over view of common antigen antibody reactions, their applications, sensitivity, advantage and disadvantage with pictorial illustrations for postgraduate and undergraduate reading

1. ANTIGEN ANTIBODY
REACTIONS

2. 
Antigen antibody reaction is a bimolecular
association in which the antigen and antibody
combines with each other specifically and in
an observable manner.

Non covalent Interaction between epitope of
antigen and
complementary determining
region of antibody.

3. Purpose :

Antibody mediated immunity in infectious
disease
or
injury,
hypersensitivity
and
autoimmune disease

In laboratory for diagnosis of infectious
disease, detecting antigen or antibody.

4. General features of antigen antibody( Ag-Ab ) reation:

Ag – Ab reactions are specific

Entire molecules and not the fragments react

No denaturation of Ag or Ab

Combination occurs at the surface. Surface Ag which is
immunologically active

Both Ag and Ab participates

Ag and Ab combines in varying proportions. Ab are generally
bivalent and Ag have valencies up to hundred

5. Strength of antigen antibody reaction:

The Ag-Ab binding includes non covalent bonds
– H bond, Ionic bond, hydrophobic interactions &
vanderwals forces.

Close fit between Ag and Ab requiring greater
degree of complementarities between Ag and Ab.

Affinity and avidity

7. 
Affinity
The combined strength of the noncovalent
interactions between a single antigen-binding
site on an antibody and a single epitope is
the affinity of the antibody for that epitope.

Strength of attraction

8. Avidity:

Strength of bond after formation of antigen antibody
complex

Binding capacity within biological systems

Interaction of an antibody molecule with an antigen
molecule at one site will increase the probability of
interaction between those two molecules at a second
site.

Strength of such multiple interactions determines
avidity

9. Stages of antigen antibody rection:

Primary reaction

Secondary reaction

Tertiary reaction

10. Primary:

Without any visible effects

Rapid, follows general law of physical
chemistry, low temperature

Reversible

Weaker intermolecular forces

11. Secondary:

Precipitation,
agglutination,
lysis
of
cell,
killing of live antigen, neutralization of toxins
& fixation of complement and immobilisation
of
motile
phagocytosis.
organisms,
enhancement
of

12. Tertiary reactions:

Chain reactions

Neutralization or destruction of injurious antigens

Humoral immunity, clinical allergy and
immunological diseases.

13. Cross reactivity:

Antibody excited by one antigen can cross react with
unrelated antigen

Different
antigens
sharing
similar
or
identical
epitope.

Affinty of cross reacting epitope is less

Most commonly among polysaccharide Ag.

Homologous
determinants
microbial
and
host
antigenic

14. Examples:

ABO Blood group antigens
Antibodies to missing antigen develops due
to exposure to cross reacting microbial antigens.

Streptococcal M antigens
Simulating myocardial and skeletal muscle
protein

Cross reacting vaccines

15. Antigen antibody reactions in serology:

Precipitation

Agglutination

Complement fixation

Enzyme immuno assay

Radioimmuno assay

Neutralization

Western blotting

Immunofluresence

16. Definition :

A soluble antigen combines with its antibody in the
presence of electrolyte at a suitable temperature and
pH,
the
Ag-Ab
complex
forms
an
insoluble
precipitate

If the precipitate remains suspended its known as
flocculation

In
liquid
or
gel
media
(agar,
polyacrylamide)

Sensitive for detection of antigens
agarose
or

17. Zone phenomenon:

Relative proportions of antigen and antibody

Increasing
amount
of
antigen
added
to
constant amount of antisera in different test
tubes.

Maximal precipitation at zone of equivalence

19. Mechanism of precipitation:
Marrach hypothesis:

Multivalent antigen combines with bivalent
antibody.

Lattice formation

In antigen or antibody excess lattice does not
enlarge.

20. Precipitation reactions in liquid:

Ring test

Slide test

Tube flocculation test
Precipitation reactions in gel:

Immunodiffusion

Electroimmunodiffusion

21. Ring test:

Layering of antigen solution over a column of
antiserum in a narrow tube

Precipitate at the junction

E.g. Ascoli’s thermoprecipitin test
Grouping of streptococci
test.
by Lancefield

22. Slide flocculation test

Antigen and antiserum placed on slide and
mixed by shaking

Appearance of floccules

23. Tube flocculation test:

Khan test for syphilis

Standardization of toxins and toxoids

Serial dillutions of toxins or toxoids added to
fixed quantity of antitoxin

Amount of toxin/ toxoid that flocculates with
fixed quantity of anti toxin is defined as 1 Lf
dose.

24. Precipitation reactions in gel:

Immunodiffussion

Agar matrix (1%)

Antibody incorporated into the agar.

Antigen diffuses into the agar containing matrix

Line of precipitation stained and preserved

Multiple antigen in reacting mixture can be identified

Detects identity, cross reactivity and non identity
between antigens

25. Modifications of immunodiffusion:

Single diffusion in one dimension

Double diffusion in one dimension

Single diffusion in two diffusion

Double diffusion in two dimension

Immunoelectrophoresis

Electroimmunodiffusion

26. Single diffusion in one dimension:
( Oudin procedure )

Antibody incorporated in agar gel and antigen
layered over it.

Line of precipitation at the advancing front of
antigen

Number of bands indicates number of antigens

28. Double diffusion in one dimension
( Oakley fulthorpe procedure)

Antibody incorporated in agar

Intervening column of plain agar

Antigen layered over it

Both antigen and antibody moves towards each
other

29. Single diffusion in two dimension
( Radial immunodiffusion / Maccni’s method)
 Antiserum incorporated in agar poured on
petridish
 Antigen is added to the wells cut on the
surface of gel
 Concentric circles of precipitation diameter of
halo gives estimate of antigen
 Estimation of immunoglobulin classes in sera
screening sera for antibodies to influenza

30. Double diffusion in 2 dimension
( Oucherlony procedure):

Most widely employed

Compare different antigen and antibody

Agar gel on slide with multiple wells

Antiserum
in
central
well
antigens in surrounding wells
and
different

32. Elek’s test for diphtheria bacillus

33. Immunoelectrophoresis:

Electrophoretic seperation of composite antigen

Immunodiffusion against its antiserum

Separate precipitation lines for individual protein

For detection of normal and abnormal proteins
present in the serum

35. Electroimmunodiffusion:
 Electrical
driving up of
antibody.

a.
b.
antigen
and
Methods:
One dimensional double electroimmuno
diffusion
One
dimensional
single
electro
immumodiffusion

36. One dimensional double electro immunodiffusion

Counter immuno electrophoresis

Precipitation lines formed in 30 min

10 times more sensitive than standard double
diffusion

Application: alpha fetoprotein in serum, specific
antigen of cryptococcus and meningococcus.

37. Counter immunoelectrophoresis

38. One Dimensional Single Electroimmunodiffusion
( Rocket Electrophoresis)

Quantitative estimation of antigens

Antiserum is incorporated into the agarose gel
on a glass slide

Antigen in increasing concentration is added to
wells punched on gel

Pattern of immunoprecipitation resembles rocket

40. Nephelometry:

Immune
complexes
are
monitored
by
spectrometry

Adding
constant
amount
of
optically
clear
specific antisera to varying amounts of antigen

Quantitative measurement of immunecomplexes
using photoelectric cell as optical density

41. 
When a particulate antigen combines with its
antibody in the presence of electrolytes at
suitable temperature and pH the complexes are
clumped or sedimented

Antibodies involed in agglutination- agglutunins

Sensitive for detection of antibodies

42. Principle of agglutination:

Antigen and antibody in optimal proportion

Large lattice formation: antibody interaction
with
multivalent
antigen
antigenic determinants.

Prozone phenomenon
with
repeating

43. Prozone – mechanism:

High antibody concentration, exceeding the
number of epitopes. Monovalent binding of
antibody with antigen failing to form cross
linkge.

44. 
Polyclonal
antibodies
containing
high
concentration of antibodies tat can bind to
antigen but do not cause agglutination.
Known as blocking antibodies ( IgG class)

45. 
Excess protein concentration on the bacteria
or antigenic particle causing electrostatic
repulsion.

46. Strength of agglutination:
Appearance
Report
• Small aggregates
+1
• Medium size aggregates
+2
• Several large aggregates
+3
• one solid aggregate
+4

47. Determination of Antibody Titre:

Highest dilution of the biological sample
which gives visible agglutination with antigen
with no agglutination at higher dillutions

Using serial dilutions

48. Factors affecting Agglutination:

Buffer pH

Relative concentration of antigen and antibody

Location and concentration of antigenic
determinant on the particle

Electrostatic interaction between particles

Electrolyte concentration

Antibody isotype

Temperature

49. Types of agglutination reactions:

Direct agglutination

Indirect agglutination

Reverse agglutination

Latex particle agglutination inhibition reactions

Quantitative agglutination reactions

Haemagglutination

Viral haemagglutination

Viral haemagglutination inhibition

50. Direct(active) agglutination:

Antigen is intrinsic component of the particle
e.g bacterium, erythrocytes

Detection of antibodies in biological fluids

E.g. antibodies to brucella, weil-felix , widal
test, blood grouping

52. Indirect (passive) agglutination:

Antigen adsorbed to the particle

Erythrocytes, charcoal, clay (bentonite),
colloidal gold are particles to which antigen is
complexed with

Testing of organisms which are very
pathogenic

53. Latex particle agglutination test:

Uniform
size
latex
particle
coated
with
antigen will agglutinate in the presence of
antibodies specific for the antigen

Samples: CSF, serum

E.g. R F, ASLO

55. Reverse (passive) agglutination:

Antibody is attached to the particle

Biological sample tested for antigen

e.g: C- reactive protein, candida species,
Neisseria gonorrhoeae.

57. Coagglutination:

It’s a type of reverse passive agglutination test

Bacteria
is
added
as
inert
particle
to
IgG
antibodies, Fab region is free for antigen to bind

Applications:
typing
Gonococcal
and
streptococcal

59. Latex particle agglutination inhibition assay

60. Haemagglutination

Agglutination of RBC s

Direct- antigen a component of blood cell

In direct- soluble antigens are complexed
with red blood cell e.g. thyroglobulin

Reverse haemagglutination- antibody is
complexed to the red cell

61. Antigens binding to red cell:

Spontaneous adsorption

Covalent binding using chemical links

Tannic acid treatment

62. Viral haemagglutination:

Non immune agglutination

Agglutinate red cells in the absence of
antibody

Dengue, rubella, influenza, mumps viruses

Haemagglutinin of influenza with human
sheep and chicken red cells

V bottom micro titer plates

Adherence of RBC s to bottom of plates.

64. Viral Haemagglutination Inhibition:

Detection of antibodies in biological sample

Competetive binding assay done in two steps

Absence of haemagglutination is positive

End point haemagglutination - reciprocal of
highest dilution of patient’s serum that
completely inhibits haemagglutination

66. Antiglobulin Tests:

Coomb’s, Mourant and Race in 1945

To detect anti Rh antibodies

Anti Rh antibodies when mixed with Rh possitive red cells,
they coat the surface of erythrocytes without agglutination

Incomplete antibodies

Coomb’s reagent – anti human antibody specific for Fc
portion of IgG.

Types- direct and indirect.

67. Direct coomb’s test:

To detect maternal IgG antibodies bound to Rh
antigen on fetal red cells

Erythroblastosis fetalis

Rh negative mother carrying Rh positive baby

Production of both IgM and IgG antibodies

IgG antibodies only crosses placenta

Sensitization of RBCs takes place in vivo

69. Indirect coomb’s test:

To detect presence of anti Rh antibodies in
the serum of Rh- ve mother carrying or given
birth to Rh + ve baby

Senstization takes place in vitro

Rh +ve RBCs are incubated with mother’s
serum
and
coomb’s test.
test
is
proceeded
as
direct

71. Heterophile Agglutination Test:

Sharing of common antigens by different
biological species e.g Forssman antigen

Weil felix test in typhus fever
Paul bunnel test in infectious mononucleosis
Cold agglutinin in Mycoplasma pneumoniae

72. 
Paul bunnel test is a mono spot test detects sheep
agglutinins in patients with infectious mononucleosis

Weil-felix:
common
antigen
between
typhus
rickettsiae and proteus bacilli

Coldagglutinins ( Ig M) – agglutination at 4°C reversed
at 37°C
-
one
week
following
Mycoplasmal
dissapears by 6 weeks
- also in Lymphomas & leukemias
-complement mediated lysis of red cells
infection,

73. Quantitative Agglutination Tests:

Soluble particle immunoassay ( SPIA)

Disperse dye immunoassay (DIA)

Immunoassay by particle counting (IMPACT)

74. Immunoassay
SPIA
Technique
Inert Particle
Detection
Indirect or
reverse
agglutination
Gold- inorganic -Qualitative,
colloidal particle color change
-Quantitative ,colorimetric
analysis
DIA
Indirect or
reverse
agglutination
Dye- organic
-Qualitative,
colloidal particle color visualized
-quantitative,
color measured
optically
photometric
analysis
IMPACT
Indirect or
reverse
agglutination
Latex particle
-quantitative
automated
particle counter

75. Introduction:






The complement fixation test (CFT) was
extensively used in syphilis serology after being
introduced by Wasserman in 1909.
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.
Complement binds to Ag-Ab complex
When the Ag is an RBC it causes lysis of RBC’s.

76. Principle:
Monitoring the formation of immune
complexes by its ability to fix and consume
complement proteins.
Sensitivity: - 0.04 mg of antibody nitrogen
- 0.1 mg of antigen

77. Components of complement fixation test:
Test system

Antigen

Patient’s serum containing antibodies

Complement – fresh guinea pig serum
Indicator system

Complement fixation diluents rich in Ca and Mg
ions

Sheep erythrocytes

Amboceptors - Antibody to sheep erythrocytes

79. Preparation of complement:

Serum obtained by cardiac puncture from
healthy adult guinea pig (250 to 300 gm)
pooled for 18 to 24 hrs

Chlamydial or paramyxoviral infections can
give rise to artefactual rections

80. Preservation of complement:

Freeze drying at – 70° C, reconstituted
solution stored at 4° C used with in one week

Richardson’s method: addition of hypertonic
sodium chloride or any salt

81. Titration of guinea pig serum:

One Minimum Hemolytic Dose ( MHD) of
complement is defined as the highest dilution
of the serum that lyses one unit volume of
washed sheep erythrocytes in the presence of
excess hemolysin within a fixed time, at a
fixed temperature (37°C)

82. 
The MHD of amboceptor is defined as least
amount of the inactivated amboceptor that
lyses one unit volume of washed sheep
erythrocyte in the presence of excess
complement within a fixed time and at a fixed
temperature

83. Patient’s serum:

5 ml of blood by venepuncture

Coagulated in a sterile stopper test tube

Serum is pipetted off and diluted to starting
dilutions

Heated to 56°C for 30 mins

Reduces nonspecific fixation and inactivates
its complement

84. Procedure:
stage 1

Serial dilution of heat inactivated test sera ( paired sera)
in buffered CF diluent done in a microtitre plate

1 volume of complement 3 HD50 (50% hemolytic dose)
is added to each well

I volume of antigen ( 2 – 4 units) at optimal peak
dilution is added to each well

Stack and cover plates and stand overnight at 4 °C

85. Stage 2

Sensitization of sheep RBCs by slowly adding
an equal volume of hemolysin and incubated
at 37°C for 10 min or at room temperature for
30 min

Plates warmed for 30 min at room
temperature and 0.025 ml of 2% sensitized
sheep cells to each well

86. 
Re-incubated for 30 min to allow lysis ,
shaking at 10, 20 and 30 min to resuspend
the cells

Allow deposition of cells

87. 1
2
3
4
A1, B1, B2, B3- NO HEMOLYSIS – POSITIVE
A2, A3, A4, B4 – HEMOLYSIS- NEGATIVE

88. Controls :

Serum control

Antigen control

Cell control

Positive serum control

89. Result :

Highest dilution of serum giving a fixation
value is taken as the titre of antibody

End point titre – dilution fixing 70 to 100 % of
complement

For antigen detection, test is done by diluting
the antigen

90. Indirect complement fixation test:

For seras not able to fix complement

Test is set up in duplicate

After first step as in routine complement fixation test, the
standard antiserum known to fix complement is added to
one set.

If test serum contained antibody,the antigen being utilized
in the first step.

Standard antiserum thus unable to fix the complement.

Haemolysis in indirect test indicate Positive result.

91. Conglutinating complement adsorption assay:

Uses horse complement

Indicator system: sheep erythrocytes mixed with bovine
serum.

Conglutinin – beta globulin, antibody to horse complement

Conglutunin causes agglutination of sheep erythrocytes if
combined with complement

If the horse complement utilised by the Ag – Ab interaction
during first step No agglutination of sensitised cells

92. Applications of complement fixation test:

Wasserman reaction for syphillis

Immune Adherence – The Ag – Ab complexes
of V.cholerae, T.pallidum adhere to RBCs,
platelets.

Treponema pallidum immobilisation test

Cytolytic or cytocidal test – vibriocidal
antibody test

93. 
First devised to quantify polypeptide
hormones by Parker in 1976

Used to measure antigen or antibody

Sensitivity: Permits measurement of analytes
up to picograms quantities

94. Methods:
a.
Solutions :- Radioimmunoprecipitation
- Competition radioprecipitation
a.
Solidphase: microtitre plate, glass,
polystyerene bead, cellulose.

95. Radioimmunoprecipitation test:

Detection of antibody in test serum

Radiolabelled antigen is reacted with test serum

Antigen antibody complex formed is precipitated
and separated from unbound labelled antigen

Comparison of radioactivity between precipitate
and supernatant

Ratio gives estimate of amount of antibody in
test serum

96. Separation of bound and unbound antigen:

Antiglobulin

Ammonium sulphate precipitation

Staphylococcal protein A
Followed by centrifugation or chromatography

98. Competition radioimmunoassay:

For antigen detection in test sample

Labelled antigen competes with unlabelled
antigen in the test sample for limited amount
of antibody reagent

Free and bound labelled antigen separated
and measured

100. 
Concentration of test antigen calculated from
ratio of bound and total labelled antigen

Compared to standard dose response curve
or calibration curve

101. Solid-phase radioimmunoassay:

Advantage – separation of antigen and
antibody easily achieved

Principle is similar to enzyme assay

Radiolabelled immunoglobulins are used

Gama or scintillation counting of bound
antiglobulin

103. Uses of RIA:

Quantification of hormones, drugs, tumour
markers, IgE and viral antigen like Hbs Ag
Advantages:
Specific, precise and highly sensitive
Disadvantages:

I125 labelled reagents have shorter half-life

Radioactive hazard

Expensive equipments

104. 
Fluorescence is a property of absorbing light
rays of one wave length (excitation) and
emitting rays with different wavelength
(emission)

Albert coons in 1944 found antibodies could
be tagged with molecules with property of
fluorescence

105. 
Fluorescent tagged antibodies locates formed
immune complexes, detected by colored light
emission when excited by light of the
appropriate wavelength

Emitted light viewed by fluorescent
microscope with a UV light source

106. Fluorochromes used :
Dye
Absorbs
Fluoresceine
isothiocyann
ate
(organic dye)
Rhodamine
(organic dye)
Blue light (490
nm)
Phycoerythri
n
(from algae)
efficient
absorber, 30
fold greater
than
fluoresceine
Emits
Yellow green
fluorescence
(517 nm)
Yellow green
Deep red
range
( 515 fluorescence
nm)
(546 nm)
red
fluorescence

107. Types:

Direct immunofluorescence

Indirect immunofluorescence

108. Direct immunofluorescence:

For detecting antigens in tissues using
specific antibody labeled with a fluorescent
dye

For identification of viruses, bacteria,
membrane bound antigens

Sensitive method for identification of rabies
viral antigen in brain smears

111. Indirect immunofluorescence:

Two antibodies are used

Primary antibody and a secondary antibody
which is a fluorescent labeled reagent

Reagent: Fluorescent labeled - secondary
antibody, complement, protein A

E.g Fluorescent Treponemal antibody test

114. Advantages of indirect over direct:

Separate fluorescent conjugate for each
antigen is not required, in turn fluorescent
tagged antiglobulin to detect human
antibodies to many antigen is employed

Sensitivity of staining increased - multiple
molecules of the fluorochrome reagent bind
to each primary antibody molecule

115. Advantages:

localization of antigens in tissue sections or in
subcellular compartments

To map the actual location of target antigens,
fluorescence microscopy is a powerful tool for
relating the molecular architecture of tissues
Disadvantages:

Non-specific fluorescence

116. Applications:

Identify subpopulations of lymphocytes, notably
the CD4 and CD8 T-cell subpopulations.

For identifying bacterial species

Detecting Ag-Ab complexes in autoimmune
disease

Detecting complement components in tissues

Localizing hormones and other cellular products
stained in situ.

117. 
Designed to automate the analysis and separation
of cells stained with fluorescent antibody.

Uses a laser beam and light detector

Cells having a fluorescently tagged antibody bound
to their cell surface antigens are excited by the
laser and emit light that is recorded by a detector
system located at a right angle to the laser beam

119. Informations obtained by flowcytometry:


Number and percentage of target cells
The distribution of cells in a sample population
according to antigen densities as determined
by fluorescence intensity

The size of cells.

Analyzing cell population - with two or even
three different fluorescent antibodies

120. Applications:

Essential tools for the detection and
classification of leukemias

Rapid measurement of T cell population in
AIDS

121. 
Viral neutralization test

Toxin neutralization test

122. Viral neutralization:

Certain viral antigens inducing neutralizing
antibodies that react with viruses resulting in
loss of infectivity

To type viruses

To titrate the level of antibody in serum

123. 
Phage neutralization – plaque inhibition test
Bacteriophages seeded on lawn culture of
susceptible bacteria, plaques of lysis
produced,specific antiphage serum inhibits
plaque formation

Neutralisation in animal, egg, or cell culture

124. 
Incubating virus with sera containing
neutralizing antibody and detecting residual
viral activity by its ability to infect cell culture
or other host system

125. Mechanism of viral neutralization:

Antibody inhibits the attachment of virus to its
receptors by steric hindrance- Extrinsic
neutralization

Antibody mediated alteration in viral
expression- Intrinsic neutralization

Antigen – antibody complex formed between
virus and antibody( lattice)pseudoneutralization

126. Toxin neutralization:

Bacterial exotoxins are immunogenic
inducing neutralizing antibodies - antitoxins

Antitoxins – protection and recovery from
disease

Toxin neutralization tests- in vivo or in vitro

127. 
Invivo test : schick’s test- ability of
circulating antitoxin to neutralize diphtheria
toxin given intradermaly

Invitro test: antistreptolysin O test, anti toxin
in human serum neutralizes the hemolytic
activity of streptolysin O

128. 
In 1966 Enzyme labeled conjugates
introduced to identify antigen in tissues

EIA includes all assays based on
measurement of enzyme labeled antigen,
antibody or hapten

Types: - Homogeneous
- Heterogeneous

129. Homogenous EIA:

No separation of bound and free fractions

Completed in one step

Used for assay of haptens

E.g: Enzyme multiplied immunoassay
technique (EMIT) for opiates, cocaine,
barbiturates

130. Heterogeneous EIA:

Separation of free and bound fraction by
centrifugation or absorption on solid phase

Multi step procedure

Enzyme Liinked Immunosorbent Assay is a
major type

131. 
Immunoabsorbent is used as absorbent specific for
one of the component of the reaction

Particulate or solidphase

Particulate -cellulose, agarose,

Solid phase -polysterene, polyvenyl, polycarbonate
tubes, microwells or membrane or discs of
polyacrylamide

Sensitivity of 0.1 to 0.01ng

132. Principle:


To use an enzyme to detect the Ag-Ab binding
The enzyme converts a colorless substrate
(chromogen) to a colored product, indicating the
presence of Ag-Ab binding

Enzymes: alkaline phosphatase, horseradish
peroxidase, and galactosidase

Substrates: para-nitrophenyl phosphate and
hydrogen peroxide

133. substrat
e
Colored
product
Different antigen in sample

134. Types of ELISA:

Non competitive
Sandwich
Indirect
Capture

Competitive ELISA

135. Sandwich ELISA:

Sensitive for detection of antigen

Antibody is immobilized on a microtitre well

Sample containing antigen is added and allowed
to react with the immobilized antibody .

Well is washed

enzyme linked antibody specific for epitope on
the antigen is added

Suitable substrate added

Coloured reaction product

E.g detection of rotavirus in stool sample

137. Indirect ELISA:

Sensitive for detection of antibody

Antigen coated microtitre well

Serum sample containing primary antibody added (
Ab1)

Washed to remove unbound Ab

Enzyme-conjugated secondary anti-isotype antibody
(Ab2)

Substrate

Colored reaction read

E.g: anti- HIV antibody

139. Capture ELISA:

Sensitive for detection of antibody responses

Especially IgM responses in early disease

For diagnosis of infectious diseases like
dengue, rubella, measles, toxoplasmosis

141. Competitive ELISA:

Sensitive for antigen detection

Antibody is first incubated in solution with a
sample containing antigen

Antigen-antibody mixture added to an
antigen coated microtiter well

The more antigen present in the sample the
less free antibody will be available to bind to
the antigen-coated well

142. 
Addition of an enzyme-conjugated secondary
antibody (Ab2) specific for the isotype of the
primary antibody

Higher the concentration of antigen in the
original sample, the lower the absorbance

Detection of antigen in a crude mixture

144. 
Modification of the ELISA

Quantitative determination of cells in a
population that are producing antibodies
specific for a given antigen or an antigen for
a specific antibody

Plates are coated with capture antigen or
antibody

146. 



Newer technique devised to detect west nile
virus antibodies
domain –III envelope protein antigen linked
with enzyme
serum samples mixed with the enzymelabelled antigen
immune complexes formed are recognized
by rheumatoid factor coated microtiter plates

147. Combines sensitivity of EIA with more
specificity
Steps: 1. Separation of ligand antigen component gel
by electrophoresis
2. Blotting of electrophorosed ligand fraction
on nitrocellulose membrane
3. Enzyme immunoassay or radio immunoassay

148. EIA or RIA is done to detect:

Antibody against various ligand fraction
bands

Probe with known antisera against specific
antigen bands

150. Types of immunoblotting:

Southern blotting : First evolved, invented by
Edwin Southern, detects DNA fragments

Western blotting : detecting specific protein
in a complex mixture.

Northern blotting : detecting mRNA

152. 

Chemical reaction emitting energy in the
form of light
Chemiluminescent compounds are used to
tag as a label for Ag-Ab reactions

Compounds – luminol, acridium

5 to 10-18 moles (5 attomoles) of
target antigen have been detected

153. 
Simple, economical & reliable

Test system- cassette containing membrane
impregnated with antibody (to specific Ag)
colloidal gold dye conjugate

Cassette having 3 windows:
- test serum
- test result
- control

154. 
Serum sample added to one window

Serum moves by capillary action

Colored band appears at second site if
antibody is present in serum which forms
antigen-antibody-conjugate complex

Colored band at 3rd window – control

Test invalid if control band is absent

155. 
Ananthanarayan and Paniker’s Text book of
Microbiology, eighth edition

Mackie and McCartney’s practical medical
microbology.

Kuby’s text book of immunology

156. 



Kuby’s text book of immunology.
Stite’s Medical immunology, tenth edition.
Ananthanarayan and Paniker’s Text book of
Microbiology, eighth edition
Mackie and McCartney’s practical medical
microbology.