ag ab reactions, raghu
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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
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