESTS
• Neutralization is an antigen-antibody reaction in which
the biological effects of viruses and toxins are
neutralized by homologous antibodies known as
neutralizing antibodies.
• These tests are broadly of two types:
• Virus neutralization tests.
• Toxin neutralization tests.
Virus neutralization tests
• Neutralization of viruses by their specific antibodies are
called virus neutralization tests. Inoculation of viruses in
cell cultures, eggs, and animals results in the replication
and growth of viruses.

Virus neutralization tests
• When virus-specific neutralizing antibodies are injected
into these systems replication and growth of viruses is
inhibited.
• This forms the basis of virus neutralization test.
• Viral hemagglutination inhibition test is an example of
virus neutralization test frequently used in the diagnosis
of viral infections such as influenza, mumps, and measles.
• If patients's serum contains antibodies against certain
viruses that have the property of agglutinating the red
blood cells, these antibodies react with the viruses and
inhibit the agglutination of the red blood cells.

Toxin neutralization tests
• Toxin neutralization tests are based on the principle that
biological action of toxin is neutralized on reacting with
specific neutralizing antibodies called antitoxins.
• Examples of neutralization tests in-vivo include
• (a) Schick test to demonstrate immunity against
diphtheria and
• (b) Clostridium welchii toxin neutralization test in guinea
pig or mice.
• In-vitro neutralization tests include
• (a) antistreptolysin o test and
• (b) Nagler reaction used for rapid detection of c. welchii.

IMMUNOFLUORESCENCE
• Fluorescence is the property of absorbing light rays of one
particular wavelength and emitting rays with a different
wavelength.
• Fluorescent dyes show up brightly under ultraviolet light as
they convert ultraviolet into visible light.
• Coons and his colleagues (1942) showed that fluorescent
dyes can be conjugated to antibodies and that such
'labeled' antibodies can be used to locate and identify
antigens in tissues.
• This 'fluorescent antibody' or immunofluorescence
technique has several diagnostic and research applications.
• The fluorescent dyes commonly used:
• Fluorescein isothiocynate exhibiting - blue-green.
• Lissamine rhodamine exhibiting orange-red fluorescence.
• This technique is more sensitive than precipitation or
complement fixation techniques.

IMMUNOFLUORESCENCE
• The technique can easily detect at concentration around 1 μg
protein/ml body fluid.
• Major disadvantage with this technique is the frequent
occurrence of non-specific fluorescence in tissues and other
materials.
• The direct method
 Direct immunofluorescence test is used to detect unknown
antigen in a cell or tissue by employing known labeled
antibody that interacts directly with unknown antigen.
 If antigen is present, it reacts with labeled antibody and the
antibody-coated antigen is observed under UV light of the
fluorescence microscope
 APPLICATIONS: Antigen detection in infectious diseases
caused by: Respiratory syncytial virus, Measles, Mumps,
Rabies and Influenza.

IMMUNOFLUORESCENCE
• The indirect method
 The indirect immunofluorescence test is used for
detection of specific antibodies in the serum and
other body fluids for serodiagnosis of many
infectious diseases.
 Indirect immunofluorescence is a two-stage process.
 In the first stage, a known antigen is fixed on a slide.
 Then the patients's serum to be tested, which is
unlabeled, is applied to the slide, followed by careful
washing.
 If the patients's serum contains antibody against the
antigen, it will combine with antigen on the slide.

IMMUNOFLUORESCENCE
The indirect method
 In the second stage, the combination of antibody
with antigen can be detected by addition of a
fluorescent dye-labeled antibody to human IgG and
examined by a fluorescence microscope.
 This test is more sensitive than direct method.
 APPLICATIONS:
 Antibodies detection in infectious diseases of:
 Toxoplasmosis.
 Amoebiasis.
 Treponema pallidum

ENZYME IMMUNO ASSAYS
• Enzyme immunoassays are commonly called as
enzyme linked immunosorbent assays or ELISA.
• This is simple and versatile technique which is as
sensitive as radioimmunoassays.
• ELISA is perhaps now the most widely employed
technique for detection of antigens, antibodies,
hormones, toxins and viruses.
• Antibodies are conjugated with enzyme by addition of
glutaraldehyde so that resulting antibody molecule has
both immunological and enzyme activities and
quantified by their ability to degrade as suitable
substrate.

• The commonly used enzymes are alkaline phosphatase
and horse radish peroxidase.
• There respective substrates are p-nitrophenyl
phosphate and O-phenyl diamine dihydrochloride.
• Enzymatic activity results in a color change which can
be assessed visibly or quantified in a simple
spectrophotometer.
• ELISA can be performed with sensitized carrier surfaces
in the form of polystyrene tubes (macro-ELISA) or
polyvinyl microtitre plates (micro-ELISA) or even beads.

MicroPlates- Shape/Form
• Two main categories:
Solid Plates
Strip Plates; Non
breakable or breakable
wells
• Color: Clear, White, Black
• Well Shape:
Flat
C bottom
Round Bottom
Star/Fin Bottom

• PRINCIPLE:
• The wells of a microtitre plate are coated with antigens.
• After thorough washing, the serum samples to be tested
are added and incubated for two hours at 37°C.
• Suitable positive and negative controls are also set up.
• The wells are washed and enzyme conjugate secondary
antibody, labeled with alkaline phosphatase, added and
incubated at 37°C for one hour.
• After washing, a suitable substrate (paranitrophenyl
phosphate) is added and held at room temperature till the
positive controls show the development of a yellow colour.
• The phosphatase enzyme splits the substrate to yield a
yellow compound

• APPLICATIONS:
 Antibody and Antigen detection in infectious agents
of:
 M. Tuberculosis
 Rickettsia
 HSV
 HIV
 HBV
 HCV
 DENGUE
 VZV
 Rota virus and etc.

RADIO IMMUNO ASSAYS
• It is an extremely sensitive technique in which antibody or
antigen is labelled with a radioactive material (I125).
• The amount of radioactive material in the antigen-antibody
complex can be measured with which concentration of
antigen or antibody can be assayed.
• This test is also called as binder-ligand assay where binder
is the component to which radioactive material is labelled
and ligand (or analyte), is the component (antigen or
antibody) which is to be assayed or detected.
• In radioimmunoassay, fixed amount of antibody and
radioactive material labelled antigen react in the presence
of unlabelled antigen (test antigen).
• After the reaction 'free' and 'bound' fractions of antigen are
separated and their radioactivity measured.
• The concentration of test antigen can be calculated from
the ratio of the bound and total antigen label using
appropriate standards.

CHEMILUMINESCENCE IMMUNOASSAY
(CLIA)
• Chemiluminescence refers to a chemical reaction
emitting energy in the form of light.
• It is just as radioactive conjugates are employed in RIA,
fluorescent conjugates in IFA and enzymes in ELISA,
chemiluminescent compounds such as luminol or
acridinium esters are used in CLIA as the label to
provide the signal during the antigen-antibody
reaction. The signal (light) can be amplified, measured
and the concentration of the analyte calculated.
• The method has been fully automated and is being
increasingly used in laboratories where the volume of
work is large.

IMMUNOELECTROBLOT TECHNIQUES
• Immunoelectroblot techniques combine the sensitivity of
enzyme immunoassay with much greater specificity.
• The technique is a combination of three separate
procedures:
• (a) Separation of ligand-antigen components by
polyacrylamide gel electrophoresis;
• (b) blotting of the electrophoresed ligand fraction on
nitrocellulose membrane strips; and
• (c) enzyme immunoassay to detect antibody in test sera
against the various ligand fraction bands; or probe with
known antisera against specific antigen bands.
• APPLICATIONS: The western blot test, considered the
definitive test for the serodiagnosis of HIV infection.

IMMUNOCHROMATOGRAPHIC TESTS
• A one-step qualitative immunochromatographic
(ICG) technique has found wide application in
serodiagnosis due to its simplicity, economy and
reliability.
• The test system is a small cassette containing a
membrane impregnated with anti - HBsAg
antibody colloidal gold dye conjugate.
• The membrane is exposed at three windows on
the cassette.
• The test serum is dropped into the first window.

• As the serum travels upstream by capillary action, a
coloured band appears at the second window (test
site) if the serum contains "HbsAg, due to the
formation of a HBsAg antibody conjugate complex.
• This is the positive reaction.
• Absence of a coloured band at the test site indicates a
negative reaction.
• Simultaneously a coloured band should appear in every
case at the third window which forms an inbuilt
control, in the absence of which the test is invalid.
• The test is claimed to be nearly a sensitive and specific
as EIA tests.

(ICG)
APPLICATIONS:
 Antibody and Antigen
detection in infectious
diseases of:
 HIV
 HBV
 HCV
 DENGUE
 VZV
 Rota virus and etc.

Flow Cytometry
• Flow cytometry is commonly used in the clinical
laboratory to identify and enumerate cells bearing a
particular antigen.
• Cells in suspension are labeled with a fluorescent tag by
either direct or indirect immunofluorescence.
• The cells are then analyzed on the flow cytometer.
• In a flow cytometer, the cells exit a flow cell and are
illuminated with a laser beam.
• The amount of laser light that is scattered off the cells as
they passes through the laser can be measured, which
gives information concerning the size of the cells.

Flow Cytometry
• In addition, the laser can excite the fluorochrome on the
cells and the fluorescent light emitted by the cells can be
measured by one or more detectors.
• In a one parameter histogram, increasing amount of
fluorescence (e.g. green fluorescence) is plotted on the x
axis and the number of cells exhibiting that amount of
fluorescence is plotted on the y axis.
• The fraction of cells that are fluorescent can be
determined by integrating the area under the curve.
• In a two parameter histogram, the x axis is one parameter
(e.g. red fluorescence) and the y axis is the second
parameter (e.g. green fluorescence).
• The number of cells is indicated by the counter and the
intensity of the color.

Figure 5.7 Flow-cytometry "dot plots." In panel A, cells stained with the red CD4 antibody account for 59% of all lymphocytes; this is a
normal sample. In panel B, there is a reduction in the number of red-staining CD4+ T cells. This is a sample from a patient with HIV
infection.PE; phycoerythrin (emits red light)FITC; fluorescent isothiocyanate (emits green light)Flow cytometric plots courtesy of John
Hewitt, Immunology, Manchester Royal Infirmary
Downloaded from: StudentConsult (on 15 December 2009 10:26 AM)
© 2005 Elsevier

Antigen & Antibody Interactions

Antigen & Antibody Interactions

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