ANTIGEN - ANTIBODY INTERACTIONSPrinciples & 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

DILUTION

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)

Single 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.

Double diffusion in two dimensions (Ouchterlony procedure)

Double diffusion in two dimensions (Ouchterlony procedure)

• 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 observedWhere 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

43

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.

PASSIVE AGGLUTINATION TESTHuman 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).

Passive hemagglutination test

PASSIVE AGGLUTINATION TESTPOLYSTYRENE 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.

Latex agglutination tests

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

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.

IMMUNOFLUORESCENCEThe 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

IMMUNOFLUORESCENCE

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.

ENZYME IMMUNO ASSAYS• 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

ENZYME IMMUNO ASSAYS• 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

ENZYME IMMUNO ASSAYS• 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.

RADIO IMMUNO ASSAYS

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.

WESTERN BLOT TEST

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.

IMMUNOCHROMATOGRAPHIC TESTS

• 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.

IMMUNOCHROMATOGRAPHIC 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

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