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About This Presentation
Serology
Slide Content
Serology Serology
PrinciplesPrinciples andand InterpretationInterpretation
in Infectious diseasesin Infectious diseases Dr.T.V.Rao Dr.T.V.Rao
MDMD
PrinciplesPrinciples andand InterpretationInterpretation
in Infectious diseasesin Infectious diseases Dr.T.V.Rao Dr.T.V.Rao
MDMD
Beginning of SerologyBeginning of Serology
►Serology as a science began in 1901. Serology as a science began in 1901.
Austrian American immunologist Austrian American immunologist Karl
Landsteiner (1868-1943) identified groups (1868-1943) identified groups
of red blood cells as A, B, and O. From that of red blood cells as A, B, and O. From that
discovery came the recognition that cells of discovery came the recognition that cells of
all types, including blood cells, cells of the all types, including blood cells, cells of the
body, and microorganisms carry proteins body, and microorganisms carry proteins
and other molecules on their surface that and other molecules on their surface that
are recognized by cells of the are recognized by cells of the immune immune
systemsystem. .
►Serology as a science began in 1901. Serology as a science began in 1901.
Austrian American immunologist Austrian American immunologist Karl
Landsteiner (1868-1943) identified groups (1868-1943) identified groups
of red blood cells as A, B, and O. From that of red blood cells as A, B, and O. From that
discovery came the recognition that cells of discovery came the recognition that cells of
all types, including blood cells, cells of the all types, including blood cells, cells of the
body, and microorganisms carry proteins body, and microorganisms carry proteins
and other molecules on their surface that and other molecules on their surface that
are recognized by cells of the are recognized by cells of the immune immune
systemsystem. .
Karl LandsteinarKarl Landsteinar (1868-1943(1868-1943))
►An Austrian physician An Austrian physician
by training, by training,
Landsteiner played an Landsteiner played an
integral part in the integral part in the
identification of blood identification of blood
groups. He groups. He
demonstrated the demonstrated the
catastrophic effect of catastrophic effect of
transfusing with the transfusing with the
wrong type of blood, wrong type of blood,
►An Austrian physician An Austrian physician
by training, by training,
Landsteiner played an Landsteiner played an
integral part in the integral part in the
identification of blood identification of blood
groups. He groups. He
demonstrated the demonstrated the
catastrophic effect of catastrophic effect of
transfusing with the transfusing with the
wrong type of blood, wrong type of blood,
Specific antibodies establishes Specific antibodies establishes
Blood grouping systemBlood grouping system
Blood grouping systemBlood grouping system
Purpose of Serological TestsPurpose of Serological Tests
►Serological tests may be performed for Serological tests may be performed for
diagnostic purposes when an infection is diagnostic purposes when an infection is
suspected, in rheumatic illnesses, and in suspected, in rheumatic illnesses, and in
many other situations, such as checking an many other situations, such as checking an
individual's blood type. Serology blood tests individual's blood type. Serology blood tests
help to diagnose patients with certain help to diagnose patients with certain
immune deficiencies associated with the immune deficiencies associated with the
lack of antibodies, such as X-linked lack of antibodies, such as X-linked
agammaglobulinemia. agammaglobulinemia.
►Serological tests may be performed for Serological tests may be performed for
diagnostic purposes when an infection is diagnostic purposes when an infection is
suspected, in rheumatic illnesses, and in suspected, in rheumatic illnesses, and in
many other situations, such as checking an many other situations, such as checking an
individual's blood type. Serology blood tests individual's blood type. Serology blood tests
help to diagnose patients with certain help to diagnose patients with certain
immune deficiencies associated with the immune deficiencies associated with the
lack of antibodies, such as X-linked lack of antibodies, such as X-linked
agammaglobulinemia. agammaglobulinemia.
SerologySerology
►The branch of The branch of
laboratory medicine laboratory medicine
that studies blood that studies blood
serum for evidence of serum for evidence of
infection and other infection and other
parameters by parameters by
evaluating antigen-evaluating antigen-
antibody reactions in antibody reactions in
vitro vitro
►The branch of The branch of
laboratory medicine laboratory medicine
that studies blood that studies blood
serum for evidence of serum for evidence of
infection and other infection and other
parameters by parameters by
evaluating antigen-evaluating antigen-
antibody reactions in antibody reactions in
vitro vitro
SerologySerology
►SerologySerology is the is the
scientific study of scientific study of
blood serum. In blood serum. In
practice, the term practice, the term
usually refers to the usually refers to the
diagnostic diagnostic
identification of identification of
antibodies in the antibodies in the
serum serum
We can detect We can detect
antigens tooantigens too
►SerologySerology is the is the
scientific study of scientific study of
blood serum. In blood serum. In
practice, the term practice, the term
usually refers to the usually refers to the
diagnostic diagnostic
identification of identification of
antibodies in the antibodies in the
serum serum
We can detect We can detect
antigens tooantigens too
Serology prerogative of Serology prerogative of
MicrobiologyMicrobiology
►It is rather curious that, although serum for It is rather curious that, although serum for
a multitude of constituents in biochemistry a multitude of constituents in biochemistry
and haematological laboratories, the term and haematological laboratories, the term
serology has come to imply almost serology has come to imply almost
exclusively the detection of antibodies in exclusively the detection of antibodies in
serum for serum for antibodies in infectious antibodies in infectious
diseasesdiseases, and terminology has become , and terminology has become
prerogative of microbiologists.prerogative of microbiologists.
MicrobiologyMicrobiology
►It is rather curious that, although serum for It is rather curious that, although serum for
a multitude of constituents in biochemistry a multitude of constituents in biochemistry
and haematological laboratories, the term and haematological laboratories, the term
serology has come to imply almost serology has come to imply almost
exclusively the detection of antibodies in exclusively the detection of antibodies in
serum for serum for antibodies in infectious antibodies in infectious
diseasesdiseases, and terminology has become , and terminology has become
prerogative of microbiologists.prerogative of microbiologists.
Immunology/ SerologyImmunology/ Serology??
Precipitation ReactionsPrecipitation Reactions
►Capillary tube precipitation (Ring Test)Capillary tube precipitation (Ring Test)
►Ouchterlony Double Diffusion (Immunodiffusion)Ouchterlony Double Diffusion (Immunodiffusion)
►Radialimmunodiffusion (RID)Radialimmunodiffusion (RID)
►Immunoelectrophoresis (IEP)Immunoelectrophoresis (IEP)
►Rocket Electroimmunodiffusion (EID)Rocket Electroimmunodiffusion (EID)
►Counterimmunoelectrophoresis (CIEP)Counterimmunoelectrophoresis (CIEP)
The above tests have moved to The above tests have moved to
BiochemistryBiochemistry
Precipitation ReactionsPrecipitation Reactions
►Capillary tube precipitation (Ring Test)Capillary tube precipitation (Ring Test)
►Ouchterlony Double Diffusion (Immunodiffusion)Ouchterlony Double Diffusion (Immunodiffusion)
►Radialimmunodiffusion (RID)Radialimmunodiffusion (RID)
►Immunoelectrophoresis (IEP)Immunoelectrophoresis (IEP)
►Rocket Electroimmunodiffusion (EID)Rocket Electroimmunodiffusion (EID)
►Counterimmunoelectrophoresis (CIEP)Counterimmunoelectrophoresis (CIEP)
The above tests have moved to The above tests have moved to
BiochemistryBiochemistry
Terms used in evaluating test Terms used in evaluating test
methodologymethodology
►SensitivitySensitivity
Analytical SensitivityAnalytical Sensitivity – ability of a test to – ability of a test to
detect very small amounts of a detect very small amounts of a substancesubstance
Clinical SensitivityClinical Sensitivity – ability of test to give – ability of test to give
positive result if patient has the disease (no positive result if patient has the disease (no
false negative results) false negative results)
methodologymethodology
►SensitivitySensitivity
Analytical SensitivityAnalytical Sensitivity – ability of a test to – ability of a test to
detect very small amounts of a detect very small amounts of a substancesubstance
Clinical SensitivityClinical Sensitivity – ability of test to give – ability of test to give
positive result if patient has the disease (no positive result if patient has the disease (no
false negative results) false negative results)
SpecificitySpecificity
►Analytical SpecificityAnalytical Specificity – ability of test to – ability of test to
detect substance without interference from detect substance without interference from
cross-reacting substances cross-reacting substances
►Clinical SpecificityClinical Specificity – ability of test to give – ability of test to give
negative result if patient does not have negative result if patient does not have
disease (no false positive results) disease (no false positive results)
►Analytical SpecificityAnalytical Specificity – ability of test to – ability of test to
detect substance without interference from detect substance without interference from
cross-reacting substances cross-reacting substances
►Clinical SpecificityClinical Specificity – ability of test to give – ability of test to give
negative result if patient does not have negative result if patient does not have
disease (no false positive results) disease (no false positive results)
AffinityAffinity
►Affinity refers to the Affinity refers to the
strength of binding strength of binding
between a single between a single
antigenic determinant antigenic determinant
and an individual and an individual
antibody combining antibody combining
site.site.
► Affinity is the Affinity is the
equilibrium constant equilibrium constant
that describes the that describes the
antigen-antibody antigen-antibody
reactionreaction
►Affinity refers to the Affinity refers to the
strength of binding strength of binding
between a single between a single
antigenic determinant antigenic determinant
and an individual and an individual
antibody combining antibody combining
site.site.
► Affinity is the Affinity is the
equilibrium constant equilibrium constant
that describes the that describes the
antigen-antibody antigen-antibody
reactionreaction
AffinityAffinity
►Antibody affinity is the strength of the Antibody affinity is the strength of the
reaction between a single antigenic reaction between a single antigenic
determinant and a single combining site on determinant and a single combining site on
the antibody. the antibody.
►It is the sum of the attractive and repulsive It is the sum of the attractive and repulsive
forces operating between the antigenic forces operating between the antigenic
determinant and the combining site .determinant and the combining site .
►Antibody affinity is the strength of the Antibody affinity is the strength of the
reaction between a single antigenic reaction between a single antigenic
determinant and a single combining site on determinant and a single combining site on
the antibody. the antibody.
►It is the sum of the attractive and repulsive It is the sum of the attractive and repulsive
forces operating between the antigenic forces operating between the antigenic
determinant and the combining site .determinant and the combining site .
AvidityAvidity
►Avidity is a measure of Avidity is a measure of
the overall strength of the overall strength of
binding of an antigen binding of an antigen
with many antigenic with many antigenic
determinants and determinants and
multivalent antibodiesmultivalent antibodies
►Avidity is influenced by Avidity is influenced by
both the valence of the both the valence of the
antibody and the valence antibody and the valence
of the antigen. of the antigen.
►Avidity is more than the Avidity is more than the
sum of the individual sum of the individual
affinities. affinities.
►Avidity is a measure of Avidity is a measure of
the overall strength of the overall strength of
binding of an antigen binding of an antigen
with many antigenic with many antigenic
determinants and determinants and
multivalent antibodiesmultivalent antibodies
►Avidity is influenced by Avidity is influenced by
both the valence of the both the valence of the
antibody and the valence antibody and the valence
of the antigen. of the antigen.
►Avidity is more than the Avidity is more than the
sum of the individual sum of the individual
affinities. affinities.
DilutionDilution
►Estimating the Estimating the
antibody by antibody by
determining the determining the
greatest degree to greatest degree to
which the serum may which the serum may
be diluted without be diluted without
losing the power to losing the power to
given an observable given an observable
effect in a mixture with effect in a mixture with
specific antigenspecific antigen
►Estimating the Estimating the
antibody by antibody by
determining the determining the
greatest degree to greatest degree to
which the serum may which the serum may
be diluted without be diluted without
losing the power to losing the power to
given an observable given an observable
effect in a mixture with effect in a mixture with
specific antigenspecific antigen
TiterTiter
►Different dilutions of Different dilutions of
serum are tested in serum are tested in
mixture with a mixture with a
constant amount of constant amount of
antigen and greatest antigen and greatest
reacting dilution is reacting dilution is
taken as the measure taken as the measure
or Titeror Titer
►Different dilutions of Different dilutions of
serum are tested in serum are tested in
mixture with a mixture with a
constant amount of constant amount of
antigen and greatest antigen and greatest
reacting dilution is reacting dilution is
taken as the measure taken as the measure
or Titeror Titer
Expression of TitersExpression of Titers
►Expressed in term of the Expressed in term of the
was in which they are was in which they are
mademade
►Dilution Dilution 1 in 8 is a 1 in 8 is a
dilution made by dilution made by
mixing one volume of mixing one volume of
serum with seven serum with seven
volumes of diluentsvolumes of diluents
(Normal Saline )(Normal Saline )
►Incorrect to express Incorrect to express
dilution as 1/8 dilution as 1/8
►Expressed in term of the Expressed in term of the
was in which they are was in which they are
mademade
►Dilution Dilution 1 in 8 is a 1 in 8 is a
dilution made by dilution made by
mixing one volume of mixing one volume of
serum with seven serum with seven
volumes of diluentsvolumes of diluents
(Normal Saline )(Normal Saline )
►Incorrect to express Incorrect to express
dilution as 1/8 dilution as 1/8
Common methods in creating Common methods in creating
dilutionsdilutions
dilutionsdilutions
Sero ConversionSero Conversion
►SeroconversionSeroconversion is is
the development of the development of
detectable specific detectable specific
antibodies to antibodies to
microorganisms in the microorganisms in the
blood serum as a blood serum as a
result of infection or result of infection or
immunization.immunization.
►SeroconversionSeroconversion is is
the development of the development of
detectable specific detectable specific
antibodies to antibodies to
microorganisms in the microorganisms in the
blood serum as a blood serum as a
result of infection or result of infection or
immunization.immunization.
Sero reversionSero reversion
►SSeroreversioneroreversion is the is the
opposite of opposite of
seroconversion. This is seroconversion. This is
when the tests can no when the tests can no
longer detect longer detect
antibodies or antigens antibodies or antigens
in a patient’s serumin a patient’s serum
►SSeroreversioneroreversion is the is the
opposite of opposite of
seroconversion. This is seroconversion. This is
when the tests can no when the tests can no
longer detect longer detect
antibodies or antigens antibodies or antigens
in a patient’s serumin a patient’s serum
Testing paired SamplesTesting paired Samples
►Testing for infectious Testing for infectious
diseases is performed on diseases is performed on
acute and convalescent acute and convalescent
specimens (about 2 weeks specimens (about 2 weeks
apart) apart) Paired sample. Paired sample.
►Must see 4-fold or 2-Must see 4-fold or 2-
tube rise in titre to be tube rise in titre to be
clinically significantclinically significant
►Testing for infectious Testing for infectious
diseases is performed on diseases is performed on
acute and convalescent acute and convalescent
specimens (about 2 weeks specimens (about 2 weeks
apart) apart) Paired sample. Paired sample.
►Must see 4-fold or 2-Must see 4-fold or 2-
tube rise in titre to be tube rise in titre to be
clinically significantclinically significant
Majority Diagnostic tests are Majority Diagnostic tests are
Serological testsSerological tests
►There are several There are several
serology techniques serology techniques
that can be used that can be used
depending on the depending on the
antibodies being antibodies being
studied. These include: studied. These include:
ELISA, agglutination, ELISA, agglutination,
precipitation, precipitation,
complement-fixation, complement-fixation,
and fluorescent and fluorescent
antibodies. antibodies.
Serological testsSerological tests
►There are several There are several
serology techniques serology techniques
that can be used that can be used
depending on the depending on the
antibodies being antibodies being
studied. These include: studied. These include:
ELISA, agglutination, ELISA, agglutination,
precipitation, precipitation,
complement-fixation, complement-fixation,
and fluorescent and fluorescent
antibodies. antibodies.
Antigen and Antibody Antigen and Antibody
reactions can be reactions can be
identified by different identified by different
methodsmethods
reactions can be reactions can be
identified by different identified by different
methodsmethods
PrecipitationPrecipitation
►PrinciplePrinciple
Soluble antigen + antibody (in proper proportions) –> Soluble antigen + antibody (in proper proportions) –>
visible precipitate visible precipitate
Lattice formation (antigen binds with Fab sites of 2 Lattice formation (antigen binds with Fab sites of 2
antibodies) antibodies)
►ExamplesExamples
Double diffusion (Ouchterlony) Double diffusion (Ouchterlony)
Single diffusion (radial immunodiffusion) Single diffusion (radial immunodiffusion)
Imunoelectrphoresis Imunoelectrphoresis
Immunofixation Immunofixation
►PrinciplePrinciple
Soluble antigen + antibody (in proper proportions) –> Soluble antigen + antibody (in proper proportions) –>
visible precipitate visible precipitate
Lattice formation (antigen binds with Fab sites of 2 Lattice formation (antigen binds with Fab sites of 2
antibodies) antibodies)
►ExamplesExamples
Double diffusion (Ouchterlony) Double diffusion (Ouchterlony)
Single diffusion (radial immunodiffusion) Single diffusion (radial immunodiffusion)
Imunoelectrphoresis Imunoelectrphoresis
Immunofixation Immunofixation
AgglutinationAgglutination
►PrinciplePrinciple
Particulate antigen + antibody –> clumping Particulate antigen + antibody –> clumping
Lattice formation (antigen binds with Fab sites of 2 Lattice formation (antigen binds with Fab sites of 2
antibodies forming bridges between antigens) antibodies forming bridges between antigens)
►ExamplesExamples
Direct agglutination (Blood Bank) Direct agglutination (Blood Bank)
Passive Hemagglutination (treat RBC's with tannic acid Passive Hemagglutination (treat RBC's with tannic acid
to allow adsorption of protein antigens) to allow adsorption of protein antigens)
Passive latex agglutination (antigen attached to latex Passive latex agglutination (antigen attached to latex
particle) particle)
►PrinciplePrinciple
Particulate antigen + antibody –> clumping Particulate antigen + antibody –> clumping
Lattice formation (antigen binds with Fab sites of 2 Lattice formation (antigen binds with Fab sites of 2
antibodies forming bridges between antigens) antibodies forming bridges between antigens)
►ExamplesExamples
Direct agglutination (Blood Bank) Direct agglutination (Blood Bank)
Passive Hemagglutination (treat RBC's with tannic acid Passive Hemagglutination (treat RBC's with tannic acid
to allow adsorption of protein antigens) to allow adsorption of protein antigens)
Passive latex agglutination (antigen attached to latex Passive latex agglutination (antigen attached to latex
particle) particle)
Neutralization reactionsNeutralization reactions
►Similar in principle and interpretation of results Similar in principle and interpretation of results
►Antibody-bindingAntibody-binding
►Hemagglutination inhibition (serum antibody reacts with Hemagglutination inhibition (serum antibody reacts with
known nonparticulate antigen –> binding occurs) known nonparticulate antigen –> binding occurs)
►Neutralization (antibody neutralizes toxin) Neutralization (antibody neutralizes toxin)
►After binding, antibody is not available to react in indicator After binding, antibody is not available to react in indicator
system system
►Results:Results:
►NO agglutination or NO hemolysis = positive reaction NO agglutination or NO hemolysis = positive reaction
►Agglutination or hemolysis = negative reaction (antibody Agglutination or hemolysis = negative reaction (antibody
not bound in originnot bound in origin
►Similar in principle and interpretation of results Similar in principle and interpretation of results
►Antibody-bindingAntibody-binding
►Hemagglutination inhibition (serum antibody reacts with Hemagglutination inhibition (serum antibody reacts with
known nonparticulate antigen –> binding occurs) known nonparticulate antigen –> binding occurs)
►Neutralization (antibody neutralizes toxin) Neutralization (antibody neutralizes toxin)
►After binding, antibody is not available to react in indicator After binding, antibody is not available to react in indicator
system system
►Results:Results:
►NO agglutination or NO hemolysis = positive reaction NO agglutination or NO hemolysis = positive reaction
►Agglutination or hemolysis = negative reaction (antibody Agglutination or hemolysis = negative reaction (antibody
not bound in originnot bound in origin
►Generally, positive control samples used in Generally, positive control samples used in
inhibition or neutralization tests show no inhibition or neutralization tests show no
reaction and negative control samples show reaction and negative control samples show
a reaction (opposite of results in direct a reaction (opposite of results in direct
agglutination testing) agglutination testing)
►Example of inhibition: Hemagglutination Example of inhibition: Hemagglutination
inhibition test for rubella inhibition test for rubella
►Example of neutralization: antistreptolysin O Example of neutralization: antistreptolysin O
test (ASO) test (ASO)
Neutralization reactionsNeutralization reactions
inhibition or neutralization tests show no inhibition or neutralization tests show no
reaction and negative control samples show reaction and negative control samples show
a reaction (opposite of results in direct a reaction (opposite of results in direct
agglutination testing) agglutination testing)
►Example of inhibition: Hemagglutination Example of inhibition: Hemagglutination
inhibition test for rubella inhibition test for rubella
►Example of neutralization: antistreptolysin O Example of neutralization: antistreptolysin O
test (ASO) test (ASO)
Neutralization reactionsNeutralization reactions
Complement fixation (CF)Complement fixation (CF)
►Antibody and antigen allowed to combine in Antibody and antigen allowed to combine in
presence of complement presence of complement
►If complement is fixed by specific antigen-antibody If complement is fixed by specific antigen-antibody
reaction, it will be unable to combine with reaction, it will be unable to combine with
indicator system indicator system
►PrecautionsPrecautions
►Serum Serum mustmust be heat-activated be heat-activated
►Stored serum becomes anti-complementary Stored serum becomes anti-complementary
►Extensive QC/standardization required Extensive QC/standardization required
►Only use for IgM antibodies Only use for IgM antibodies
►Antibody and antigen allowed to combine in Antibody and antigen allowed to combine in
presence of complement presence of complement
►If complement is fixed by specific antigen-antibody If complement is fixed by specific antigen-antibody
reaction, it will be unable to combine with reaction, it will be unable to combine with
indicator system indicator system
►PrecautionsPrecautions
►Serum Serum mustmust be heat-activated be heat-activated
►Stored serum becomes anti-complementary Stored serum becomes anti-complementary
►Extensive QC/standardization required Extensive QC/standardization required
►Only use for IgM antibodies Only use for IgM antibodies
Imunoelectrphoresis (IEP)Imunoelectrphoresis (IEP)
QualitativeQualitative
►A serum sample is A serum sample is
electrophoresed through electrophoresed through
an agar medium.an agar medium.
►A trough is cut in the agar A trough is cut in the agar
and filled with Ab.and filled with Ab.
►A precipitin arc is then A precipitin arc is then
formed.formed.
►Because Ag diffuses Because Ag diffuses
radially and Ab from a radially and Ab from a
trough diffuses, the trough diffuses, the
reactants meet in optimal reactants meet in optimal
proportions for proportions for
precipitation.precipitation.
QualitativeQualitative
►A serum sample is A serum sample is
electrophoresed through electrophoresed through
an agar medium.an agar medium.
►A trough is cut in the agar A trough is cut in the agar
and filled with Ab.and filled with Ab.
►A precipitin arc is then A precipitin arc is then
formed.formed.
►Because Ag diffuses Because Ag diffuses
radially and Ab from a radially and Ab from a
trough diffuses, the trough diffuses, the
reactants meet in optimal reactants meet in optimal
proportions for proportions for
precipitation.precipitation.
Serology can be done on various Serology can be done on various
speciemnsspeciemns
►Some serological tests are not limited to Some serological tests are not limited to
blood serum, but can also be performed on blood serum, but can also be performed on
other bodily fluids such as semen and other bodily fluids such as semen and
saliva, which have (roughly) similar saliva, which have (roughly) similar
properties to serum.properties to serum.
►Serological tests may also be used Serological tests may also be used
forensically,forensically, generally to link a perpetrator generally to link a perpetrator
to a piece of evidence (e.g., linking a rapist to a piece of evidence (e.g., linking a rapist
to a semen sample).to a semen sample).
speciemnsspeciemns
►Some serological tests are not limited to Some serological tests are not limited to
blood serum, but can also be performed on blood serum, but can also be performed on
other bodily fluids such as semen and other bodily fluids such as semen and
saliva, which have (roughly) similar saliva, which have (roughly) similar
properties to serum.properties to serum.
►Serological tests may also be used Serological tests may also be used
forensically,forensically, generally to link a perpetrator generally to link a perpetrator
to a piece of evidence (e.g., linking a rapist to a piece of evidence (e.g., linking a rapist
to a semen sample).to a semen sample).
Enzyme immunoassay Enzyme immunoassay
(EIA/ELISA(EIA/ELISA) )
►Sandwich technique”Sandwich technique”
►Monoclonal or polyclonal antibody adsorbed on solid Monoclonal or polyclonal antibody adsorbed on solid
surface (bead or microtiter plate) surface (bead or microtiter plate)
►Add patient serum; if antigen is present in serum, it binds Add patient serum; if antigen is present in serum, it binds
to antibody coated bead or plate to antibody coated bead or plate
►Add excess labelled antibody (antibody conjugate); forms Add excess labelled antibody (antibody conjugate); forms
antigen-antibody-labelled antibody “sandwich” (antibody in antigen-antibody-labelled antibody “sandwich” (antibody in
conjugate is directed against another epitope of antigen conjugate is directed against another epitope of antigen
being tested) being tested)
►Add substrate, incubate, and read absorbance Add substrate, incubate, and read absorbance
►Washing required between each step Washing required between each step
►Absorbance is directly proportional to antigen Absorbance is directly proportional to antigen
concentration concentration
(EIA/ELISA(EIA/ELISA) )
►Sandwich technique”Sandwich technique”
►Monoclonal or polyclonal antibody adsorbed on solid Monoclonal or polyclonal antibody adsorbed on solid
surface (bead or microtiter plate) surface (bead or microtiter plate)
►Add patient serum; if antigen is present in serum, it binds Add patient serum; if antigen is present in serum, it binds
to antibody coated bead or plate to antibody coated bead or plate
►Add excess labelled antibody (antibody conjugate); forms Add excess labelled antibody (antibody conjugate); forms
antigen-antibody-labelled antibody “sandwich” (antibody in antigen-antibody-labelled antibody “sandwich” (antibody in
conjugate is directed against another epitope of antigen conjugate is directed against another epitope of antigen
being tested) being tested)
►Add substrate, incubate, and read absorbance Add substrate, incubate, and read absorbance
►Washing required between each step Washing required between each step
►Absorbance is directly proportional to antigen Absorbance is directly proportional to antigen
concentration concentration
ELISA methods takes overELISA methods takes over
►Enzyme-linked immunosorbent assayEnzyme-linked immunosorbent assay , also , also
called called ELISAELISA, , enzyme immunoassayenzyme immunoassay or or EIAEIA, is , is
a biochemical technique used mainly in a biochemical technique used mainly in
immunology to detect the presence of an antibody immunology to detect the presence of an antibody
or an antigen in a sample. The ELISA has been or an antigen in a sample. The ELISA has been
used as a diagnostic tool in medicine used as a diagnostic tool in medicine
►Because the ELISA can be performed to evaluate Because the ELISA can be performed to evaluate
either the presence of antigen or the presence of either the presence of antigen or the presence of
antibody in a sample antibody in a sample
►Enzyme-linked immunosorbent assayEnzyme-linked immunosorbent assay , also , also
called called ELISAELISA, , enzyme immunoassayenzyme immunoassay or or EIAEIA, is , is
a biochemical technique used mainly in a biochemical technique used mainly in
immunology to detect the presence of an antibody immunology to detect the presence of an antibody
or an antigen in a sample. The ELISA has been or an antigen in a sample. The ELISA has been
used as a diagnostic tool in medicine used as a diagnostic tool in medicine
►Because the ELISA can be performed to evaluate Because the ELISA can be performed to evaluate
either the presence of antigen or the presence of either the presence of antigen or the presence of
antibody in a sample antibody in a sample
ELISA Most popular technological ELISA Most popular technological
advance in Laboratory Medicineadvance in Laboratory Medicine
►ELISA methods can ELISA methods can
detect any infectious detect any infectious
disease provided if we disease provided if we
have antibodies and have antibodies and
antigen to any antigen to any
infection, enzyme or infection, enzyme or
any substanceany substance
advance in Laboratory Medicineadvance in Laboratory Medicine
►ELISA methods can ELISA methods can
detect any infectious detect any infectious
disease provided if we disease provided if we
have antibodies and have antibodies and
antigen to any antigen to any
infection, enzyme or infection, enzyme or
any substanceany substance
Serology applications Serology applications
inin....
►HIV testing HIV testing
►Serum HCG Serum HCG
(pregnancy) (pregnancy)
►Tests for hepatitis Tests for hepatitis
antigens and antigens and
antibodies antibodies
►Antibodies to bacteria Antibodies to bacteria
►Hepatitis SerologyHepatitis Serology
inin....
►HIV testing HIV testing
►Serum HCG Serum HCG
(pregnancy) (pregnancy)
►Tests for hepatitis Tests for hepatitis
antigens and antigens and
antibodies antibodies
►Antibodies to bacteria Antibodies to bacteria
►Hepatitis SerologyHepatitis Serology
NephelometryNephelometry
►ProcedureProcedure
Serum substance reacts with specific antisera and forms Serum substance reacts with specific antisera and forms
insoluble complexes insoluble complexes
Light is passed through suspension Light is passed through suspension
Scattered (reflected) light is proportional to number of Scattered (reflected) light is proportional to number of
insoluble complexes; compare to standards insoluble complexes; compare to standards
►ExamplesExamples
Complement component concentration Complement component concentration
Antibody concentration (IgG, IgM, IgA, etc.) Antibody concentration (IgG, IgM, IgA, etc.)
►ImmunofluorescenceImmunofluorescence
►ProcedureProcedure
Serum substance reacts with specific antisera and forms Serum substance reacts with specific antisera and forms
insoluble complexes insoluble complexes
Light is passed through suspension Light is passed through suspension
Scattered (reflected) light is proportional to number of Scattered (reflected) light is proportional to number of
insoluble complexes; compare to standards insoluble complexes; compare to standards
►ExamplesExamples
Complement component concentration Complement component concentration
Antibody concentration (IgG, IgM, IgA, etc.) Antibody concentration (IgG, IgM, IgA, etc.)
►ImmunofluorescenceImmunofluorescence
ImmunofluorescenceImmunofluorescence
►Direct – add fluorescein-labeled antibody to Direct – add fluorescein-labeled antibody to
patient tissue, wash, and examine under patient tissue, wash, and examine under
fluorescent microscope fluorescent microscope
►Indirect – add patient serum to tissue containing Indirect – add patient serum to tissue containing
known antigen, wash, add labeled antiglobulin, known antigen, wash, add labeled antiglobulin,
wash, and examine under fluorescent microscope wash, and examine under fluorescent microscope
►ExamplesExamples
Testing for Antinuclear Antibodies (ANA) Testing for Antinuclear Antibodies (ANA)
Fluorescent Treponemal Antibody Test (FTA-Abs) Fluorescent Treponemal Antibody Test (FTA-Abs)
►Direct – add fluorescein-labeled antibody to Direct – add fluorescein-labeled antibody to
patient tissue, wash, and examine under patient tissue, wash, and examine under
fluorescent microscope fluorescent microscope
►Indirect – add patient serum to tissue containing Indirect – add patient serum to tissue containing
known antigen, wash, add labeled antiglobulin, known antigen, wash, add labeled antiglobulin,
wash, and examine under fluorescent microscope wash, and examine under fluorescent microscope
►ExamplesExamples
Testing for Antinuclear Antibodies (ANA) Testing for Antinuclear Antibodies (ANA)
Fluorescent Treponemal Antibody Test (FTA-Abs) Fluorescent Treponemal Antibody Test (FTA-Abs)
Fluorescence polarization Fluorescence polarization
immunoassay (FPIA)immunoassay (FPIA)
►PrinciplePrinciple
►Add reagent antibody and fluorescent-tagged antigen to Add reagent antibody and fluorescent-tagged antigen to
patient serum patient serum
►Positive testPositive test
Antigen present in patient serum binds to reagent leaving most Antigen present in patient serum binds to reagent leaving most
tagged antigen unbound tagged antigen unbound
Unbound labeled antigens rotate quickly reducing amount of Unbound labeled antigens rotate quickly reducing amount of
polarized light produced polarized light produced
►Negative testNegative test
If no antigen present in patient serum, tagged antigen binds to If no antigen present in patient serum, tagged antigen binds to
reagent antibody reagent antibody
Tagged antigen-antibody complexes rotate slowly giving off Tagged antigen-antibody complexes rotate slowly giving off
increased polarized light increased polarized light
immunoassay (FPIA)immunoassay (FPIA)
►PrinciplePrinciple
►Add reagent antibody and fluorescent-tagged antigen to Add reagent antibody and fluorescent-tagged antigen to
patient serum patient serum
►Positive testPositive test
Antigen present in patient serum binds to reagent leaving most Antigen present in patient serum binds to reagent leaving most
tagged antigen unbound tagged antigen unbound
Unbound labeled antigens rotate quickly reducing amount of Unbound labeled antigens rotate quickly reducing amount of
polarized light produced polarized light produced
►Negative testNegative test
If no antigen present in patient serum, tagged antigen binds to If no antigen present in patient serum, tagged antigen binds to
reagent antibody reagent antibody
Tagged antigen-antibody complexes rotate slowly giving off Tagged antigen-antibody complexes rotate slowly giving off
increased polarized light increased polarized light
Flow cytometryFlow cytometry
►Method of choice for T- and B-cell analysis (lymphocyte Method of choice for T- and B-cell analysis (lymphocyte
phenotyping) phenotyping)
►PrinciplePrinciple
►Incubate specimen with 1 or 2 monoclonal antibodies Incubate specimen with 1 or 2 monoclonal antibodies
tagged with fluorochrome tagged with fluorochrome
►Single cells pass through incident light of instrument Single cells pass through incident light of instrument
(laser) which excites fluorochrome and results in emitted (laser) which excites fluorochrome and results in emitted
light of different wavelength light of different wavelength
►Intensity of fluorescence measured to detect cells Intensity of fluorescence measured to detect cells
possessing surface markers for the specific monoclonal possessing surface markers for the specific monoclonal
antibodies that were employed antibodies that were employed
►Forward light scatter indicates cell size or volume Forward light scatter indicates cell size or volume
►90° side-scattered light indicates granula90° side-scattered light indicates granula
►Method of choice for T- and B-cell analysis (lymphocyte Method of choice for T- and B-cell analysis (lymphocyte
phenotyping) phenotyping)
►PrinciplePrinciple
►Incubate specimen with 1 or 2 monoclonal antibodies Incubate specimen with 1 or 2 monoclonal antibodies
tagged with fluorochrome tagged with fluorochrome
►Single cells pass through incident light of instrument Single cells pass through incident light of instrument
(laser) which excites fluorochrome and results in emitted (laser) which excites fluorochrome and results in emitted
light of different wavelength light of different wavelength
►Intensity of fluorescence measured to detect cells Intensity of fluorescence measured to detect cells
possessing surface markers for the specific monoclonal possessing surface markers for the specific monoclonal
antibodies that were employed antibodies that were employed
►Forward light scatter indicates cell size or volume Forward light scatter indicates cell size or volume
►90° side-scattered light indicates granula90° side-scattered light indicates granula
Common uses Flow cytometryCommon uses Flow cytometry
►DNA analysis DNA analysis
►Reticulocyte counts Reticulocyte counts
►Leukaemia/lymphoma Leukaemia/lymphoma
classification classification
►CD 4 cell estimations in CD 4 cell estimations in
AIDS/HIV patients.AIDS/HIV patients.
►DNA analysis DNA analysis
►Reticulocyte counts Reticulocyte counts
►Leukaemia/lymphoma Leukaemia/lymphoma
classification classification
►CD 4 cell estimations in CD 4 cell estimations in
AIDS/HIV patients.AIDS/HIV patients.
Other Applications of Other Applications of
agglutination tests in Serologyagglutination tests in Serology
i. i. Determination of blood types or antibodies Determination of blood types or antibodies
to blood group antigensto blood group antigens..
ii. ii. To assess bacterial infectionsTo assess bacterial infections
e.ge.g. A rise in titer of an antibody to a particular . A rise in titer of an antibody to a particular
bacterium indicates an infection with that bacterial bacterium indicates an infection with that bacterial
typetype. . NN..BB. . a fourfold rise in titer is generally taken a fourfold rise in titer is generally taken
as a significant rise in antibody titer.as a significant rise in antibody titer.
agglutination tests in Serologyagglutination tests in Serology
i. i. Determination of blood types or antibodies Determination of blood types or antibodies
to blood group antigensto blood group antigens..
ii. ii. To assess bacterial infectionsTo assess bacterial infections
e.ge.g. A rise in titer of an antibody to a particular . A rise in titer of an antibody to a particular
bacterium indicates an infection with that bacterial bacterium indicates an infection with that bacterial
typetype. . NN..BB. . a fourfold rise in titer is generally taken a fourfold rise in titer is generally taken
as a significant rise in antibody titer.as a significant rise in antibody titer.
Georges-Fernand-Isidor Georges-Fernand-Isidor
Widal Widal
►Widal in 1896, and Widal in 1896, and
Widal & Sicard in Widal & Sicard in
1896 described the 1896 described the
Widal reaction, and Widal reaction, and
this test has proved this test has proved
of value in cases of value in cases
where positive where positive
cultures have been cultures have been
unobtainableunobtainable
Widal Widal
►Widal in 1896, and Widal in 1896, and
Widal & Sicard in Widal & Sicard in
1896 described the 1896 described the
Widal reaction, and Widal reaction, and
this test has proved this test has proved
of value in cases of value in cases
where positive where positive
cultures have been cultures have been
unobtainableunobtainable
Widal test a Popular test in Widal test a Popular test in
diagnosis of Typhoid Feverdiagnosis of Typhoid Fever
►The The Widal testWidal test is a is a
presumptive presumptive
serological test for serological test for
Enteric fever or Enteric fever or
Undulant fever. In Undulant fever. In
case of case of SalmonellaSalmonella
infections, it is a infections, it is a
demonstration of demonstration of
agglutinating agglutinating antibodies antibodies
against antigens O-somatic against antigens O-somatic
and H-flagellar in the and H-flagellar in the
blood.blood.
diagnosis of Typhoid Feverdiagnosis of Typhoid Fever
►The The Widal testWidal test is a is a
presumptive presumptive
serological test for serological test for
Enteric fever or Enteric fever or
Undulant fever. In Undulant fever. In
case of case of SalmonellaSalmonella
infections, it is a infections, it is a
demonstration of demonstration of
agglutinating agglutinating antibodies antibodies
against antigens O-somatic against antigens O-somatic
and H-flagellar in the and H-flagellar in the
blood.blood.
Widal test is century old ,Widal test is century old ,
Is it loosing importance ?Is it loosing importance ?
►In this reaction antibodies react with antigens on In this reaction antibodies react with antigens on
the surface of particulate objects and cause the the surface of particulate objects and cause the
objects to clump together, or agglutinate. These objects to clump together, or agglutinate. These
reactions were the earliest to be adapted to reactions were the earliest to be adapted to
diagnostic laboratory. Widal test is used for diagnostic laboratory. Widal test is used for
diagnosis of typhoid fever. This test, developed by diagnosis of typhoid fever. This test, developed by
Georges Fernand I. Widal (French physician) in Georges Fernand I. Widal (French physician) in
1896, is now supplemented by more sophisticated 1896, is now supplemented by more sophisticated
procedures. procedures.
Is it loosing importance ?Is it loosing importance ?
►In this reaction antibodies react with antigens on In this reaction antibodies react with antigens on
the surface of particulate objects and cause the the surface of particulate objects and cause the
objects to clump together, or agglutinate. These objects to clump together, or agglutinate. These
reactions were the earliest to be adapted to reactions were the earliest to be adapted to
diagnostic laboratory. Widal test is used for diagnostic laboratory. Widal test is used for
diagnosis of typhoid fever. This test, developed by diagnosis of typhoid fever. This test, developed by
Georges Fernand I. Widal (French physician) in Georges Fernand I. Widal (French physician) in
1896, is now supplemented by more sophisticated 1896, is now supplemented by more sophisticated
procedures. procedures.
Widal testWidal test – – A standard tube A standard tube
agglutination testagglutination test
►Test can be performed by the tube dilution Test can be performed by the tube dilution
technique which permits, the assay of antibody technique which permits, the assay of antibody
titre. In this, a constant amount of the antigen is titre. In this, a constant amount of the antigen is
added to a series of tubes containing serum added to a series of tubes containing serum
dilutions. After mixing, the tubes are incubated at dilutions. After mixing, the tubes are incubated at
a particular temperature and the highest dilution a particular temperature and the highest dilution
of serum showing visible agglutination is of serum showing visible agglutination is
determined. determined.
agglutination testagglutination test
►Test can be performed by the tube dilution Test can be performed by the tube dilution
technique which permits, the assay of antibody technique which permits, the assay of antibody
titre. In this, a constant amount of the antigen is titre. In this, a constant amount of the antigen is
added to a series of tubes containing serum added to a series of tubes containing serum
dilutions. After mixing, the tubes are incubated at dilutions. After mixing, the tubes are incubated at
a particular temperature and the highest dilution a particular temperature and the highest dilution
of serum showing visible agglutination is of serum showing visible agglutination is
determined. determined.
Agglutination how it appear Agglutination how it appear
after reactivityafter reactivity
►O O
agglutination agglutination
is granularis granular
►H H
agglutination agglutination
is loose and is loose and
floccularfloccular
after reactivityafter reactivity
►O O
agglutination agglutination
is granularis granular
►H H
agglutination agglutination
is loose and is loose and
floccularfloccular
Principle of the TestPrinciple of the Test
►A classic example of the agglutination A classic example of the agglutination
reaction is seen in the reaction is seen in the widal testwidal test for for
diagnosis of typhoid fever. In this test the diagnosis of typhoid fever. In this test the
antibody content of the patient's serum, is antibody content of the patient's serum, is
measured by adding a constant amount of measured by adding a constant amount of
antigen (Salmonella typhi) to the serially antigen (Salmonella typhi) to the serially
diluted serum. diluted serum.
►A classic example of the agglutination A classic example of the agglutination
reaction is seen in the reaction is seen in the widal testwidal test for for
diagnosis of typhoid fever. In this test the diagnosis of typhoid fever. In this test the
antibody content of the patient's serum, is antibody content of the patient's serum, is
measured by adding a constant amount of measured by adding a constant amount of
antigen (Salmonella typhi) to the serially antigen (Salmonella typhi) to the serially
diluted serum. diluted serum.
Reading the Widal TestReading the Widal Test
►Read the results by viewing the tubes under Read the results by viewing the tubes under
good light against the dark background with good light against the dark background with
x2 magnifying lensx2 magnifying lens
►Do not shake tubes before reading the Do not shake tubes before reading the
resultsresults
►Read titers as greatest dilutions giving Read titers as greatest dilutions giving
visible agglutinations.visible agglutinations.
►Limiting agglutination is 1in 200 the titer is Limiting agglutination is 1in 200 the titer is
200 not to be reported as 1/200.200 not to be reported as 1/200.
►Read the results by viewing the tubes under Read the results by viewing the tubes under
good light against the dark background with good light against the dark background with
x2 magnifying lensx2 magnifying lens
►Do not shake tubes before reading the Do not shake tubes before reading the
resultsresults
►Read titers as greatest dilutions giving Read titers as greatest dilutions giving
visible agglutinations.visible agglutinations.
►Limiting agglutination is 1in 200 the titer is Limiting agglutination is 1in 200 the titer is
200 not to be reported as 1/200.200 not to be reported as 1/200.
Interpretation of Widal testInterpretation of Widal test
►Test results need to be Test results need to be
interpreted carefully in interpreted carefully in
the light of past history the light of past history
of enteric fever, of enteric fever,
typhoid vaccination, typhoid vaccination,
general level of general level of
antibodies in the antibodies in the
populations in endemic populations in endemic
areas of the world. areas of the world.
►Test results need to be Test results need to be
interpreted carefully in interpreted carefully in
the light of past history the light of past history
of enteric fever, of enteric fever,
typhoid vaccination, typhoid vaccination,
general level of general level of
antibodies in the antibodies in the
populations in endemic populations in endemic
areas of the world. areas of the world.
Testing in Typhoid carriersTesting in Typhoid carriers
►Many known carriers of typhoid bacilli Many known carriers of typhoid bacilli
possess antibody against the Vi possess antibody against the Vi
(virulence) antigen of S. typhi. This is a (virulence) antigen of S. typhi. This is a
surface antigen easily lost during surface antigen easily lost during
cultivation(Vi tires seem to correlate cultivation(Vi tires seem to correlate
better with the carrier state than do O better with the carrier state than do O
or H titres). For this reason, Felix et al. or H titres). For this reason, Felix et al.
suggested the use of Vi agglutination suggested the use of Vi agglutination
for detection of carriers.for detection of carriers.
►Many known carriers of typhoid bacilli Many known carriers of typhoid bacilli
possess antibody against the Vi possess antibody against the Vi
(virulence) antigen of S. typhi. This is a (virulence) antigen of S. typhi. This is a
surface antigen easily lost during surface antigen easily lost during
cultivation(Vi tires seem to correlate cultivation(Vi tires seem to correlate
better with the carrier state than do O better with the carrier state than do O
or H titres). For this reason, Felix et al. or H titres). For this reason, Felix et al.
suggested the use of Vi agglutination suggested the use of Vi agglutination
for detection of carriers.for detection of carriers.
Importance of Vi antibodiesImportance of Vi antibodies
►Many known carriers of typhoid bacilli Many known carriers of typhoid bacilli
possess antibody against the Vi possess antibody against the Vi
(virulence) antigen of S. typhi. This is a (virulence) antigen of S. typhi. This is a
surface antigen easily lost during surface antigen easily lost during
cultivation(Vi tires seem to correlate cultivation(Vi tires seem to correlate
better with the carrier state than do O better with the carrier state than do O
or H titres). For this reason, Felix et al. or H titres). For this reason, Felix et al.
suggested the use of Vi agglutination suggested the use of Vi agglutination
for detection of carriers.for detection of carriers.
►Many known carriers of typhoid bacilli Many known carriers of typhoid bacilli
possess antibody against the Vi possess antibody against the Vi
(virulence) antigen of S. typhi. This is a (virulence) antigen of S. typhi. This is a
surface antigen easily lost during surface antigen easily lost during
cultivation(Vi tires seem to correlate cultivation(Vi tires seem to correlate
better with the carrier state than do O better with the carrier state than do O
or H titres). For this reason, Felix et al. or H titres). For this reason, Felix et al.
suggested the use of Vi agglutination suggested the use of Vi agglutination
for detection of carriers.for detection of carriers.
Prozone phenomenon in Prozone phenomenon in
Agglutination testsAgglutination tests
Prozone effect Prozone effect - - OccasionallyOccasionally, , it is observed that it is observed that
when the concentration of antibody is high when the concentration of antibody is high ((ii..ee. .
lower dilutionslower dilutions)), there is no agglutination and , there is no agglutination and
then, as the sample is diluted, agglutination then, as the sample is diluted, agglutination
occurs.occurs.
The lack of agglutination at high concentrations of The lack of agglutination at high concentrations of
antibodies is called theantibodies is called the prozoneprozone effect effect. . Lack of Lack of
agglutination in the prozone is due to antibody agglutination in the prozone is due to antibody
excess resulting in very small complexes that do excess resulting in very small complexes that do
not clump to form visible agglutinationnot clump to form visible agglutination
Agglutination testsAgglutination tests
Prozone effect Prozone effect - - OccasionallyOccasionally, , it is observed that it is observed that
when the concentration of antibody is high when the concentration of antibody is high ((ii..ee. .
lower dilutionslower dilutions)), there is no agglutination and , there is no agglutination and
then, as the sample is diluted, agglutination then, as the sample is diluted, agglutination
occurs.occurs.
The lack of agglutination at high concentrations of The lack of agglutination at high concentrations of
antibodies is called theantibodies is called the prozoneprozone effect effect. . Lack of Lack of
agglutination in the prozone is due to antibody agglutination in the prozone is due to antibody
excess resulting in very small complexes that do excess resulting in very small complexes that do
not clump to form visible agglutinationnot clump to form visible agglutination
Causes Of False-positive Widal Causes Of False-positive Widal
Agglutination TestsAgglutination Tests
►Previous immunization with Salmonella Previous immunization with Salmonella
antigenantigen..
►CrossCross--reaction with non – typhoidal reaction with non – typhoidal
SalmonellaSalmonella..
►Variability and poorly standardized Variability and poorly standardized
commercial commercial antigen preparationantigen preparation..
►Infection with malaria Infection with malaria
►other Enterobacteriaceae charring the same other Enterobacteriaceae charring the same
ss--LPS LPS ..
Agglutination TestsAgglutination Tests
►Previous immunization with Salmonella Previous immunization with Salmonella
antigenantigen..
►CrossCross--reaction with non – typhoidal reaction with non – typhoidal
SalmonellaSalmonella..
►Variability and poorly standardized Variability and poorly standardized
commercial commercial antigen preparationantigen preparation..
►Infection with malaria Infection with malaria
►other Enterobacteriaceae charring the same other Enterobacteriaceae charring the same
ss--LPS LPS ..
Causes of Negative Widal Causes of Negative Widal
Agglutination TestAgglutination Test
►The carrier stateThe carrier state
►An inadequate inoculum of bacterial antigen An inadequate inoculum of bacterial antigen
in the host to induce antibody productionin the host to induce antibody production
►Technical difficulty or errors in the Technical difficulty or errors in the
performance of the test.performance of the test.
►Previous antibiotic treatmentPrevious antibiotic treatment
►Variability in the preparation of Variability in the preparation of
commercial antigens.commercial antigens.
Agglutination TestAgglutination Test
►The carrier stateThe carrier state
►An inadequate inoculum of bacterial antigen An inadequate inoculum of bacterial antigen
in the host to induce antibody productionin the host to induce antibody production
►Technical difficulty or errors in the Technical difficulty or errors in the
performance of the test.performance of the test.
►Previous antibiotic treatmentPrevious antibiotic treatment
►Variability in the preparation of Variability in the preparation of
commercial antigens.commercial antigens.
Declining importance of Widal Declining importance of Widal
testtest
►The value of the salmonella agglutination The value of the salmonella agglutination
tests has declined as the incidence of tests has declined as the incidence of
typhoid fever has decreased, at least in the typhoid fever has decreased, at least in the
developed world, the general use of developed world, the general use of
vaccines has increased, and ever increasing vaccines has increased, and ever increasing
-numbers of antigenically related serotypes -numbers of antigenically related serotypes
of Salmonella have been recognised.of Salmonella have been recognised.
testtest
►The value of the salmonella agglutination The value of the salmonella agglutination
tests has declined as the incidence of tests has declined as the incidence of
typhoid fever has decreased, at least in the typhoid fever has decreased, at least in the
developed world, the general use of developed world, the general use of
vaccines has increased, and ever increasing vaccines has increased, and ever increasing
-numbers of antigenically related serotypes -numbers of antigenically related serotypes
of Salmonella have been recognised.of Salmonella have been recognised.
Serology - Importance of repeated Serology - Importance of repeated
teststests
Criteria for diagnosing Primary InfectionCriteria for diagnosing Primary Infection
►4 fold or more increase in titre of IgG or total antibody between 4 fold or more increase in titre of IgG or total antibody between
acute and convalescent seraacute and convalescent sera
►Presence of IgMPresence of IgM
►SeroconversionSeroconversion
►A single high titre of IgG (or total antibody) - very unreliableA single high titre of IgG (or total antibody) - very unreliable
Criteria for diagnosing ReinfectionCriteria for diagnosing Reinfection
►Four fold or more increase in titre of IgG or total Four fold or more increase in titre of IgG or total
antibody between acute and convalescent seraantibody between acute and convalescent sera
►Absence or slight increase in IgMAbsence or slight increase in IgM
teststests
Criteria for diagnosing Primary InfectionCriteria for diagnosing Primary Infection
►4 fold or more increase in titre of IgG or total antibody between 4 fold or more increase in titre of IgG or total antibody between
acute and convalescent seraacute and convalescent sera
►Presence of IgMPresence of IgM
►SeroconversionSeroconversion
►A single high titre of IgG (or total antibody) - very unreliableA single high titre of IgG (or total antibody) - very unreliable
Criteria for diagnosing ReinfectionCriteria for diagnosing Reinfection
►Four fold or more increase in titre of IgG or total Four fold or more increase in titre of IgG or total
antibody between acute and convalescent seraantibody between acute and convalescent sera
►Absence or slight increase in IgMAbsence or slight increase in IgM
Typical Serological Profile After Acute Typical Serological Profile After Acute
InfectionInfection
Note that during Reinfections, IgM may be absent or present at a low level transientlyNote that during Reinfections, IgM may be absent or present at a low level transiently
InfectionInfection
Note that during Reinfections, IgM may be absent or present at a low level transientlyNote that during Reinfections, IgM may be absent or present at a low level transiently
Antigen – Antibody Antigen – Antibody
reactions presenting reactions presenting
with precipitationwith precipitation
reactions presenting reactions presenting
with precipitationwith precipitation
Precipitation CurvePrecipitation Curve
Precipitation CurvePrecipitation Curve
Measurement of Precipitation by Measurement of Precipitation by
LightLight
►Antigen-antibody complexes, when formed Antigen-antibody complexes, when formed
at a high rate, will precipitate out of a at a high rate, will precipitate out of a
solution resulting in a turbid or cloudy solution resulting in a turbid or cloudy
appearance.appearance.
►Turbidimetry measures the turbidity or Turbidimetry measures the turbidity or
cloudiness of a solution by measuring cloudiness of a solution by measuring
amount of light directly passing through a amount of light directly passing through a
solution.solution.
►Nephelometry indirect measurement, measures amount of Nephelometry indirect measurement, measures amount of
light scattered by the antigen-antibody complexes.light scattered by the antigen-antibody complexes.
LightLight
►Antigen-antibody complexes, when formed Antigen-antibody complexes, when formed
at a high rate, will precipitate out of a at a high rate, will precipitate out of a
solution resulting in a turbid or cloudy solution resulting in a turbid or cloudy
appearance.appearance.
►Turbidimetry measures the turbidity or Turbidimetry measures the turbidity or
cloudiness of a solution by measuring cloudiness of a solution by measuring
amount of light directly passing through a amount of light directly passing through a
solution.solution.
►Nephelometry indirect measurement, measures amount of Nephelometry indirect measurement, measures amount of
light scattered by the antigen-antibody complexes.light scattered by the antigen-antibody complexes.
Screening Tests for SyphilisScreening Tests for Syphilis
► Serologic methods are divided into two Serologic methods are divided into two
classes. One class, the nontreponemal tests, classes. One class, the nontreponemal tests,
detects antibodies to lipoidal antigens detects antibodies to lipoidal antigens
present in either the host or T. pallidum; present in either the host or T. pallidum;
examples are the examples are the Venereal Disease Venereal Disease
Research Laboratory and rapid plasma Research Laboratory and rapid plasma
reagin and tests. reagin and tests.
► Serologic methods are divided into two Serologic methods are divided into two
classes. One class, the nontreponemal tests, classes. One class, the nontreponemal tests,
detects antibodies to lipoidal antigens detects antibodies to lipoidal antigens
present in either the host or T. pallidum; present in either the host or T. pallidum;
examples are the examples are the Venereal Disease Venereal Disease
Research Laboratory and rapid plasma Research Laboratory and rapid plasma
reagin and tests. reagin and tests.
Serological Diagnosis Of SyphilisSerological Diagnosis Of Syphilis
I.I.Specific Anti- Specific Anti-
treponemal treponemal
AntibodyAntibody
II.II.Anti – treponemal Anti – treponemal
AntibodyAntibody
III.III.Reagin Antibody Reagin Antibody
(VDRL and RPR(VDRL and RPR))
Associated with higher Associated with higher
false positivesfalse positives
I.I.Specific Anti- Specific Anti-
treponemal treponemal
AntibodyAntibody
II.II.Anti – treponemal Anti – treponemal
AntibodyAntibody
III.III.Reagin Antibody Reagin Antibody
(VDRL and RPR(VDRL and RPR))
Associated with higher Associated with higher
false positivesfalse positives
Indication for testing for Indication for testing for
SyphilisSyphilis
Pregnant women Pregnant women
sexual contacts or sexual contacts or
partners of patients partners of patients
diagnosed with diagnosed with
syphilis syphilis
children born to children born to
mothers with syphilis mothers with syphilis
patients with HIV patients with HIV
infection infection
SyphilisSyphilis
Pregnant women Pregnant women
sexual contacts or sexual contacts or
partners of patients partners of patients
diagnosed with diagnosed with
syphilis syphilis
children born to children born to
mothers with syphilis mothers with syphilis
patients with HIV patients with HIV
infection infection
Tests For Reagin AntibodyTests For Reagin Antibody
►A large numbers of tests for ReaginA large numbers of tests for Reagin::
►VDRLVDRL ((Venereal Diseases Reference LaboratoryVenereal Diseases Reference Laboratory).).
►RPR RPR ((Rapid Plasma ReaginRapid Plasma Reagin))
►ARTART ((Automated Reagin TestAutomated Reagin Test))
Good sensitive screening Good sensitive screening
Titre falls rapidly with treatmentTitre falls rapidly with treatment
►Reagin titre falls with treatmentReagin titre falls with treatment..
►A large numbers of tests for ReaginA large numbers of tests for Reagin::
►VDRLVDRL ((Venereal Diseases Reference LaboratoryVenereal Diseases Reference Laboratory).).
►RPR RPR ((Rapid Plasma ReaginRapid Plasma Reagin))
►ARTART ((Automated Reagin TestAutomated Reagin Test))
Good sensitive screening Good sensitive screening
Titre falls rapidly with treatmentTitre falls rapidly with treatment
►Reagin titre falls with treatmentReagin titre falls with treatment..
VDRLVDRL – A standard test for Syphilis – A standard test for Syphilis
►NONTREPONEMAL ANTIGEN TESTS. NONTREPONEMAL ANTIGEN TESTS.
Nontreponemal antigen tests are used as Nontreponemal antigen tests are used as
screeners. They measure the presence of reagin, screeners. They measure the presence of reagin,
which is an antibody formed in reaction to syphilis. which is an antibody formed in reaction to syphilis.
In the venereal disease research laboratory In the venereal disease research laboratory
(VDRL) test, a sample of the patient's blood is (VDRL) test, a sample of the patient's blood is
mixed with cardiolipin and cholesterol. If the mixed with cardiolipin and cholesterol. If the
mixture forms clumps or masses of matter, the mixture forms clumps or masses of matter, the
test is considered reactive or positive. The serum test is considered reactive or positive. The serum
sample can be diluted several times to determine sample can be diluted several times to determine
the concentration of reagin in the patient's blood. the concentration of reagin in the patient's blood.
►NONTREPONEMAL ANTIGEN TESTS. NONTREPONEMAL ANTIGEN TESTS.
Nontreponemal antigen tests are used as Nontreponemal antigen tests are used as
screeners. They measure the presence of reagin, screeners. They measure the presence of reagin,
which is an antibody formed in reaction to syphilis. which is an antibody formed in reaction to syphilis.
In the venereal disease research laboratory In the venereal disease research laboratory
(VDRL) test, a sample of the patient's blood is (VDRL) test, a sample of the patient's blood is
mixed with cardiolipin and cholesterol. If the mixed with cardiolipin and cholesterol. If the
mixture forms clumps or masses of matter, the mixture forms clumps or masses of matter, the
test is considered reactive or positive. The serum test is considered reactive or positive. The serum
sample can be diluted several times to determine sample can be diluted several times to determine
the concentration of reagin in the patient's blood. the concentration of reagin in the patient's blood.
Screening tests should be Screening tests should be
reported with cautionsreported with cautions
►Reactivity in these tests generally indicates Reactivity in these tests generally indicates
host tissue damage that may not be host tissue damage that may not be
specific for syphilisspecific for syphilis. Because these tests . Because these tests
are easy and inexpensive to perform, they are easy and inexpensive to perform, they
are commonly used for screening, and with are commonly used for screening, and with
proper clinical signs they are suggestive of proper clinical signs they are suggestive of
syphilis. The other class of test, the syphilis. The other class of test, the
Treponemal tests, uses specific Treponemal Treponemal tests, uses specific Treponemal
antigens. antigens.
reported with cautionsreported with cautions
►Reactivity in these tests generally indicates Reactivity in these tests generally indicates
host tissue damage that may not be host tissue damage that may not be
specific for syphilisspecific for syphilis. Because these tests . Because these tests
are easy and inexpensive to perform, they are easy and inexpensive to perform, they
are commonly used for screening, and with are commonly used for screening, and with
proper clinical signs they are suggestive of proper clinical signs they are suggestive of
syphilis. The other class of test, the syphilis. The other class of test, the
Treponemal tests, uses specific Treponemal Treponemal tests, uses specific Treponemal
antigens. antigens.
Combination of testes are Combination of testes are
desirabledesirable
►Syphilis Syphilis serodiagnosis serodiagnosis
relies on a combination relies on a combination
of nonspecific of nonspecific
screening tests screening tests
(antilipoidal antibodies) (antilipoidal antibodies)
and and Treponema Treponema
pallidumpallidum-specific tests -specific tests
(anti-(anti-TT. . pallidumpallidum
antibodies). antibodies).
desirabledesirable
►Syphilis Syphilis serodiagnosis serodiagnosis
relies on a combination relies on a combination
of nonspecific of nonspecific
screening tests screening tests
(antilipoidal antibodies) (antilipoidal antibodies)
and and Treponema Treponema
pallidumpallidum-specific tests -specific tests
(anti-(anti-TT. . pallidumpallidum
antibodies). antibodies).
Measurement of Precipitation by Measurement of Precipitation by
LightLight
►Antigen-antibody complexes, when formed Antigen-antibody complexes, when formed
at a high rate, will precipitate out of a at a high rate, will precipitate out of a
solution resulting in a turbid or cloudy solution resulting in a turbid or cloudy
appearance.appearance.
►Turbidimetry measures the turbidity or Turbidimetry measures the turbidity or
cloudiness of a solution by measuring cloudiness of a solution by measuring
amount of light directly passing through a amount of light directly passing through a
solution.solution.
►Nephelometry indirect measurement, measures amount of Nephelometry indirect measurement, measures amount of
light scattered by the antigen-antibody complexeslight scattered by the antigen-antibody complexes..
LightLight
►Antigen-antibody complexes, when formed Antigen-antibody complexes, when formed
at a high rate, will precipitate out of a at a high rate, will precipitate out of a
solution resulting in a turbid or cloudy solution resulting in a turbid or cloudy
appearance.appearance.
►Turbidimetry measures the turbidity or Turbidimetry measures the turbidity or
cloudiness of a solution by measuring cloudiness of a solution by measuring
amount of light directly passing through a amount of light directly passing through a
solution.solution.
►Nephelometry indirect measurement, measures amount of Nephelometry indirect measurement, measures amount of
light scattered by the antigen-antibody complexeslight scattered by the antigen-antibody complexes..
Confirmation is warrantedConfirmation is warranted
►Confirmation of infection requires a reactive Confirmation of infection requires a reactive
Treponemal test. Examples of the Treponemal Treponemal test. Examples of the Treponemal
tests are the microhemagglutination assay for tests are the microhemagglutination assay for
antibodies to T. pallidum and the fluorescent antibodies to T. pallidum and the fluorescent
treponemal antibody absorption test. These tests treponemal antibody absorption test. These tests
are more expensive and complicated to perform are more expensive and complicated to perform
than the nontreponemal tests. On the horizon are than the nontreponemal tests. On the horizon are
a number of direct antigen, enzyme-linked a number of direct antigen, enzyme-linked
immunosorbent assay, and PCR technique immunosorbent assay, and PCR technique
►Confirmation of infection requires a reactive Confirmation of infection requires a reactive
Treponemal test. Examples of the Treponemal Treponemal test. Examples of the Treponemal
tests are the microhemagglutination assay for tests are the microhemagglutination assay for
antibodies to T. pallidum and the fluorescent antibodies to T. pallidum and the fluorescent
treponemal antibody absorption test. These tests treponemal antibody absorption test. These tests
are more expensive and complicated to perform are more expensive and complicated to perform
than the nontreponemal tests. On the horizon are than the nontreponemal tests. On the horizon are
a number of direct antigen, enzyme-linked a number of direct antigen, enzyme-linked
immunosorbent assay, and PCR technique immunosorbent assay, and PCR technique
Non reactiveNon reactive and and Reactive Reactive VDRL VDRL
TestsTests
TestsTests
Rapid plasma reaginRapid plasma reagin
►The rapid plasma The rapid plasma
reagin (RPR) test reagin (RPR) test
works on the same works on the same
principle as the VDRL. principle as the VDRL.
It is available as a kit. It is available as a kit.
The patient's serum is The patient's serum is
mixed with cardiolipin mixed with cardiolipin
on a plastic-coated on a plastic-coated
card that can be card that can be
examined with the examined with the
naked eye. naked eye.
►The rapid plasma The rapid plasma
reagin (RPR) test reagin (RPR) test
works on the same works on the same
principle as the VDRL. principle as the VDRL.
It is available as a kit. It is available as a kit.
The patient's serum is The patient's serum is
mixed with cardiolipin mixed with cardiolipin
on a plastic-coated on a plastic-coated
card that can be card that can be
examined with the examined with the
naked eye. naked eye.
Agglutination++Agglutination++ RPR RPR Agglutination+Agglutination+
Agglutination+:Agglutination+: ve ve RPR RPR AgglutinationAgglutination : :
--
--
Biological false positivesBiological false positives
►Biological False Positive AntibodyBiological False Positive Antibody ((BFPBFP) ) Reagin Reagin
AntibodyAntibody: : associated with other diseases associated with other diseases ((BFPBFP))
B.B.AcuteAcute::
►PneumoniaPneumonia
►Vaccination with live attenuated virusesVaccination with live attenuated viruses..
►MalariaMalaria
►PregnancyPregnancy
C.C.ChronicChronic::
►Leprosy – the only infectionLeprosy – the only infection
►Reagin titer falls rapidly with treatmentReagin titer falls rapidly with treatment
►Biological False Positive AntibodyBiological False Positive Antibody ((BFPBFP) ) Reagin Reagin
AntibodyAntibody: : associated with other diseases associated with other diseases ((BFPBFP))
B.B.AcuteAcute::
►PneumoniaPneumonia
►Vaccination with live attenuated virusesVaccination with live attenuated viruses..
►MalariaMalaria
►PregnancyPregnancy
C.C.ChronicChronic::
►Leprosy – the only infectionLeprosy – the only infection
►Reagin titer falls rapidly with treatmentReagin titer falls rapidly with treatment
Serological Diagnosis Of SyphilisSerological Diagnosis Of Syphilis
Test for specific Anti Test for specific Anti - - treponemal Antibodytreponemal Antibody
•Absorbed fluorescent treponemal antibody Absorbed fluorescent treponemal antibody
((FTA - ABsFTA - ABs))
•Treponema Pallidum Immobilization Test Treponema Pallidum Immobilization Test
((TPITPI))
•Most sensitiveMost sensitive
•Utilize living Treponema maintained by passage in Utilize living Treponema maintained by passage in
rabbits testesrabbits testes. .
•Expensive Expensive
•Potentially hazardousPotentially hazardous..
•Not done in the present contest as Technically demandingNot done in the present contest as Technically demanding
Test for specific Anti Test for specific Anti - - treponemal Antibodytreponemal Antibody
•Absorbed fluorescent treponemal antibody Absorbed fluorescent treponemal antibody
((FTA - ABsFTA - ABs))
•Treponema Pallidum Immobilization Test Treponema Pallidum Immobilization Test
((TPITPI))
•Most sensitiveMost sensitive
•Utilize living Treponema maintained by passage in Utilize living Treponema maintained by passage in
rabbits testesrabbits testes. .
•Expensive Expensive
•Potentially hazardousPotentially hazardous..
•Not done in the present contest as Technically demandingNot done in the present contest as Technically demanding
Doing a quantization test RPRDoing a quantization test RPR
Other Serological Methods in Other Serological Methods in
Diagnosis Of SyphilisDiagnosis Of Syphilis
Treponema pallidum haemagglutination Treponema pallidum haemagglutination
((TPHATPHA)) testtest..
A.A.Sheep, chicken or turkey RBCs. Sensitized by Sheep, chicken or turkey RBCs. Sensitized by
attaching killed Treponema pallidumattaching killed Treponema pallidum..
B.B.Agglutinate by presence of antibodyAgglutinate by presence of antibody
C.C.Less sensitive than FTA – AbsLess sensitive than FTA – Abs
D.D.Less reliable in the diagnosis of primary syphilisLess reliable in the diagnosis of primary syphilis..
E.E.Sometimes false positiveSometimes false positive
Diagnosis Of SyphilisDiagnosis Of Syphilis
Treponema pallidum haemagglutination Treponema pallidum haemagglutination
((TPHATPHA)) testtest..
A.A.Sheep, chicken or turkey RBCs. Sensitized by Sheep, chicken or turkey RBCs. Sensitized by
attaching killed Treponema pallidumattaching killed Treponema pallidum..
B.B.Agglutinate by presence of antibodyAgglutinate by presence of antibody
C.C.Less sensitive than FTA – AbsLess sensitive than FTA – Abs
D.D.Less reliable in the diagnosis of primary syphilisLess reliable in the diagnosis of primary syphilis..
E.E.Sometimes false positiveSometimes false positive
Treponema Palladium Treponema Palladium
Hemagglutination testHemagglutination test
TPHATPHA
Hemagglutination testHemagglutination test
TPHATPHA
Other Serological Tests for Other Serological Tests for
SyphilisSyphilis
►Anti – Treponemal AntibodyAnti – Treponemal Antibody
►Anti-Treponemal ABs group detected by Anti-Treponemal ABs group detected by
Reiter Protein Complement Fixation TestReiter Protein Complement Fixation Test
(RPCFT)(RPCFT)
A.A.Appears later than specific ABsAppears later than specific ABs
B.B.Some syphilis patient do not produce the form Some syphilis patient do not produce the form
of ABs of ABs
C.C.Used is limited.Used is limited.
SyphilisSyphilis
►Anti – Treponemal AntibodyAnti – Treponemal Antibody
►Anti-Treponemal ABs group detected by Anti-Treponemal ABs group detected by
Reiter Protein Complement Fixation TestReiter Protein Complement Fixation Test
(RPCFT)(RPCFT)
A.A.Appears later than specific ABsAppears later than specific ABs
B.B.Some syphilis patient do not produce the form Some syphilis patient do not produce the form
of ABs of ABs
C.C.Used is limited.Used is limited.
Detection by FTA-ABS IgG and Detection by FTA-ABS IgG and
IgMIgM
►In the FTA-ABS tests, In the FTA-ABS tests,
the patient's blood the patient's blood
serum is mixed with a serum is mixed with a
preparation that preparation that
prevents interference prevents interference
from antibodies to from antibodies to
other treponemal other treponemal
infections. infections.
IgMIgM
►In the FTA-ABS tests, In the FTA-ABS tests,
the patient's blood the patient's blood
serum is mixed with a serum is mixed with a
preparation that preparation that
prevents interference prevents interference
from antibodies to from antibodies to
other treponemal other treponemal
infections. infections.
FTA abs IgG and IgM detection continues to FTA abs IgG and IgM detection continues to
be a confirmatory test in diagnosis of Syphilisbe a confirmatory test in diagnosis of Syphilis
►The test serum is added to a slide The test serum is added to a slide
containing containing T. pallidumT. pallidum. In a positive . In a positive
reaction, syphilitic antibodies in the blood reaction, syphilitic antibodies in the blood
coat the spirochetes on the slide. The slide coat the spirochetes on the slide. The slide
is then stained with fluorescein, which is then stained with fluorescein, which
causes the coated spirochetes to fluoresce causes the coated spirochetes to fluoresce
when the slide is viewed under ultraviolet when the slide is viewed under ultraviolet
(UV) light.. (UV) light..
be a confirmatory test in diagnosis of Syphilisbe a confirmatory test in diagnosis of Syphilis
►The test serum is added to a slide The test serum is added to a slide
containing containing T. pallidumT. pallidum. In a positive . In a positive
reaction, syphilitic antibodies in the blood reaction, syphilitic antibodies in the blood
coat the spirochetes on the slide. The slide coat the spirochetes on the slide. The slide
is then stained with fluorescein, which is then stained with fluorescein, which
causes the coated spirochetes to fluoresce causes the coated spirochetes to fluoresce
when the slide is viewed under ultraviolet when the slide is viewed under ultraviolet
(UV) light.. (UV) light..
Principle of Fluorescent MethodPrinciple of Fluorescent Method
Active Treponema Pallidum Active Treponema Pallidum
InfectionInfection
1.1.Positive Specific Tests ePositive Specific Tests e..gg. . TPHATPHA
2.2.Positive Positive ( ( ≥1≥1/ / 88) ) of nonof non--specific test specific test
((VDRLVDRL))
•TPITPI--T T ((Treponema Pallidum Immobilization Treponema Pallidum Immobilization
TestTest) )
•FTA –T FTA –T ((Fluorescent Treponema TestFluorescent Treponema Test))
•Sometimes needed for confirmationSometimes needed for confirmation..
InfectionInfection
1.1.Positive Specific Tests ePositive Specific Tests e..gg. . TPHATPHA
2.2.Positive Positive ( ( ≥1≥1/ / 88) ) of nonof non--specific test specific test
((VDRLVDRL))
•TPITPI--T T ((Treponema Pallidum Immobilization Treponema Pallidum Immobilization
TestTest) )
•FTA –T FTA –T ((Fluorescent Treponema TestFluorescent Treponema Test))
•Sometimes needed for confirmationSometimes needed for confirmation..
Emergin Methods in Diagnosis of Emergin Methods in Diagnosis of
SyphilisSyphilis
► Currently, ELISA, Currently, ELISA,
Western blot, and PCR Western blot, and PCR
testing are being testing are being
studied as additional studied as additional
diagnostic tests, diagnostic tests,
particularly for particularly for
congenital syphilis and congenital syphilis and
neurosyphilis. neurosyphilis.
SyphilisSyphilis
► Currently, ELISA, Currently, ELISA,
Western blot, and PCR Western blot, and PCR
testing are being testing are being
studied as additional studied as additional
diagnostic tests, diagnostic tests,
particularly for particularly for
congenital syphilis and congenital syphilis and
neurosyphilis. neurosyphilis.
SPINAL FLUID TESTS in Syphilis.SPINAL FLUID TESTS in Syphilis.
. Testing of cerebrospinal fluid (CSF) is an . Testing of cerebrospinal fluid (CSF) is an
important part of patient monitoring as well important part of patient monitoring as well
as a diagnostic test. The VDRL and FTA-ABS as a diagnostic test. The VDRL and FTA-ABS
tests can be performed on CSF as well as on tests can be performed on CSF as well as on
blood. An abnormally high white cell count blood. An abnormally high white cell count
and elevated protein levels in the CSF, and elevated protein levels in the CSF,
together with positive VDRL results, suggest together with positive VDRL results, suggest
a possible diagnosis of neurosyphilis. a possible diagnosis of neurosyphilis.
. Testing of cerebrospinal fluid (CSF) is an . Testing of cerebrospinal fluid (CSF) is an
important part of patient monitoring as well important part of patient monitoring as well
as a diagnostic test. The VDRL and FTA-ABS as a diagnostic test. The VDRL and FTA-ABS
tests can be performed on CSF as well as on tests can be performed on CSF as well as on
blood. An abnormally high white cell count blood. An abnormally high white cell count
and elevated protein levels in the CSF, and elevated protein levels in the CSF,
together with positive VDRL results, suggest together with positive VDRL results, suggest
a possible diagnosis of neurosyphilis. a possible diagnosis of neurosyphilis.
CSF testing is indicated only inCSF testing is indicated only in……
►CSF testing is not used CSF testing is not used
for routine screening. for routine screening.
It is used most It is used most
frequently for infants frequently for infants
with congenital with congenital
syphilis, HIV-positive syphilis, HIV-positive
patients, and patients patients, and patients
of any age who are not of any age who are not
responding to penicillin responding to penicillin
treatment. treatment.
►CSF testing is not used CSF testing is not used
for routine screening. for routine screening.
It is used most It is used most
frequently for infants frequently for infants
with congenital with congenital
syphilis, HIV-positive syphilis, HIV-positive
patients, and patients patients, and patients
of any age who are not of any age who are not
responding to penicillin responding to penicillin
treatment. treatment.
Biological false reactive VDRL test among the Biological false reactive VDRL test among the
HIV infected patientsHIV infected patients
►Fewer reports on the biological false positive VDRL in HIV Fewer reports on the biological false positive VDRL in HIV
individuals are documented. In this work, the author individuals are documented. In this work, the author
studied the rate of biological false reactive VDRL among studied the rate of biological false reactive VDRL among
the HIV-infected patients. Of interest, in this study, the the HIV-infected patients. Of interest, in this study, the
rate is significantly lower (by Fishers exact test) than a rate is significantly lower (by Fishers exact test) than a
recent previous report among prostitutes in India (10/94, recent previous report among prostitutes in India (10/94,
about 10.6 %). In the general population, the biological about 10.6 %). In the general population, the biological
false positive VDRL generally returns to negative within 14 false positive VDRL generally returns to negative within 14
weeks, without other clinical significance.weeks, without other clinical significance.
►
VirojWiwanitkitVirojWiwanitkit
HIV infected patientsHIV infected patients
►Fewer reports on the biological false positive VDRL in HIV Fewer reports on the biological false positive VDRL in HIV
individuals are documented. In this work, the author individuals are documented. In this work, the author
studied the rate of biological false reactive VDRL among studied the rate of biological false reactive VDRL among
the HIV-infected patients. Of interest, in this study, the the HIV-infected patients. Of interest, in this study, the
rate is significantly lower (by Fishers exact test) than a rate is significantly lower (by Fishers exact test) than a
recent previous report among prostitutes in India (10/94, recent previous report among prostitutes in India (10/94,
about 10.6 %). In the general population, the biological about 10.6 %). In the general population, the biological
false positive VDRL generally returns to negative within 14 false positive VDRL generally returns to negative within 14
weeks, without other clinical significance.weeks, without other clinical significance.
►
VirojWiwanitkitVirojWiwanitkit
Rickettsiae and SerologyRickettsiae and Serology
►RickettsiaeRickettsiae is a genus of motile, Gram-negative, is a genus of motile, Gram-negative,
non-spore forming, highly pleomorphic bacteria non-spore forming, highly pleomorphic bacteria
that can present as cocci (0.1 μm in diameter), that can present as cocci (0.1 μm in diameter),
rods (1–4 μm long) or thread-like (10 μm long). rods (1–4 μm long) or thread-like (10 μm long).
Obligate intracellular parasites Obligate intracellular parasites
►Because of this, Because of this, RickettsiaeRickettsiae cannot live in artificial cannot live in artificial
nutrient environments and are grown either in nutrient environments and are grown either in
tissue or embryo cultures (typically, chicken tissue or embryo cultures (typically, chicken
embryos are used). embryos are used).
►Still we have to dependent on Weil Felix testStill we have to dependent on Weil Felix test
►RickettsiaeRickettsiae is a genus of motile, Gram-negative, is a genus of motile, Gram-negative,
non-spore forming, highly pleomorphic bacteria non-spore forming, highly pleomorphic bacteria
that can present as cocci (0.1 μm in diameter), that can present as cocci (0.1 μm in diameter),
rods (1–4 μm long) or thread-like (10 μm long). rods (1–4 μm long) or thread-like (10 μm long).
Obligate intracellular parasites Obligate intracellular parasites
►Because of this, Because of this, RickettsiaeRickettsiae cannot live in artificial cannot live in artificial
nutrient environments and are grown either in nutrient environments and are grown either in
tissue or embryo cultures (typically, chicken tissue or embryo cultures (typically, chicken
embryos are used). embryos are used).
►Still we have to dependent on Weil Felix testStill we have to dependent on Weil Felix test
Weil and Felix contribute for Weil and Felix contribute for
testingtesting
►In 1915, Weil and Felix showed that serum In 1915, Weil and Felix showed that serum
of patients infected with any member of the of patients infected with any member of the
typhus group of diseases contains typhus group of diseases contains
agglutinins for one or more strains of O X agglutinins for one or more strains of O X
Proteus. In cases of typhus fever the Proteus. In cases of typhus fever the
reaction usually appears before the sixth reaction usually appears before the sixth
day and reaches its height in the second day and reaches its height in the second
week. week.
testingtesting
►In 1915, Weil and Felix showed that serum In 1915, Weil and Felix showed that serum
of patients infected with any member of the of patients infected with any member of the
typhus group of diseases contains typhus group of diseases contains
agglutinins for one or more strains of O X agglutinins for one or more strains of O X
Proteus. In cases of typhus fever the Proteus. In cases of typhus fever the
reaction usually appears before the sixth reaction usually appears before the sixth
day and reaches its height in the second day and reaches its height in the second
week. week.
Weil-Felix reaction – A Weil-Felix reaction – A
Heterophile agglutination TestHeterophile agglutination Test
►A A Weil-Felix reactionWeil-Felix reaction is a type of is a type of
agglutination test in which patients serum is agglutination test in which patients serum is
tested for agglutinins to O antigen of certain tested for agglutinins to O antigen of certain
non-motile Proteus and rickettsial non-motile Proteus and rickettsial
strains(OX19, OX2, OXk) strains(OX19, OX2, OXk)
►OX19, OX2 are strains of OX19, OX2 are strains of Proteus vulgaris.Proteus vulgaris.
OXk is the strain of OXk is the strain of Proteus mirabilis.Proteus mirabilis.
Heterophile agglutination TestHeterophile agglutination Test
►A A Weil-Felix reactionWeil-Felix reaction is a type of is a type of
agglutination test in which patients serum is agglutination test in which patients serum is
tested for agglutinins to O antigen of certain tested for agglutinins to O antigen of certain
non-motile Proteus and rickettsial non-motile Proteus and rickettsial
strains(OX19, OX2, OXk) strains(OX19, OX2, OXk)
►OX19, OX2 are strains of OX19, OX2 are strains of Proteus vulgaris.Proteus vulgaris.
OXk is the strain of OXk is the strain of Proteus mirabilis.Proteus mirabilis.
Weil-Felix a Heterophile Weil-Felix a Heterophile
agglutination testagglutination test
►The agglutination reactions, The agglutination reactions,
based on antigens common to based on antigens common to
both organisms, determine the both organisms, determine the
presence and type of rickettsial presence and type of rickettsial
infectioninfection
►Because Rickettsiae are both Because Rickettsiae are both
fastidious and hazardous, few fastidious and hazardous, few
laboratories undertake their laboratories undertake their
isolation and diagnostic isolation and diagnostic
identification identification
►Weil-Felix test that is based on Weil-Felix test that is based on
the cross-reactive antigens of the cross-reactive antigens of
OX-19 and OX-2 strains of OX-19 and OX-2 strains of
Proteus vulgarisProteus vulgaris. .
agglutination testagglutination test
►The agglutination reactions, The agglutination reactions,
based on antigens common to based on antigens common to
both organisms, determine the both organisms, determine the
presence and type of rickettsial presence and type of rickettsial
infectioninfection
►Because Rickettsiae are both Because Rickettsiae are both
fastidious and hazardous, few fastidious and hazardous, few
laboratories undertake their laboratories undertake their
isolation and diagnostic isolation and diagnostic
identification identification
►Weil-Felix test that is based on Weil-Felix test that is based on
the cross-reactive antigens of the cross-reactive antigens of
OX-19 and OX-2 strains of OX-19 and OX-2 strains of
Proteus vulgarisProteus vulgaris. .
Interpretations in Interpretations in Weil-Felix Weil-Felix
reactionreaction
►Sera from endemic typhus agglutinate OX19, OX2.Sera from endemic typhus agglutinate OX19, OX2.
Tick borne spotted fever agglutinate OX19, OX2. Tick borne spotted fever agglutinate OX19, OX2.
►Scrub Typhus agglutinate OXk strain Scrub Typhus agglutinate OXk strain
►Test is negative in rickettsialpox, trench fever and Test is negative in rickettsialpox, trench fever and
Q-fever.Q-fever.
False positive reaction may occur in urinary or False positive reaction may occur in urinary or
other Proteus infectionsother Proteus infections
Test may be negative in 50 percent scrub typhus Test may be negative in 50 percent scrub typhus
reactionreaction
►Sera from endemic typhus agglutinate OX19, OX2.Sera from endemic typhus agglutinate OX19, OX2.
Tick borne spotted fever agglutinate OX19, OX2. Tick borne spotted fever agglutinate OX19, OX2.
►Scrub Typhus agglutinate OXk strain Scrub Typhus agglutinate OXk strain
►Test is negative in rickettsialpox, trench fever and Test is negative in rickettsialpox, trench fever and
Q-fever.Q-fever.
False positive reaction may occur in urinary or False positive reaction may occur in urinary or
other Proteus infectionsother Proteus infections
Test may be negative in 50 percent scrub typhus Test may be negative in 50 percent scrub typhus
Weil-Felix testWeil-Felix test
indicated in when patients present indicated in when patients present
with rasheswith rashes
►Test for diagnosis of Test for diagnosis of
typhus and certain typhus and certain
other rickettsial other rickettsial
diseases. The blood diseases. The blood
serum of a patient with serum of a patient with
suspected rickettsial suspected rickettsial
disease is tested disease is tested
against certain strains against certain strains
of (OX-2, OX-19, OX-of (OX-2, OX-19, OX-
K).. K)..
indicated in when patients present indicated in when patients present
with rasheswith rashes
►Test for diagnosis of Test for diagnosis of
typhus and certain typhus and certain
other rickettsial other rickettsial
diseases. The blood diseases. The blood
serum of a patient with serum of a patient with
suspected rickettsial suspected rickettsial
disease is tested disease is tested
against certain strains against certain strains
of (OX-2, OX-19, OX-of (OX-2, OX-19, OX-
K).. K)..
Weil Felix test and Weil Felix test and
Concentration CampsConcentration Camps
Concentration CampsConcentration Camps
Weil-Felix test positivity saves Weil-Felix test positivity saves
from from NazisNazis
►In Poland, during World War II, where a pair of In Poland, during World War II, where a pair of
quick-thinking doctors used a little-known quick-thinking doctors used a little-known
organism to keep the Nazis at bay.organism to keep the Nazis at bay.
The microorganisms is Proteus OX19. . Its one The microorganisms is Proteus OX19. . Its one
remarkable feature is that human antibodies for remarkable feature is that human antibodies for
Proteus OX19 cross-react with the antibodies for Proteus OX19 cross-react with the antibodies for
Ricksettia – the bacterium responsible for the Ricksettia – the bacterium responsible for the
deadly disease typhus. Blood from a patient deadly disease typhus. Blood from a patient
infected with Proteus Ox19 will give a false-infected with Proteus Ox19 will give a false-
positive in the most common typhus screening positive in the most common typhus screening
method, the method, the Weil-Felix test.Weil-Felix test.
from from NazisNazis
►In Poland, during World War II, where a pair of In Poland, during World War II, where a pair of
quick-thinking doctors used a little-known quick-thinking doctors used a little-known
organism to keep the Nazis at bay.organism to keep the Nazis at bay.
The microorganisms is Proteus OX19. . Its one The microorganisms is Proteus OX19. . Its one
remarkable feature is that human antibodies for remarkable feature is that human antibodies for
Proteus OX19 cross-react with the antibodies for Proteus OX19 cross-react with the antibodies for
Ricksettia – the bacterium responsible for the Ricksettia – the bacterium responsible for the
deadly disease typhus. Blood from a patient deadly disease typhus. Blood from a patient
infected with Proteus Ox19 will give a false-infected with Proteus Ox19 will give a false-
positive in the most common typhus screening positive in the most common typhus screening
method, the method, the Weil-Felix test.Weil-Felix test.
How they made Weil-Felix test How they made Weil-Felix test
PositivePositive
►While the Polish doctors could, and did, While the Polish doctors could, and did,
inject a number of other people with inject a number of other people with
Proteus to induce positive Weil-Felix results, Proteus to induce positive Weil-Felix results,
an on-site Nazi medical team could well an on-site Nazi medical team could well
have proved their undoing. Fortunately, have proved their undoing. Fortunately,
ingenuity and a good dose of hospitality and ingenuity and a good dose of hospitality and
alcohol prevented them from being alcohol prevented them from being
uncovereduncovered. . ( ( From the British From the British
Medical Journal )Medical Journal )
PositivePositive
►While the Polish doctors could, and did, While the Polish doctors could, and did,
inject a number of other people with inject a number of other people with
Proteus to induce positive Weil-Felix results, Proteus to induce positive Weil-Felix results,
an on-site Nazi medical team could well an on-site Nazi medical team could well
have proved their undoing. Fortunately, have proved their undoing. Fortunately,
ingenuity and a good dose of hospitality and ingenuity and a good dose of hospitality and
alcohol prevented them from being alcohol prevented them from being
uncovereduncovered. . ( ( From the British From the British
Medical Journal )Medical Journal )
Other Emerging Other Emerging
Serological TestsSerological Tests
Serological TestsSerological Tests
Co-agglutinationCo-agglutination
►Co agglutination is similar to the latex Co agglutination is similar to the latex
agglutination technique for detecting agglutination technique for detecting
antigen (described above). Protein A, a antigen (described above). Protein A, a
uniformly distributed cell wall component of uniformly distributed cell wall component of
Staphylococcus aureus, is able to bind to Staphylococcus aureus, is able to bind to
the Fc region of most IgG isotype antibodies the Fc region of most IgG isotype antibodies
leaving the Fab region free to interact with leaving the Fab region free to interact with
antigens present in the applied specimens. antigens present in the applied specimens.
The visible agglutination of the S. Aureus The visible agglutination of the S. Aureus
particles indicates the antigen-antibody particles indicates the antigen-antibody
reactionsreactions
►Co agglutination is similar to the latex Co agglutination is similar to the latex
agglutination technique for detecting agglutination technique for detecting
antigen (described above). Protein A, a antigen (described above). Protein A, a
uniformly distributed cell wall component of uniformly distributed cell wall component of
Staphylococcus aureus, is able to bind to Staphylococcus aureus, is able to bind to
the Fc region of most IgG isotype antibodies the Fc region of most IgG isotype antibodies
leaving the Fab region free to interact with leaving the Fab region free to interact with
antigens present in the applied specimens. antigens present in the applied specimens.
The visible agglutination of the S. Aureus The visible agglutination of the S. Aureus
particles indicates the antigen-antibody particles indicates the antigen-antibody
reactionsreactions
Co agglutination TestCo agglutination Test
Agglutination test in Agglutination test in
which inert particles which inert particles
(latex beads or heat-(latex beads or heat-
killed killed S aureus S aureus Cowan Cowan
1 strain with protein A) 1 strain with protein A)
are coated with are coated with
antibody to any of a antibody to any of a
variety of antigens and variety of antigens and
then used to detect then used to detect
the antigen in the antigen in
specimens or in specimens or in
isolated bacteria. isolated bacteria.
Agglutination test in Agglutination test in
which inert particles which inert particles
(latex beads or heat-(latex beads or heat-
killed killed S aureus S aureus Cowan Cowan
1 strain with protein A) 1 strain with protein A)
are coated with are coated with
antibody to any of a antibody to any of a
variety of antigens and variety of antigens and
then used to detect then used to detect
the antigen in the antigen in
specimens or in specimens or in
isolated bacteria. isolated bacteria.
ChemiluminescenceChemiluminescence
►ChemiluminescenceChemiluminescence is is
the emission of light the emission of light
with limited emission with limited emission
of heat of heat
(luminescence), as the (luminescence), as the
result of a chemical result of a chemical
reaction. reaction.
►ChemiluminescenceChemiluminescence is is
the emission of light the emission of light
with limited emission with limited emission
of heat of heat
(luminescence), as the (luminescence), as the
result of a chemical result of a chemical
reaction. reaction.
Chemiluminescent Immunoenzymatic Chemiluminescent Immunoenzymatic
AssayAssay
►Process for the quantitative and qualitative Process for the quantitative and qualitative
determination of antigens, antibodies and their determination of antigens, antibodies and their
complexes by means of a chemiluminescing complexes by means of a chemiluminescing
labelling substance activated or excited to labelling substance activated or excited to
chemiluminescence's by an analytical reagent. By chemiluminescence's by an analytical reagent. By
means of a serological reaction, initially an means of a serological reaction, initially an
antigen/antibody complex is formed which is antigen/antibody complex is formed which is
treated with a chemiluminescing conjugate treated with a chemiluminescing conjugate
containing chemiluminescing triphenylmethane containing chemiluminescing triphenylmethane
dyes and the chemiluminescence of the dyes and the chemiluminescence of the
chemiluminescing complex formed is measured. chemiluminescing complex formed is measured.
AssayAssay
►Process for the quantitative and qualitative Process for the quantitative and qualitative
determination of antigens, antibodies and their determination of antigens, antibodies and their
complexes by means of a chemiluminescing complexes by means of a chemiluminescing
labelling substance activated or excited to labelling substance activated or excited to
chemiluminescence's by an analytical reagent. By chemiluminescence's by an analytical reagent. By
means of a serological reaction, initially an means of a serological reaction, initially an
antigen/antibody complex is formed which is antigen/antibody complex is formed which is
treated with a chemiluminescing conjugate treated with a chemiluminescing conjugate
containing chemiluminescing triphenylmethane containing chemiluminescing triphenylmethane
dyes and the chemiluminescence of the dyes and the chemiluminescence of the
chemiluminescing complex formed is measured. chemiluminescing complex formed is measured.
Recent testing AdvancesRecent testing Advances
►The ToRC IgG kit simultaneously detects The ToRC IgG kit simultaneously detects
IgG class antibodies to Toxoplasmosis IgG class antibodies to Toxoplasmosis
gondii, rubella and cytomegalovirus (CMV).gondii, rubella and cytomegalovirus (CMV).
►The HSV-1 and HSV-2 IgG kit utilises type-The HSV-1 and HSV-2 IgG kit utilises type-
specific proteins to simultaneously detect specific proteins to simultaneously detect
and differentiate IgG class antibodies to the and differentiate IgG class antibodies to the
two most common herpes subtypes, HSV-1 two most common herpes subtypes, HSV-1
and HSV-2. and HSV-2.
►The ToRC IgG kit simultaneously detects The ToRC IgG kit simultaneously detects
IgG class antibodies to Toxoplasmosis IgG class antibodies to Toxoplasmosis
gondii, rubella and cytomegalovirus (CMV).gondii, rubella and cytomegalovirus (CMV).
►The HSV-1 and HSV-2 IgG kit utilises type-The HSV-1 and HSV-2 IgG kit utilises type-
specific proteins to simultaneously detect specific proteins to simultaneously detect
and differentiate IgG class antibodies to the and differentiate IgG class antibodies to the
two most common herpes subtypes, HSV-1 two most common herpes subtypes, HSV-1
and HSV-2. and HSV-2.
False Positive Serological TestsFalse Positive Serological Tests
1.1.Cross reacting antibodyCross reacting antibody
2.2.Cross reactivation of latent organism Cross reactivation of latent organism ((Influenza Influenza
Virus A infection activate CMV IgM – productionVirus A infection activate CMV IgM – production
3.3.Presence of Rheumatoid factorsPresence of Rheumatoid factors
RF RF = = IgM IgM
RF RF + + IgGIgG = = ComplexedComplexed
== False positive organismFalse positive organism--
specific IgM Antibodyspecific IgM Antibody
1.1.Cross reacting antibodyCross reacting antibody
2.2.Cross reactivation of latent organism Cross reactivation of latent organism ((Influenza Influenza
Virus A infection activate CMV IgM – productionVirus A infection activate CMV IgM – production
3.3.Presence of Rheumatoid factorsPresence of Rheumatoid factors
RF RF = = IgM IgM
RF RF + + IgGIgG = = ComplexedComplexed
== False positive organismFalse positive organism--
specific IgM Antibodyspecific IgM Antibody
False Negative Serologic TestFalse Negative Serologic Test
1.1.Immune system not intactImmune system not intact
2.2.Delay in Antibody response Delay in Antibody response ((Lyme disease Lyme disease
- Legionnaire’s Disease- Legionnaire’s Disease))
3.3.Competition for Antigen binding site of Competition for Antigen binding site of
antibodyantibody))
IgM binds to the Antigen IgG siteIgM binds to the Antigen IgG site
IgG binds to the Antigen IgM siteIgG binds to the Antigen IgM site
6.6.Prozone PhenomenaProzone Phenomena
1.1.Immune system not intactImmune system not intact
2.2.Delay in Antibody response Delay in Antibody response ((Lyme disease Lyme disease
- Legionnaire’s Disease- Legionnaire’s Disease))
3.3.Competition for Antigen binding site of Competition for Antigen binding site of
antibodyantibody))
IgM binds to the Antigen IgG siteIgM binds to the Antigen IgG site
IgG binds to the Antigen IgM siteIgG binds to the Antigen IgM site
6.6.Prozone PhenomenaProzone Phenomena
Usefulness of Serological Usefulness of Serological
ResultsResults
►How useful a serological result is depends on How useful a serological result is depends on
the individual virus. the individual virus.
►For example, for viruses such as rubella and For example, for viruses such as rubella and
hepatitis A, the onset of clinical symptoms hepatitis A, the onset of clinical symptoms
coincide with the development of antibodies. coincide with the development of antibodies.
The detection of IgM or rising titers of IgG in The detection of IgM or rising titers of IgG in
the serum of the patient would indicate active the serum of the patient would indicate active
disease.disease.
ResultsResults
►How useful a serological result is depends on How useful a serological result is depends on
the individual virus. the individual virus.
►For example, for viruses such as rubella and For example, for viruses such as rubella and
hepatitis A, the onset of clinical symptoms hepatitis A, the onset of clinical symptoms
coincide with the development of antibodies. coincide with the development of antibodies.
The detection of IgM or rising titers of IgG in The detection of IgM or rising titers of IgG in
the serum of the patient would indicate active the serum of the patient would indicate active
disease.disease.
Rota Virus - wether serology Rota Virus - wether serology
useful ?useful ?
►However, many viruses often However, many viruses often
produce clinical disease produce clinical disease
before the appearance of before the appearance of
antibodies such as respiratory antibodies such as respiratory
and diarrheal viruses. So in and diarrheal viruses. So in
this case, any serological this case, any serological
diagnosis would be diagnosis would be
retrospective and therefore retrospective and therefore
will not be that useful. will not be that useful.
►Acute presence of Antigen is Acute presence of Antigen is
much useful in Diagnosismuch useful in Diagnosis
useful ?useful ?
►However, many viruses often However, many viruses often
produce clinical disease produce clinical disease
before the appearance of before the appearance of
antibodies such as respiratory antibodies such as respiratory
and diarrheal viruses. So in and diarrheal viruses. So in
this case, any serological this case, any serological
diagnosis would be diagnosis would be
retrospective and therefore retrospective and therefore
will not be that useful. will not be that useful.
►Acute presence of Antigen is Acute presence of Antigen is
much useful in Diagnosismuch useful in Diagnosis
Antibody detection is definitive Antibody detection is definitive
DiagnosisDiagnosis
►There are also viruses There are also viruses
which produce clinical which produce clinical
disease months or years disease months or years
after seroconversion e.g. after seroconversion e.g.
HIV and rabies. In the HIV and rabies. In the
case of these viruses, the case of these viruses, the
mere presence of mere presence of
antibody is sufficient to antibody is sufficient to
make a definitive make a definitive
diagnosis.diagnosis.
DiagnosisDiagnosis
►There are also viruses There are also viruses
which produce clinical which produce clinical
disease months or years disease months or years
after seroconversion e.g. after seroconversion e.g.
HIV and rabies. In the HIV and rabies. In the
case of these viruses, the case of these viruses, the
mere presence of mere presence of
antibody is sufficient to antibody is sufficient to
make a definitive make a definitive
diagnosis.diagnosis.
Problems with SerologyProblems with Serology
►Long period of time required for diagnosis for paired Long period of time required for diagnosis for paired
acute and convalescent sera.acute and convalescent sera.
►Mild local infections such as HSV genitalis may not Mild local infections such as HSV genitalis may not
produce a detectable humoral immune response.produce a detectable humoral immune response.
►Extensive antigenic cross-reactivity between related Extensive antigenic cross-reactivity between related
viruses e.g. HSV and VZV, Japanese B encephalitis and viruses e.g. HSV and VZV, Japanese B encephalitis and
Dengue, may lead to false positive results. Dengue, may lead to false positive results.
►Long period of time required for diagnosis for paired Long period of time required for diagnosis for paired
acute and convalescent sera.acute and convalescent sera.
►Mild local infections such as HSV genitalis may not Mild local infections such as HSV genitalis may not
produce a detectable humoral immune response.produce a detectable humoral immune response.
►Extensive antigenic cross-reactivity between related Extensive antigenic cross-reactivity between related
viruses e.g. HSV and VZV, Japanese B encephalitis and viruses e.g. HSV and VZV, Japanese B encephalitis and
Dengue, may lead to false positive results. Dengue, may lead to false positive results.
Problems with SerologyProblems with Serology
Other Health condtions interfereOther Health condtions interfere
►Immunocompromised patients often give a reduced Immunocompromised patients often give a reduced
or absent humoral immune response.or absent humoral immune response.
►Patients with infectious mononucleosis and those Patients with infectious mononucleosis and those
with connective tissue diseases such as SLE may with connective tissue diseases such as SLE may
react non-specifically giving a false positive result.react non-specifically giving a false positive result.
►Patients given blood or blood products may give a Patients given blood or blood products may give a
false positive result due to the transfer of antibodyfalse positive result due to the transfer of antibody
Other Health condtions interfereOther Health condtions interfere
►Immunocompromised patients often give a reduced Immunocompromised patients often give a reduced
or absent humoral immune response.or absent humoral immune response.
►Patients with infectious mononucleosis and those Patients with infectious mononucleosis and those
with connective tissue diseases such as SLE may with connective tissue diseases such as SLE may
react non-specifically giving a false positive result.react non-specifically giving a false positive result.
►Patients given blood or blood products may give a Patients given blood or blood products may give a
false positive result due to the transfer of antibodyfalse positive result due to the transfer of antibody
AutomationsAutomations
►One of the first successful One of the first successful
attempts to automateattempts to automate
►antibody tests was made antibody tests was made
by Weitz (1967) at the by Weitz (1967) at the
Lister Institute, London. Lister Institute, London.
The apparatus developed The apparatus developed
by Weitz (Fig. 3) allowed by Weitz (Fig. 3) allowed
the performance of up to the performance of up to
12 titrations in a single 12 titrations in a single
operation, with even less operation, with even less
manipulation than that manipulation than that
required for a single test required for a single test
done by a more done by a more
conventional technique. conventional technique.
►One of the first successful One of the first successful
attempts to automateattempts to automate
►antibody tests was made antibody tests was made
by Weitz (1967) at the by Weitz (1967) at the
Lister Institute, London. Lister Institute, London.
The apparatus developed The apparatus developed
by Weitz (Fig. 3) allowed by Weitz (Fig. 3) allowed
the performance of up to the performance of up to
12 titrations in a single 12 titrations in a single
operation, with even less operation, with even less
manipulation than that manipulation than that
required for a single test required for a single test
done by a more done by a more
conventional technique. conventional technique.
Created as part ofCreated as part of
Dr.T.V.Rao MD’s Dr.T.V.Rao MD’s ‘e’ learning ‘e’ learning
ProgrammeProgramme
EmailEmail
[email protected]@gmail.com
Dr.T.V.Rao MD’s Dr.T.V.Rao MD’s ‘e’ learning ‘e’ learning
ProgrammeProgramme
EmailEmail
[email protected]@gmail.com
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