Antigen – Antibody reactions

Dr. Pendru Raghunath ReddyAssistant Professor of MicrobiologyDr. VRK Women’s Medical College

Antigens and antibodies combine with each other Antigens and antibodies combine with each other specifically and in an observable mannerspecifically and in an observable manner

In the body, they form the basis of antibody mediated In the body, they form the basis of antibody mediated immunity in infectious diseases, or hypersensitivity and immunity in infectious diseases, or hypersensitivity and autoimmune diseasesautoimmune diseases

Antigen – antibody reactions in vitro are known as Antigen – antibody reactions in vitro are known as serological reactionsserological reactions

In laboratory, they help in diagnosis of infections, in In laboratory, they help in diagnosis of infections, in epidemiological surveys, in the identification of infectious epidemiological surveys, in the identification of infectious agents, enzymesagents, enzymes

Stages of Ag – Ab reactionsStages of Ag – Ab reactions

Primary stagePrimary stage

Initial interaction between Ag & Ab – invisibleInitial interaction between Ag & Ab – invisible

Rapid, occurs at low temperatures & obeys the general Rapid, occurs at low temperatures & obeys the general laws of physical chemistry & thermodynamicslaws of physical chemistry & thermodynamics

Reaction is reversibleReaction is reversible

Ag & Ab is bound to each other by weak Van der Waal’s Ag & Ab is bound to each other by weak Van der Waal’s forces, Ionic bonds & Hydrogen bondingforces, Ionic bonds & Hydrogen bonding

Ag-Ab interactionsAg-Ab interactionsBonds:Bonds: HydrogenHydrogen IonicIonic Hydrophobic interactionsHydrophobic interactions Van der Waals forcesVan der Waals forces

Each bond is weak; many Each bond is weak; many are are strongstrong

To “hold” they must be close To “hold” they must be close requiring high amts of requiring high amts of complementarity!complementarity!

Secondary stageSecondary stage

Demonstrable events – Precipitation, agglutination, lysis of Demonstrable events – Precipitation, agglutination, lysis of cells, killing of live antigens, neutralization of toxins, cells, killing of live antigens, neutralization of toxins, complement fixation, immobilization of motile organisms & complement fixation, immobilization of motile organisms & enhancement of phagocytosis.enhancement of phagocytosis.

PrecipitinPrecipitin – Ab participate in precipitation – Ab participate in precipitation

AgglutininAgglutinin - Ab participate in agglutination - Ab participate in agglutination

PrecipitinogenPrecipitinogen – Ag participate in precipitation – Ag participate in precipitation

AgglutinogenAgglutinogen - Ag participate in agglutination - Ag participate in agglutination

Tertiary stageTertiary stage

Includes neutralization or destruction of injurious agents Includes neutralization or destruction of injurious agents or tissue damageor tissue damage

Also includes humoral immunity against infectious Also includes humoral immunity against infectious diseases as well as clinical allergy & other immunological diseases as well as clinical allergy & other immunological diseasesdiseases

GENERAL FEATURES OF Ag – Ab GENERAL FEATURES OF Ag – Ab REACTIONSREACTIONS

1.1. The reaction is specificThe reaction is specific

2.2. Entire molecules react and not the fragmentsEntire molecules react and not the fragments

3.3. There is no denaturation of the antigen or antibody during the There is no denaturation of the antigen or antibody during the reactionreaction

4.4. The combination occurs at the surface. So surface antigens are The combination occurs at the surface. So surface antigens are immunologically relevantimmunologically relevant

5.5. The combination is firm but reversible. The firmness is influenced by The combination is firm but reversible. The firmness is influenced by the affinity & avidity of the reactionthe affinity & avidity of the reaction

6.6. Antigens & antibodies can combine in varying proportions. Both Ags Antigens & antibodies can combine in varying proportions. Both Ags & Abs are multivalent & Abs are multivalent

Affinity = ∑ attractive and repulsive forces

Ab

Ag

High Affinity

Ab

Ag

Low Affinity

Affinity• Refers to the intensity of attraction between the

antigen & antibody molecules. It is the function of closeness of fit between the epitope & antigen binding region of its Ab

Avidity

• Strength of the bond after the formation Ag-Ab complexes

• The overall strength of binding between an Ag with many determinants and multivalent Abs

Keq = 104

Affinity 106

Avidity

1010

Avidity

SpecificitySpecificity

The ability of an individual antibody combining site to The ability of an individual antibody combining site to react with only one antigenic determinantreact with only one antigenic determinant

The ability of a population of antibody molecules to react The ability of a population of antibody molecules to react with only one antigenwith only one antigen

Cross Reactivity• The ability of an individual Ab combining site to

react with more than one antigenic determinant.• The ability of a population of Ab molecules to

react with more than one Ag

Anti-A Ab

Ag A

Anti-A Ab

Ag B

Shared epitope

Anti-A Ab

Ag C

Similar epitope

Cross reactions

Factors Affecting Measurement of Ag/Ab Reactions

• Affinity

• Avidity

• Ag:Ab ratio

• Physical form of Ag

Ab excess Ag excess

Equivalence – Lattice formation

Types of Antigen – Antibody ReactionsTypes of Antigen – Antibody Reactions1.1. Precipitation reactionPrecipitation reaction

2.2. Agglutination reactionAgglutination reaction

3.3. Neutralization reactionNeutralization reaction

4.4. Opsonisation Opsonisation

Serological tests based on Ag – Ab reactionsSerological tests based on Ag – Ab reactions1.1. Complement fixation testComplement fixation test

2.2. ImmunofluorescenceImmunofluorescence

3.3. RadioimmunoassayRadioimmunoassay

4.4. Enzyme immunoassayEnzyme immunoassay

PRECIPITATION REACTIONPRECIPITATION REACTION

PRINCIPLE PRINCIPLE

When a soluble Ag combines with its Ab in the When a soluble Ag combines with its Ab in the presence of electrolytes (NaCl) at a suitable presence of electrolytes (NaCl) at a suitable temperature & pH, the Ag-Ab complex forms an temperature & pH, the Ag-Ab complex forms an insoluble precipitate.insoluble precipitate.

When instead of sedimenting, the precipitate remains When instead of sedimenting, the precipitate remains suspended as floccules – suspended as floccules – Flocculation reactionFlocculation reaction

Precipitation can take place in liquid media or in gels Precipitation can take place in liquid media or in gels such as agar, agarose or polyacrylamide.such as agar, agarose or polyacrylamide.

ZONE PHENOMENONZONE PHENOMENON

The amount of precipitate formed is greatly influenced by The amount of precipitate formed is greatly influenced by the relative proportions of Ags & Absthe relative proportions of Ags & Abs

If increasing quantities of Ags are added to the same If increasing quantities of Ags are added to the same amount of antiserum in different tubes, precipitation is amount of antiserum in different tubes, precipitation is found to occur most rapidly & abundantly in the middle found to occur most rapidly & abundantly in the middle tubestubes Preceding tubes – Ab excess (Preceding tubes – Ab excess (ProzoneProzone)) Middle tubes – Ag & Ab in equivalent proportions Middle tubes – Ag & Ab in equivalent proportions

((Zone of equivalenceZone of equivalence)) Later tubes – Ag excess (Later tubes – Ag excess (Post zonePost zone))

Mechanism of precipitationMechanism of precipitation

Marrack (1934) proposed the lattice hypothesis – Marrack (1934) proposed the lattice hypothesis – mechanism of precipitationmechanism of precipitation

The multivalent antigens combine with bivalent Abs in The multivalent antigens combine with bivalent Abs in varying proportions, depending on the Ag – Ab ratio on varying proportions, depending on the Ag – Ab ratio on the reacting mixturethe reacting mixture

Precipitation results when a large lattice is formed Precipitation results when a large lattice is formed consisting of alternating Ag & Abconsisting of alternating Ag & Ab

Marrack’s hypothesis

Applications of Precipitation reactionApplications of Precipitation reaction

It can be carried out as either a quantitative or qualitative testIt can be carried out as either a quantitative or qualitative test

Sensitive for the detection of AgsSensitive for the detection of Ags

1.1. Identification of bacteria eg: Lancefield’s grouping of Identification of bacteria eg: Lancefield’s grouping of StreptococcusStreptococcus

2.2. Detection of antibody for diagnostic purposesDetection of antibody for diagnostic purposes

eg: VDRL in syphiliseg: VDRL in syphilis

Types of precipitation reactions

1.Ring test

2.Flocculation test

3.Immunodiffusion

4.Electroimmunodiffusion

RING TESTRING TEST Consists of layering Ag solution over a column of Consists of layering Ag solution over a column of

antisera in a narrow tubeantisera in a narrow tube

Eg: Ascolis thermoprecipitin test, Grouping of Eg: Ascolis thermoprecipitin test, Grouping of Streptococci Streptococci by Lancefield techniqueby Lancefield technique

Flocculation testFlocculation test

Slide testSlide test

When a drop of Ag & antiserum is placed on a slide & mixed by When a drop of Ag & antiserum is placed on a slide & mixed by shaking, floccules will appearshaking, floccules will appear

Eg: VDRL test & RPR test for syphilis Eg: VDRL test & RPR test for syphilis

Tube testTube test

The Kahn test (tube flocculation) for syphilisThe Kahn test (tube flocculation) for syphilis

This is also employed for the standardization of toxins & This is also employed for the standardization of toxins & toxoidstoxoids

Serial dilutions of toxin/toxoid are added to the tubes Serial dilutions of toxin/toxoid are added to the tubes containing a fixed quantity of antitoxincontaining a fixed quantity of antitoxin

The amount of toxin that flocculates optimally with one The amount of toxin that flocculates optimally with one unit of the antitoxin – Lf doseunit of the antitoxin – Lf dose

IMMUNODIFFUSION (precipitation in gel)IMMUNODIFFUSION (precipitation in gel)

Advantages of immunodiffusion:Advantages of immunodiffusion:

Reaction is visible as a distinct band of precipitationReaction is visible as a distinct band of precipitation

Stable, can be stained for preservationStable, can be stained for preservation

Indicates identity, cross reactions, non identity between Indicates identity, cross reactions, non identity between different Agsdifferent Ags

Various immunodiffusion testsVarious immunodiffusion tests1. 1. Single diffusion in one dimension (Oudin Single diffusion in one dimension (Oudin

procedure)procedure) Ab is incorporated in agar gel in a test tube & Ag Ab is incorporated in agar gel in a test tube & Ag

solution is layered over itsolution is layered over it

Ag diffuses downward through the agar gel – forming a Ag diffuses downward through the agar gel – forming a

line of precipitationline of precipitation..

2. Double diffusion in one dimension (Oakley 2. Double diffusion in one dimension (Oakley Fulthorpe procedureFulthorpe procedure))

Ab is incorporated in agar gelAb is incorporated in agar gel

Above which is placed a column of plain agarAbove which is placed a column of plain agar

The Ag is layered over itThe Ag is layered over it

The Ag & Ab move towards each other through the The Ag & Ab move towards each other through the intervening column of plain agar & form the precipitateintervening column of plain agar & form the precipitate

3. 3. Single diffusion in two dimensions (Radial Single diffusion in two dimensions (Radial immunodiffusionimmunodiffusion))

Here the antisera is incorporated in a gel & poured on a Here the antisera is incorporated in a gel & poured on a flat surfaceflat surface

Wells are cut on the surface to which Ag is addedWells are cut on the surface to which Ag is added

It diffuses radially from the well & forms ring shaped It diffuses radially from the well & forms ring shaped bands of precipitation concentrically around the wellbands of precipitation concentrically around the well

Radial Immunodiffusion (Mancini)Radial Immunodiffusion (Mancini)

InterpretationInterpretation Diameter of ring is Diameter of ring is

proportional to the proportional to the concentrationconcentration

QuantitativeQuantitative Ig levelsIg levels

• Method– Ab in gel– Ag in a well

Ag Concentration

Dia

met

er2

AgAgAgAg

Ab in gel

Uses

1. It has been widely employed for estimation of immunoglobulin classes i.e. IgG, IgM, IgA in sera

2. It has also been used for screening sera for antibodies to influenza viruses

44. . Double diffusion in two dimensions (Ouchterlony Double diffusion in two dimensions (Ouchterlony procedureprocedure))

Helps to compare different antisera & antigens directlyHelps to compare different antisera & antigens directly

Agar gel is poured on a slide & wells are cut Agar gel is poured on a slide & wells are cut

Antiserum – central wellAntiserum – central well

Different Ags in the surrounding wellsDifferent Ags in the surrounding wells

Reaction of identity

Partial identity

Lack of relatedness

Elek’s gel precipitation

5. 5. ImmunoelectrophoresisImmunoelectrophoresis

This involves the electrophoretic separation of composite Ag into This involves the electrophoretic separation of composite Ag into its constituent proteins, followed by immunodiffusion against its its constituent proteins, followed by immunodiffusion against its antiserum – separate precipitin linesantiserum – separate precipitin lines

It is performed on an agarose gel with an Ag well & Ab trough cut It is performed on an agarose gel with an Ag well & Ab trough cut on iton it

The test serum is placed in the antigen well & electrophoresed The test serum is placed in the antigen well & electrophoresed for about 1 hourfor about 1 hour

Ab against human serum is placed in the trough & diffusion allowed Ab against human serum is placed in the trough & diffusion allowed for 18 – 24 hrsfor 18 – 24 hrs

Immunoelectrophoresis

Uses

1. By this technique, a number of antigens can be identified in human serum

2. It is particularly useful for detection of normal and abnormal serum proteins like myeloma proteins

ELECTROIMMUNODIFFUSIONELECTROIMMUNODIFFUSION

The development of precipitin lines can be speeded up The development of precipitin lines can be speeded up by electrically driving the Ag & Abby electrically driving the Ag & Ab

Two types Two types

1.1. Counterimmunoelectrophoresis (One dimensional Counterimmunoelectrophoresis (One dimensional double electroimmunodiffusion)double electroimmunodiffusion)

2.2. Rocket electrophoresis (One dimensional single Rocket electrophoresis (One dimensional single electroimmunodiffusion)electroimmunodiffusion)

1.1. Counterimmunoelectrophoresis (CIE)Counterimmunoelectrophoresis (CIE)

This involves simultaneous electrophoresis of Ag & Ab in This involves simultaneous electrophoresis of Ag & Ab in gel in opposite directions resulting in precipitation at a point gel in opposite directions resulting in precipitation at a point between thembetween them

Used only when Ag and Ab have opposite chargesUsed only when Ag and Ab have opposite charges

Produce precipitation lines within 30 minsProduce precipitation lines within 30 mins

Clinical application: detecting Ags like alphafetoprotein in Clinical application: detecting Ags like alphafetoprotein in serum, Ags of Cryptococcus & Meningococcus in the CSFserum, Ags of Cryptococcus & Meningococcus in the CSF

It is also applied for detecting hepatitis B antigens and It is also applied for detecting hepatitis B antigens and antibodiesantibodies

Ag Ab- +

2. 2. Rocket electrophoresisRocket electrophoresis Used for quantitative estimation of AgsUsed for quantitative estimation of Ags

The antiserum to the Ag to be quantitated is incorporated in The antiserum to the Ag to be quantitated is incorporated in agarose gel on a slideagarose gel on a slide

Ag in increasing concentrations, is placed in wells punched in Ag in increasing concentrations, is placed in wells punched in the set gelthe set gel

The Ag is electrophoresed into the Ab containing agaroseThe Ag is electrophoresed into the Ab containing agarose

The pattern of immunoprecipitation resembles a The pattern of immunoprecipitation resembles a ROCKETROCKET

The length of these rocket like structures corresponds to The length of these rocket like structures corresponds to the concentration of the antigenthe concentration of the antigen

Rocket electrophoresis

Laurell’s two dimensional electrophoresisLaurell’s two dimensional electrophoresis

Variant of rocket electrophoresisVariant of rocket electrophoresis

Used to quantitate each of the several Ags in a mixtureUsed to quantitate each of the several Ags in a mixture

In the first stage, the Ag mixture is electrophoretically In the first stage, the Ag mixture is electrophoretically separated separated

In second stage, electrophoresis is done perpendicular to In second stage, electrophoresis is done perpendicular to that of first stage to get rocket like precipitationthat of first stage to get rocket like precipitation

Agglutination

When particulate antigen combines with its antibody in the presence of electrolytes at an optimal temperature and pH, resulting in visible clumping of particles

More sensitive than precipitation for the detection of antibodies

The agglutination reaction takes place better with IgM antibody

Lattice formation hypothesis holds good for aggltination too

Blocking antibodies inhibit the agglutination by the complete antibody added subsequently

Types of agglutination reactions

1.Side agglutination test

2.Tube agglutination test

3.The antiglobulin (Coombs) test

4.Heterophile agglutination test

5.Passive agglutination test

Slide agglutination test

A uniform suspension of antigen is made in a drop of saline on a slide and a drop of the appropriate antiserum is added

Reaction is facilitated by mixing the antigen and the antiserum with a wire loop or by gently rocking the slide

Clumping occurs instantly or within seconds when agglutination test is positive

A control consisting of antigen suspension in saline, without adding antiserum must be included on the same slide

Uses

1. It is a routine procedure to identify the bacterial strains isolated from clinical specimens (eg: Identification of Salmonella species)

2. It is also used for blood grouping and cross matching

Tube agglutination test

What is the titer of Ab?

The titer is customarily reported as the reciprocal of the highest dilution of Ab that causes an obvious agglutination

No agglutinationAgglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 40

Tube Agglutination TestTube Agglutination Test

Uses

Used for serological diagnosis of

1.Enteric fever (Widal test)

2.Typhus fever (Weil-Felix reaction)

3.Infectious mononucleosis (Paul-Bunnel test)

4.Brucellosis (SAT)

5.Primary atypical pneumonia (Streptococcus MG agglutination test)

Problems related to tube agglutination

1.Prozone phenomenon

2.Blocking antibodies

Blocking or incomplete antibodies may be detected by performing the test in hypertonic (5%) saline or albumin saline

Antiglobulin (Coombs) test is more reliable for detecting these antibodies

The antiglobulin (Coombs test)

Originally devised by Coombs, Mourant and Race (1945) for thedetection of incomplete anti-Rh antibodies

There are two types of Coombs test

1.Direct Coombs test

2.Indirect Coombs test

+ ↔

Patient’s RBCs Coombs Reagent(Antiglobulin)

Direct Coombs test

Patient’s Serum

TargetRBCs

+ ↔Step 1

+ ↔

Coombs Reagent(Antiglobulin)

Step 2

Indirect Coombs test

The only difference between the two is that the sensitisation of the erythrocytes with incomplete antibodies takes place in vivo in direct type whereas it occurs in vitro in indirect type

Uses of Coombs test

1.For detection of anti-Rh antibodies

2.For demonstration of any type of incomplete antibody (eg: Brucellosis)

Heterophile agglutination test

Heterophile antibodies have a property to react with microorganisms or cells of unrelated species due to common antigenic sharing

i) Weil-Felix reaction

Some proteus (OX19, OX2, and OXK) strains are agglutinated by sera of patients with rickettsial infections

This is due to antigenic sharing between these Proteus strains and Rickettsial species

ii) Paul-Bunnel test

Sheep erythrocytes are agglutinated by sera of infectious -mononucleosis’

iii) Streptococcus MG agglutination test

It is positive in primary atypical pneumonia

Passive agglutination test

A precipitation reaction can be converted into agglutination test by attaching soluble antigens to the surface of carrier particles such as latex particles, bentonite and red blood cells

Such tests are called passive agglutination tests

When instead of antigen, the antibody is adsorbed on the carrier particles for estimation of antigens, it is known as reversed passive agglutination

Latex agglutination test

Polystyrene latex particles (0.8 – 1 µm in diameter) are widely employed to adsorb several types of antigens

This test is convenient, rapid and specific

Used for detection of hepatitis B antigen, ASO, CRP, RA factor, HCG and many other antigens

Latex agglutination tile is used to perform this test

Haemagglutination test

Erythrocytes sensitised with antigen are used for detection of antibodies

Rose-Waaler test for detection of RA factor in patient serum

The antigen used for the test is sheep red blood cells sensitised with rabbit antisheep erythrocyte antibody (amboceptor)

Coagglutination

Some strains of Staphylococcus aureus (especially Cowan 1 strain) possess protein A on their surface

When specific IgG molecule is coated on these strains, Fc portion of IgG molecule binds to protein A whereas antigen combining Fab terminal reamains free

When the corresponding antigen is mixed with these coated cells, Fab terminal binds to antigen resulting in agglutination

This test is used for detection of bacterial antigens in blood, urine and CSF (eg: Gonocooci, Streptococcus pyogenes and Haemophilus influenzae)

Complement fixation test

Complement is a protein (globulin) present in normal serum

Whole complement system is made up of nine components: C1 to C9

Complement proteins are heat labile and are destroyed by heating at 56°C for 20 – 30 minutes

Principle

The antigen-antibody complexes have ability to fix complement

This reaction has no visible effect

To detect the fixation of complement, an indicator system consisting of sheep erythrocytes coated with amboceptor is used

Components of CFTComponents of CFT

Test SystemTest System Antigen:Antigen: It may be soluble or particulate. It may be soluble or particulate.

Antibody: Antibody: Human serum (May or may not contain Antibody Human serum (May or may not contain Antibody towards specific Antigen)towards specific Antigen)

Complement:Complement: It is pooled serum obtained from 4 to 5 guinea It is pooled serum obtained from 4 to 5 guinea pigs. It should be fresh or specially preserved as the pigs. It should be fresh or specially preserved as the complement activity is heat labile (stored at -30 °C in small complement activity is heat labile (stored at -30 °C in small fractions). The complement activity should be initially fractions). The complement activity should be initially standardized before using in the teststandardized before using in the test

Indicator System (Haemolytic system)Indicator System (Haemolytic system) Erythrocytes: Erythrocytes: Sheep RBCSheep RBC

Amboceptor (Hemolysin):Amboceptor (Hemolysin): Rabbit antibody to sheep red Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.under standard immunization protocol.

Controls

Antigen and serum controls are included in the test

Complement control is used to ensure that the desired amount has been added

Cell control to make sure that sensitised erythrocytes do not undergo lysis in the absence of complement

Positive TestPositive Test

Step 1:Step 1:

At 37°CAt 37°C

Antigen + Antibody + Complement Antigen + Antibody + Complement Complement gets fixed Complement gets fixed

(from serum)(from serum) 1 Hour 1 Hour

Step 2:Step 2:

At 37°CAt 37°C

Fixed Complement complex + Haemolytic system No HaemolysisFixed Complement complex + Haemolytic system No Haemolysis

1 Hour (1 Hour (Test Test Positive)Positive)

Negative Test

Step 1:

At 37°C

Antigen + Antibody absent + Complement Complement not fixed

1 Hour

Step 2:

At 37°C

Free Complement + Haemolytic system Haemolysis

1 Hour (Test Negative)

Results and Interpretations:Results and Interpretations:

No haemolysis is considered as a No haemolysis is considered as a positive testpositive test

Haemolysis of erythrocytes indicative of a Haemolysis of erythrocytes indicative of a negative testnegative test

1 2 3 41 2 3 4

AA

BB

Microtiter plate showing Haemolysis (Well A3, A4 and B4) Microtiter plate showing Haemolysis (Well A3, A4 and B4) and No Haemolysis (Well A1, B1, B2, and B3)and No Haemolysis (Well A1, B1, B2, and B3)

Indirect complement fixation test

Certain avian (eg: duck, parrot) and mammalian (eg: horse, cat) sera cannot fix guinea pig complement

Test is done in duplicate and after the first step, the standard antiserum known to fix complement is added in one set

Positive test

First step: Antigen + test serum (positive for antibody) + guinea pig complement

Second step: Standard antiserum cannot react with antigen because has been used up by antibody in the first step, hence, complement is free

Indicator system: Haemolysis occurs because complement is free

Negative test

First step: Antigen + test serum (negative for antibody) + guinea pig complement

Second step: Standard antiserum will react with antigen and fix the free complement

Indicator system: No haemolysis because complement is not free

Conglutination

This is an alternative method for systems which do not fix guinea pig complement

The indicator system is sheep erythrocytes sensitised with bovine serum

Bovine serum contains a a beta globulin component named conglutinin

Conglutinin can cause agglutination of sensitised sheep erythrocytes, if these are combined with complement

No agglutination – Positive result Agglutination – Negative result

Complement dependent serological tests

1.Immobilisation test

2.Immune adherence

3.Cytolytic or cytocidal tests

Neutralisation test

Opsonisation

Immunofluorescence

Fluorescence is the property of certain dyes which absorb rays of one particular wavelength (UV light) and emit rays with a different wavelength (visible light)

Coons and his colleagues showed that fluorescent dyes can be conjugated to antibodies and these labelled antibodies can be used to detect antigens in tissues

The commonly used fluorescent dyes are

i)Fluorescin isothiocyanate (Blue green fluorescence)

ii)Lissamine rhodamine (orange red fluorescence)

Immunofluorescence test is of two types

1.Drect immunofluorescence test

2.Indirect immunofluorescence test

Direct immunofluorescence test

Uses

1. It is commonly employed for detection of bacteria, viruses or other antigens in blood, CSF, urine, faeces, tissues and other specimens

2. It is a sensitive method to diagnose rabies by detection of the rabies virus antigens in brain smears

Disadvantage

Separate specific fluorescent labelled antibody has to be prepared against each antigen to be tested

Indirect immunofluorescence test

Advantages

A single antihuman globulin fluorescent conjugate can be employed for detection of antibody to any antigen

All antibodies are globulin in nature, therefore, antihuman globulin attaches to all antibodies

This has overcome the disadvantage of direct immuno- -fluorescence test

Radioimmunoassay (RIA)

Berson and Yallow (1959) first described

Since then it has been utilised for quantitation of hormones, drugs, HBsAg, IgE and viral antigens

Radioimmunoassay is widely-used because of its great sensitivity Using antibodies of high affinity, it is possible to detect a few picograms (10−12 g) of antigen

The greater the specificity of the antiserum, the greater the specificity of the assay

FIGURE 6-9

A solid-phase radioimmunoassay (RIA) to detect hepatitis B virus in blood samples & A standard curve to determine the conc. of HBsAg in unknown serum.

The principle involves competitive binding of radiolabeled Ag and unlabeled Ag to the limited supply of a high affinity Ab

Disadvantages

Radiation hazards: Uses radiolabelled reagents

Requires specially trained persons

Labs require special license to handle radioactive material

Requires special arrangements for

Requisition, storage of radioactive material radioactive waste disposal

Enzyme Linked Immunosorbent Assay (ELISA)

Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample

Term was coined by Engvall and Pearlmann in 1971

Similar to RIA, except no radiolabel

Very sensitive, pg/mL

Different types of ELISAs

1.Indirect ELISA

2.Sandwich ELISA

3.Competitive ELISA

4.Cassette or cylinder ELISA

Indirect ELISA

Two commonly used enzymes

1.Horseradish peroxidase (HRP)

2.Alkaline phosphatase (AP)

Sandwich ELISA

Two antibodies required; must recognize different epitopes

1st Antibody is referred to as capture Ab

2nd antibody as detection Ab

Competitive ELISA

The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabelled)

The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal).

Cassette or cylinder ELISA

It is a simple modification of ELISA for testing one or few samples of sera at a time

The test is rapid (about ten minutes) as compared with the 2 - 4 hours taken for conventional ELISA

Uses of ELISA

Used for detection of antigens and antibodies for various microorganisms

1)Detection of HIV antibodies in serum

2)Detection of mycobacterial antibodies in tuberculosis

3)Detection of rotavirus in faeces

4)Detection of hepatitis B markers in serum

5)Detection of enterotoxin of E. coli in faeces

Western blotting (Immunoblotting)

Western Blot for detection of HIV antibodyWestern Blot for detection of HIV antibody

HIV-1 Western BlotHIV-1 Western Blot Lane1: Positive ControlLane1: Positive Control Lane 2: Negative ControlLane 2: Negative Control Sample A: NegativeSample A: Negative Sample B: IndeterminateSample B: Indeterminate Sample C: PositiveSample C: Positive