Ag-Ab reactions for MBBS students

1. Antigen – Antibody reactions
Dr. Pendru Raghunath Reddy
Assistant Professor of Microbiology
Dr. VRK Women’s Medical College

2.  Antigens and antibodies combine with each otherAntigens 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 mediatedIn the body, they form the basis of antibody mediated
immunity in infectious diseases, or hypersensitivity andimmunity in infectious diseases, or hypersensitivity and
autoimmune diseasesautoimmune diseases
 Antigen – antibody reactions in vitro are known asAntigen – antibody reactions in vitro are known as
serological reactionsserological reactions
 In laboratory, they help in diagnosis of infections, inIn laboratory, they help in diagnosis of infections, in
epidemiological surveys, in the identification of infectiousepidemiological surveys, in the identification of infectious
agents, enzymesagents, enzymes

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

4. Ag-Ab interactionsAg-Ab interactions
Bonds:Bonds:
 HydrogenHydrogen
 IonicIonic
 Hydrophobic interactionsHydrophobic interactions
 Van der Waals forcesVan der Waals forces
Each bond is weak; manyEach bond is weak; many
areare
strongstrong
To “hold” they must be closeTo “hold” they must be close
 requiring high amts ofrequiring high amts of
complementarity!complementarity!

5. Secondary stageSecondary stage
 Demonstrable events – Precipitation, agglutination, lysis ofDemonstrable 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

6. Tertiary stageTertiary stage
 Includes neutralization or destruction of injurious agentsIncludes neutralization or destruction of injurious agents
or tissue damageor tissue damage
 Also includes humoral immunity against infectiousAlso includes humoral immunity against infectious
diseases as well as clinical allergy & otherdiseases as well as clinical allergy & other
immunological diseasesimmunological diseases

7. GENERAL FEATURES OF Ag – AbGENERAL 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 duringThere is no denaturation of the antigen or antibody during
the reactionthe reaction
4.4. The combination occurs at the surface. So surface antigensThe combination occurs at the surface. So surface antigens
are immunologically relevantare immunologically relevant
5.5. The combination is firm but reversible. The firmness isThe combination is firm but reversible. The firmness is
influenced by the affinity & avidity of the reactioninfluenced by the affinity & avidity of the reaction
6.6. Antigens & antibodies can combine in varying proportions.Antigens & antibodies can combine in varying proportions.
Both Ags & Abs are multivalentBoth Ags & Abs are multivalent

8. 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

9. 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

10. SpecificitySpecificity
 The ability of an individual antibody combining site toThe 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 reactThe ability of a population of antibody molecules to react
with only one antigenwith only one antigen

11. 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

12. Factors Affecting Measurement of
Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation

13. Types of Antigen – Antibody ReactionsTypes of Antigen – Antibody Reactions
1.1. Precipitation reactionPrecipitation reaction
2.2. Agglutination reactionAgglutination reaction
3.3. Neutralization reactionNeutralization reaction
4.4. OpsonisationOpsonisation
Serological tests based on Ag – Ab reactionsSerological tests based on Ag – Ab reactions
1.1. Complement fixation testComplement fixation test
2.2. ImmunofluorescenceImmunofluorescence
3.3. RadioimmunoassayRadioimmunoassay
4.4. Enzyme immunoassayEnzyme immunoassay

14. PRECIPITATION REACTIONPRECIPITATION REACTION
PRINCIPLEPRINCIPLE
When a soluble Ag combines with its Ab in theWhen a soluble Ag combines with its Ab in the
presence of electrolytes (NaCl) at a suitablepresence of electrolytes (NaCl) at a suitable
temperature & pH, the Ag-Ab complex forms antemperature & pH, the Ag-Ab complex forms an
insoluble precipitate.insoluble precipitate.
 When instead of sedimenting, the precipitateWhen instead of sedimenting, the precipitate
remains suspended as floccules –remains suspended as floccules – FlocculationFlocculation
reactionreaction
 Precipitation can take place in liquid media or inPrecipitation can take place in liquid media or in
gels such as agar, agarose or polyacrylamide.gels such as agar, agarose or polyacrylamide.

15. ZONE PHENOMENONZONE PHENOMENON
 The amount of precipitate formed is greatly influenced byThe 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 sameIf increasing quantities of Ags are added to the same
amount of antiserum in different tubes, precipitation isamount of antiserum in different tubes, precipitation is
found to occur most rapidly & abundantly in the middlefound to occur most rapidly & abundantly in the middle
tubestubes

Preceding tubes – Ab excess (Preceding tubes – Ab excess (ProzoneProzone))

Middle tubes – Ag & Ab in equivalent proportionsMiddle tubes – Ag & Ab in equivalent proportions
((Zone of equivalenceZone of equivalence))

Later tubes – Ag excess (Later tubes – Ag excess (Post zonePost zone))

17. 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 inThe multivalent antigens combine with bivalent Abs in
varying proportions, depending on the Ag – Ab ratio onvarying proportions, depending on the Ag – Ab ratio on
the reacting mixturethe reacting mixture
 Precipitation results when a large lattice is formedPrecipitation results when a large lattice is formed
consisting of alternating Ag & Abconsisting of alternating Ag & Ab

18. Marrack’s hypothesis

19. 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 ofIdentification 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

20. Types of precipitation reactions
1.Ring test
2.Flocculation test
3.Immunodiffusion
4.Electroimmunodiffusion

21. RING TESTRING TEST
 Consists of layering Ag solution over a column ofConsists of layering Ag solution over a column of
antisera in a narrow tubeantisera in a narrow tube
Eg: Ascolis thermoprecipitin test, Grouping ofEg: Ascolis thermoprecipitin test, Grouping of
StreptococciStreptococci by Lancefield techniqueby Lancefield technique

22. Flocculation testFlocculation test
Slide testSlide test
 When a drop of Ag & antiserum is placed on a slide &When a drop of Ag & antiserum is placed on a slide &
mixed by shaking, floccules will appearmixed by shaking, floccules will appear
 Eg: VDRL test & RPR test for syphilisEg: VDRL test & RPR test for syphilis

23. 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 tubesSerial 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 oneThe amount of toxin that flocculates optimally with one
unit of the antitoxin – Lf doseunit of the antitoxin – Lf dose

24. 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 betweenIndicates identity, cross reactions, non identity between
different Agsdifferent Ags

25. Various immunodiffusion testsVarious immunodiffusion tests
1.1. Single diffusion in one dimension (OudinSingle diffusion in one dimension (Oudin
procedure)procedure)
 Ab is incorporated in agar gel in a test tube & AgAb 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 aAg diffuses downward through the agar gel – forming a
line of precipitationline of precipitation..

26. 2. Double diffusion in one dimension (Oakley2. 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 theThe Ag & Ab move towards each other through the
intervening column of plain agar & form the precipitateintervening column of plain agar & form the precipitate

28. 3.3. Single diffusion in two dimensions (RadialSingle diffusion in two dimensions (Radial
immunodiffusionimmunodiffusion))
 Here the antisera is incorporated in a gel & poured on aHere 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 shapedIt diffuses radially from the well & forms ring shaped
bands of precipitation concentrically around the wellbands of precipitation concentrically around the well

30. Radial Immunodiffusion (Mancini)Radial Immunodiffusion (Mancini)
 InterpretationInterpretation

Diameter of ring isDiameter of ring is
proportional to theproportional to the
concentrationconcentration
 QuantitativeQuantitative

Ig levelsIg levels
• Method
– Ab in gel
– Ag in a well
Ag Concentration
Diameter2
AgAgAgAg
Ab in gel

31. 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

32. 44.. Double diffusion in two dimensions (OuchterlonyDouble 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 cutAgar 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

33. Reaction of identity
Partial identity
Lack of relatedness

34. Elek’s gel precipitation

35. 5.5. ImmunoelectrophoresisImmunoelectrophoresis
 This involves the electrophoretic separation of composite AgThis involves the electrophoretic separation of composite Ag
into its constituent proteins, followed by immunodiffusioninto its constituent proteins, followed by immunodiffusion
against its antiserum – separate precipitin linesagainst its antiserum – separate precipitin lines
 It is performed on an agarose gel with an Ag well & Ab troughIt is performed on an agarose gel with an Ag well & Ab trough
cut on itcut on it
 The test serum is placed in the antigen well & electrophoresedThe 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 & diffusionAb against human serum is placed in the trough & diffusion
allowed for 18 – 24 hrsallowed for 18 – 24 hrs

37. Immunoelectrophoresis

38. 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

39. ELECTROIMMUNODIFFUSIONELECTROIMMUNODIFFUSION
 The development of precipitin lines can be speeded upThe development of precipitin lines can be speeded up
by electrically driving the Ag & Abby electrically driving the Ag & Ab
 Two typesTwo types
1.1. Counterimmunoelectrophoresis (One dimensionalCounterimmunoelectrophoresis (One dimensional
double electroimmunodiffusion)double electroimmunodiffusion)
2.2. Rocket electrophoresis (One dimensional singleRocket electrophoresis (One dimensional single
electroimmunodiffusion)electroimmunodiffusion)

40. 1.1. Counterimmunoelectrophoresis (CIE)Counterimmunoelectrophoresis (CIE)
 This involves simultaneous electrophoresis of Ag & Ab inThis involves simultaneous electrophoresis of Ag & Ab in
gel in opposite directions resulting in precipitation at a pointgel 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 inClinical 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 andIt is also applied for detecting hepatitis B antigens and
antibodiesantibodies

41. Ag Ab
- +

42. 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 inThe 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 punchedAg in increasing concentrations, is placed in wells punched
in the set gelin the set gel
 The Ag is electrophoresed into the Ab containing agaroseThe Ag is electrophoresed into the Ab containing agarose
 The pattern of immunoprecipitation resembles aThe pattern of immunoprecipitation resembles a ROCKETROCKET
 The length of these rocket like structures corresponds toThe length of these rocket like structures corresponds to
the concentration of the antigenthe concentration of the antigen

43. Rocket electrophoresis

44. 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 electrophoreticallyIn the first stage, the Ag mixture is electrophoretically
separatedseparated
 In second stage, electrophoresis is done perpendicular toIn second stage, electrophoresis is done perpendicular to
that of first stage to get rocket like precipitationthat of first stage to get rocket like precipitation

45. 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

46. 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

47. 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

48. 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

49. Tube agglutination test

50. 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

51. 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

52. 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)

53. 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

54. The antiglobulin (Coombs test)
Originally devised by Coombs, Mourant and Race (1945) for the
detection of incomplete anti-Rh antibodies
There are two types of Coombs test
1.Direct Coombs test
2.Indirect Coombs test

55. + ↔
Patient’s RBCs Coombs Reagent
(Antiglobulin)
Direct Coombs test

56. Patient’s
Serum
Target
RBCs
+ ↔
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

57. Uses of Coombs test
1.For detection of anti-Rh antibodies
2.For demonstration of any type of incomplete antibody
(eg: Brucellosis)

58. 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

59. 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

60. 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

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

62.  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

63. 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)

65. 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)

67. 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

68. 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

70. 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 AntibodyHuman serum (May or may not contain Antibody
towards specific Antigen)towards specific Antigen)
 Complement:Complement: It is pooled serum obtained from 4 to 5It is pooled serum obtained from 4 to 5
guinea pigs. It should be fresh or specially preserved as theguinea pigs. It should be fresh or specially preserved as the
complement activity is heat labile (stored at -30 °C in smallcomplement activity is heat labile (stored at -30 °C in small
fractions). The complement activity should be initiallyfractions). 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 redRabbit antibody to sheep red
cells prepared by inoculating sheep erythrocytes into rabbitcells prepared by inoculating sheep erythrocytes into rabbit
under standard immunization protocol.under standard immunization protocol.

71. 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

72. Positive TestPositive Test
 Step 1:Step 1:
At 37°CAt 37°C
Antigen + Antibody + ComplementAntigen + Antibody + Complement Complement gets fixedComplement gets fixed
(from serum)(from serum) 1 Hour1 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 Positive)Test Positive)

73. 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)

74. Results and Interpretations:Results and Interpretations:
 No haemolysis is considered as aNo haemolysis is considered as a positive testpositive test
 Haemolysis of erythrocytes indicative of aHaemolysis 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)

75. 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

76. 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

77. 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

78. 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

79. Complement dependent serological tests
1.Immobilisation test
2.Immune adherence
3.Cytolytic or cytocidal tests
Neutralisation test
Opsonisation

80. 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)

81. Immunofluorescence test is of two types
1.Drect immunofluorescence test
2.Indirect immunofluorescence test

82. Direct immunofluorescence test

83. 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

84. Indirect immunofluorescence test

85. 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

86. 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

87. 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

88. 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

89. 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

90. Different types of ELISAs
1.Indirect ELISA
2.Sandwich ELISA
3.Competitive ELISA
4.Cassette or cylinder ELISA

91. Indirect ELISA
Two commonly used enzymes
1.Horseradish peroxidase (HRP)
2.Alkaline phosphatase (AP)

93. Sandwich ELISA
 Two antibodies required; must recognize different epitopes
 1st
Antibody is referred to as capture Ab
 2nd
antibody as detection Ab

94. Competitive ELISA

95.  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).

96. 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

98. 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

99. Western blotting (Immunoblotting)

100. 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