Serological assays

Up dating on different
Serological Assays

Dr.Tariq Mustafa Mohamed Ali
Al Ain Veterinary Laboratory
Animal Health section,Agriculture sector
Department of Municipalities and Agriculture

Serological assays

Antigen (Ag) + Antibody(AB)

Antigen (Ag):
A molecule which elicits a specific immune
response when introduced into an animal.
1. Generally large molecules (>10,000 daltons
in molecular weight).
2. Structurally complex (proteins are usually
very antigenic).
3. Accessible to the immune system .
4. Foreign (not recognizable as "self").

Antigenic structure of Brucella
A- Surface antigen (OM)
 Lipopolysaccharide (LPS) It is either S-LPS or R-LPS
Constitute the major Ag interact in SAT, CFT, RB , MRT
and represents the A &M epitops
 OMP consists of 2 Ag
1. NH 25-27 K ( Not existed in the Rough strain)
2. Poly B 36 – 37 K ( Not existed in the Rough strain)
3. NA 31 K ( Existed in B. melitensis )
B- Internal Antigens free from LPS
 More than 20 protenious Ag
(NH and Poly B and A2 ( one of internal Ags) used for
differentiation between Vaccinated and infected animals ).

Antigenic structure of Brucella

Examples for virus antigens

 Foot and mouth
disease viral antigens
 146 S (VP1,2,3 & 4).
 75 S ( VP1, 2 &VP0)
 12 S ( Vp 1, 2 &3)

Antibody (Ab):
 A glycoprotein produced in response to an
antigen that is specific for the antigen and
binds to it via non-covalent interactions.
 The term “Immunoglobulin" is often used
interchangeably with "antibody".
 Immunoglobulins (Ig) come in different forms
(IgA, IgD, IgE, IgG, IgM) that reflect their
structure.

Main classes of antibodies ( IgG, IgA,
IgD, IgE, and IgM).

Monoclonal antibodies
 Typically made by fusing myeloma cells with
the spleen cells from a mouse that has been
immunized with the desired antigen to
produce what is called Hybridoma cell

Antigen / Antibody Reactions
 (A) The hinge region of an
antibody molecule opens
and closes to allow better
binding between the
antibody and antigenic
determinants on the surface
of an antigen. (B) Hinge
flexibility also facilitates the
cross-linking of antigens into
large antigen-antibody
complexes.

Requirements of valid diagnostic
assays :
 Selection of optimal reagent concentration
and protocols parameters
 Use the test of optimal Sensitivity and
Specificity to the target disease.
 Use the test of good Repeatability and
Reproducibility
 Applied in the Reference animal population
 Use the test of high prediction or efficiency

Sensitivity of Diagnostic methods

 It is the proportion of True positive (TP) that
are detected by this method
 Increase sensitivity accompanied with a
proportion of false positive (FP) .
 A test with 90% sensitivity implies 10% will
be false negative.
 Sensitivity = TP/ (TP+FN) X 100

Specificity of Diagnostic methods
 It is the proportion of true negative (TN) that
are detected by this method
 Increase specificity accompanied with a
proportion of false negative ( FN ).
 A test with 90% specificity implies 10% will be
false positive .
 Specificity = TN/ (TN+FP) X 100

Factors yielded false results

 Cross reaction with similar epitops
 Presence of non specific inhibitors in the tested
serum.
 Some animals show natural or induced tolerance
(BVD).
 Non specific inhibitors (Anti complementary serum).
 Kind of AB ( Excess IgG 1 block IgG 2 in brucella
testing.
 Using unsuitable test (Incomplete antibody).
 Improper timing for testing the animals (cows for
brucellosis by CFT before abortion might give FN)

Diagnostic objectives of serological
tests .
 Detection of Target Antigen.
 Study the relation between the field
isolate and reference isolates.
 Detection of rising antibody titers
between acute and convalescent stage
of infection.

Antibody response to infection and
reinfection

Serological assays

It is either
 Qualitative
 Quantitative test

( Dilution takes place to the unknown
object )

Agglutination tests
 Card or plate Agglutination
 Rose Bengal test (RB).
 Serum Agglutination test (SAT).
 Micro-agglutination (MAT)
 Latex Agglutination (LA)

Principals of Agglutination tests
 The antigen is reacted with the antibody
inducing (clumping) of the antigen.

Application of agglutination tests
 Typing of different isolates as in case
of E.coli ,salmonella, Pasteurella
spp.etc..
 Diagnosis of Brucellosis :
 Rose Bengal test .
 Milk ring test.
 SAT
 Micro agglutination test

Principals of Latex Agglutination
(LA)
 LA tests are similar in principle to bacterial
agglutination .
 Latex particles could be coated with antibody
or antigen and will agglutinate when mixed
with the corresponding antigen or antibody.
 Used for Identification of many
(Streptococcus ,salmonella typing and
toxoplasmosis)

Disadvantages of quantitative
agglutination tests
 Prozone Phenomena :
Lack of agglutination at high concentrations of
antibodies due to excess antibody yielded a very
small complexes that do not clump to form visible
agglutination.

Agglutination but use erythrocytes
as targets or carrier
 Indirect Hemagglutination (IHA)( Coomb
test).

 Hemagglutination inhibition tests (HI).

 Passive hemagglutination (PHA)

Principals of Hemagglutination
 Detect antibody in the mare colostrum in
Equine infectious anemia by Coomb’s test
(Antiglobulin test)

Principals of Hamagglutination
Inhibition Test
It measures the ability of antibodies
to inhibit the hem-agglutinating
activity of fixed amount of virus

In the absence of anti-virus
antibodies

Erythrocytes

Virus
Virus agglutination of erythrocytes

Heamagglutination Inhibition

Anti-virus
Erythrocytes antibodies

Virus

Viruses unable to bind to
the erythrocytes

Criteria of HI

 Diagnosis of haemagglutining viruses such
as Influenza ,Newcastle disease etc .
 Evaluating the immune response of
vaccinated animals or birds.
 It is serotype specific

Principals of Passive Hemagglutination
(PHA)
 Passive hemagglutination is a classical
immunological test in which antigen is linked
chemically to preserved red blood cells (RBC)
using tannic acid, glutraldehyde or CRCl3 .

 The labeled cells are then used to detect the
appropriate antibody in a simple
hemagglutination test.

End point
The results are reported as the reciprocal of the maximal dilution
that gives 50% visible agglutination

Serum

Precipitation tests

 Agar gel immunodiffusion tests “Double
immunodiffusion” (AGID).
 Single radial immunodiffusion (SRID)
 Immunoelectrophoresis (IE)
 Countercurrent immunoelectrophoresis
(CCIE)
 Immunochromatography

Principals of Precipitation tests
 The serum antibody and the antigen
preparations are placed in holes
punched into into agar or agarose .
 The antigen as well as Antibodies
diffuses into the gel, and at the
equivalence zone they form a
precipitation line.

Principals of Radial Immunodiffusion(Mancini)
(SRID)
 In SRID assay the antibody is incorporated
into the agar gel as it is poured and different
dilutions of the antigen are placed in holes
punched into the agar to generate a standard
curve.
 As the antigen diffuses into the gel, it reacts
with the antibody and at the equivalence point
a ring of precipitation is formed.

Single radial immunodiffusion
(SRID)

Principals of
Immunoelectrophoresis
 In immunoelectrophoresis, both antigen
preparation and antibody are placed in a
well punched out of an agar gel and
exposed to weak electric current for a
period of time .
 As the Ag and Ab diffuse into the agar a
precipitin lines are produced at the
equivalence zone when an antigen/antibody
reaction occurs

Countercurrent electrophoresis CIE
 The antigen preparation into the wells
situated on the cathode side of each pair
Antigens flow towards the anode (+).

Countercurrent electrophoresis CIE

Using labeled antibodies

A.Radio immune assay
B.Immunofluorescence
C.Enzyme linked immunosorbent
assays (ELISA)

Principals of RADIOIMMUNOASSAY
(RIA) test
 Radioactively labeled antibody competes with the
animal’s unlabeled antibody for binding sites on a
known amount of antigen.
 A reduction in radioactivity of the antigen-animal
antibody complex compared with the radioactive
counts measured in the control test with no antibody
is used to quantitate the amount of serum antibody
bound to the antigen.

Application of RADIOIMMUNOASSAY
(RIA)
 Radioimmunoassay (RIA) is primarily used to
measure antigens (notably certain hormones
or proteins) in serum samples
 This automated method of detecting antigens
is usually performed in a chemistry
laboratory.
 Of limited application in Veterinary diagnostic
laboratory

Principals of Immunofluorescence (IF) test

 Immunofluorescence (IF) depends on the
coupling of intracellular viral or bacterial
antigens with fluorescein isothiocyanate
(FITC) conjugated specific antibodies .
 The reaction is detected by expose the
reaction site to ultraviolet (UV) or blue light.
Yielding apple green fluorescence

Principal steps in direct
immunofluorescence

Principal steps in indirect
immunofluorescence (IIF)

2 - Principals of Enzyme-Linked
Immuno Sorbent Assay (ELISA).
 In a simple terms, antigen is fixed to the
solid plate surface, and then the serum in
question is added .
 The mixture is washed away followed by
addition of anti-species Ab conjugated with
an enzyme .
 In the final step a substance is added that
the enzyme can convert to some detectable
signal.

ELISA for Antigen or antibody

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Indirect ELISA (i ELISA) :

 Plate is coated with captured antibody
 Sample is added, and any antigen present binds to the
captured antibody then washed
 GP HIS is added, and binds to antigen if exists .
 Conjugated Rabbit anti GP Ig is followed and binds to
detecting antibody
 The substrate is added and is converted by enzyme to
detectable form.

Competitive ELISA (cELISA) for
detection of RP antibodies.
 ELISA plates coated with RP Ag.
 Add tested serum samples and RP Mab in blocking
buffer then Incubate plates with continuous shaking
for 1 H at 37oC
 Add HRPO conjugated Anti mouse Ab followed by
incubation for 1 H at 37oC.
 Add the substrate/chromogen and allow colour to
develop for ten minutes.
 Stop colour development
 Read plates on ELISA reader at an absorbance of
492 nm.

Standard tests
 Complement Fixation test (CFT).
 Serum neutralization tests (SNT).

 Protective tests (PD50)

Principals of Complement Fixation
Test (CFT)
 The test consists of 2 Ag/AB reactions. One of them
is the target Ag or serum against the corresponding
refrence Serum or Ag .
 The 2 nd Ag/Ab called hemolytic system (SRBCs +
Rabbit antisheep RbCs “hemolysine”) .
 The complement is activated by Ag-Ab complex.
 If C’ consumed in the first reaction . The HS is not
affected and the HS not been lysed. And vise is
versa.

Principals of Complement Fixation
Test (CFT)

Criteria of CFT
 Tested serum should be inactivated at 56°C for 30
minutes.
 Complement and haemolysin should be titrated first.
 All Reagents used in the test should be tested for
QC(C’C, RBCs C, Haemolysin C, Serum C)
 100 % and 0% lysis) should be included in the test.
 Complement and haemolysin titration should precede
the test proper.
 CFT could be done slow or rapid test
 ICFT could be applied.

Complement (C’) titration

Each C’ dil 0% 100%

VB 0.1 0.2 0.1

Tested Ag or
0.1 0.1 0.1
Serum

C’ dilution 0.1 0 0.1 (1/10)

HS 0.2 0.2 0.2

Each
Ag C SC C’ C HS C
Ag dil
VB 0 0.1 0.2 0.2 0.3
Tested
Ag 0.1
HIS
(1/10) 0.1 0.1
C’ (2-
4Unit)
0.1 0.1 0.1 0.1(1/10)

Incubate at 37 oC for 30 m
HS
0.2 0.2 0.2 0.2 0.2

Incubate at 37 oC for 15 m

What does a CFT look like?

Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement
fixation obtained with acute and convalescent phase serum specimens,
respectively. (2-fold serum dilutions were used) The observed 4-fold increase is
significant and indicates recent infection.

Principle of Serum Neutralization
(SNT)
 It demonstrate the amount of antibodies
necessary to inhibit the activity of known
amount of virus particles(10 2) .
 If it done in tissue culture (TC) tube , the
cytopathic effect (CPE) was recorded.
 If it done in embryonated eggs death is
recorded .

SNT assay
 The test is done using test tubes, microtiter plate ,
2 oz bottle, embryonated eggs , Mice etc…
 The target serum is exposed to 4-fold dilutions
followed by the addition of an equal volume of
virus suspension diluted to contain approximately
100 ID50 .Following an incubation of serum + virus
1 - 2 hours at room temperature .Inoculate the
mixture of each dilution into odd number of target
host .Incubated at 37°C, and observed daily for
development of viral CPE.

Criteria of SNT
 The end point is the reciprocal serum dilution
yielded 50% CPE in case of tissue culture.
 This test is considered the most reliable of all
serologic procedures, being less prone to variation
and less subjective in its interpretation.
 SNT tests are almost always performed using
cell cultures.

Principle of Protection Tests
 It has the same principal of SNT but in vivo.
 The target host is immunized with several
Vaccine dilution followed by challenge with
constant virus concentration at 3 weeks post
vaccination.
 Protection tests are used for Vaccine Evaluation
 An example of a protection test once used is that
for FMD vaccine evaluation.

Calculation of PD50
No. +ve /Total
Vaccine dilution Positive%
No. of animals
Undiluted 1/5 20%
1/4 2/5 40%
1/16 3/5 60%
1/64 4/5 80%
1/256 5/5 100%
m =xk +1/2d- d (-Spi /100)

Immune Electron Microscopy

Classical Immune electron microscopy (IEM) - the sample is
treated with specific anti-sera before being put up for EM.
Viral particles present will be agglutinated and thus congregate
together by the antibody.

Solid phase immune electron microscopy (SPIEM) - the grid is
coated with specific anti-sera. Virus particles present in the
sample will be absorbed onto the grid by the antibody.

Uses of Immune Electron
Microscopy
At least 106 virus particles per ml required for visualization,
The magnification power should be 50,000 - 60,000 X
Viruses may be detected in the following specimens.

 Feces Rotavirus, and corona virus &
Adenovirus, Calicivirus , Parvoviruses

 Skin scrapings Papillomavirus, ORF , Pox

Problems with Electron Microscopy
 Expensive equipment .

 Expensive maintenance .

 Require experienced observer .

 Sensitivity is often low .

Fluorescence polarization assay
(FPA)
 The assay works on the principle that
molecules in solution randomly rotate at a
rate inversely proportional to their size.
 A small molecule labeled with a fluorochrome
will depolarize plane polarized light at a more
rapid rate than a large molecule such as Ag-
Ab complex.

(FPA)
 Fluorescence polarization provides a direct readout of the
extent of FITC conjugated Ag binding to serum AB.
 In positive cases ; the FITC conjugated Ag bind to the Ab
molecules leads to formation of large , slowly rotating
molecules and high fluorescence polarization.
 In negative cases ; When no reaction takes place with
FITC conjugated antigen, the FITC conjugated Ag remain
as small, rapidly rotating molecules resulting in low
fluorescence polarization.

(FPA)

Western blotting
 Western blotting is based on the principles of
immunochromatography where proteins were
separated into poly acrylamide gel according to
the isoelectric point and molecular weight.
 Immunoblotting is performed chiefly in diagnostic
laboratories to identify the desirable protein
antigens in complex mixtures.

Criteria of Western blotting

 The separated protein bands were detected by :
1. Colorimetric detection or
2. Chemiluminescent detection or
3. Radioactive detection or
4. Fluorescent detection

Western Blot

Western Blot
 Lane1: Positive Control
 Lane 2: Negative Control
 Sample A: Negative
 Sample B: Indeterminate
 Sample C: Positive

Immunoblotting
 Viral antigens are detected with a
polyclonal or a MAb onto nitrocellulose
paper.
 After incubation, the protein bands
(immune complexes) are visualised with
peroxidase-conjugated protein and a
colour reagent.
 A colour develops in the bands where
antibody binds to the antigen.

Immunochromatography
 Test principle.
 Antibodies were attached to two different zones on a
nitrocellulose membrane .
 Purified monoclonal antibody against nucleoprotein (anti-N
Mab) that had been identified as IgG2a subtype by using
Mab isotyping kit was attached to the test zone at the
concentration of 0.75 μg/strip, and purified goat anti-mouse
IgG (was attached to the control zone at the concentration of
0.70 μg/strip.
 The 30 nm colloidal gold conjugated antibodies were dried
on the glass fiber
 The test strip was assembled in the following order (Sample
pad , gold pad, nitrocellulose paper, and absorption pad
(cellulose paper).
 All pads are overlapped to enable migration of sample

Diagram of the test strip for the detection of anti-canine parvovirus antibody.
Serum is added to the sample pad where serum antibodies can interact with
CPV. Addition of buffer enables the complex to migrate along the test strip
where gold-conjugated antibodies are captured by the immobilized anti-porcine
or anti-canine IgG.

Quantitative application of the test
 The intensity of the
color developed at the T
line (T) correlated with
the HI assay-
determined titer of the
reference serum
sample shown on each
test strip

Diagram of the test strip for the
detection of Rabies virus
Saliva is added to the
sample pad.
sample pad► gold pad
Mab against
nucleoprotein ► go
through nitrocellulose
paper to test line
(Rabies Mab) ► Control
line ( goat anti-mouse)
and absorption pad
(cellulose paper).
 All pads overlapped to
enable migration of
sample

DETECTION OF NUCLEIC
ACIDS GENOME
 Polymerase Chain Reaction (PCR)
 Reverse transcryptase Polymerase Chain Reaction
(RT-PCR).
 Restriction Fragment length polymorphism
(RFLP).
 Nucleic acid hybridization (DNA probe) .
 Microarray Analysis
 DNA sequencing.

Polymerase Chain Reaction (PCR)
 The amplification of DNA by the PCR is
accomplished via a succession of
incubation steps at different temperatures.
 PCR can amplify copies of a small region
of 100-400 or more base pairs into millions
of copies.

Basic steps of PCR
 Separation of DNA strands (Denaturation) By
heating to 95 oC .
 Activate primer’s links (Annealing) By heating
to 55 oC .
 Activate polymerase activity in 3’ to 5’
direction (Extension) By heating to 72 oC, to
synthesis new strand .

Chemical structure of nucleotide

Identification of PCR products

Criteria of PCR
 PCR is a highly sensitive procedure for detecting
infectious agents in host tissues and vectors, even when a
small number of host cells are infected
 PCR is very useful in the diagnosis of chronic-persistent
infections such as :
 Bovine leukemia virus

 Caprine arthritis/encephalitis virus, etc.)

 Latent infections by IBR
 PCR could be used for testing vaccines contamination.
 It does not differentiate between viable and nonviable
organisms.

Criteria of PCR
 Extremely liable to contamination
 Require high degree of operator skill
 Not easy to set up a quantitative assay.
 A positive result may be difficult to interpret,
especially with latent viruses such as CMV, where
any seropositive person will have virus present in
their blood irrespective whether they have disease
or not.

Restriction fragment length
polymorphisms (RFLP)
 Detect differences in the genomes of closely
related microbial species
 DNA is extracted and clipped into fragments of
specific nucleotide sequences with restriction
endonucleases.
 The resultant DNA fragments are then separated
in agarose gel by electrophoresis and visualised
with ethidium bromide.
 The fragments can then be hybridised with
complementary DNA (cDNA) tagged with 32P to
determine the differences or similarities in the
genomes.

Criteria of FRLP
Uses :
 Used to detect differences in the genomes of
closely related microbial species or compare
field isolates of a given virus.
 A distinct limitation of the method is that the
presence of a mutation cannot be detected
unless that mutation happens to fall within the
recognition sequence of the restriction
endonuclease being used for digestion of the
DNA.

Solid-phase of NA hybridization
assays
Principals of the test
 The double-stranded nucleic acid of a virus is
denatured with alkali to separate strands.
 The single strands of nucleic acid are attached to
a solid support, usually a nylon or nitrocellulose
membrane, to prevent the strands from
reannealing.
 The nucleic acid attaches to the membrane by its
sugar-phosphate backbone; the nitrogenous
bases are thus projecting outward.

Nucleic Acid probe
 A probe (single-stranded DNA or RNA molecule of known
origin - containing the nucleotide sequence specific to that
of the target virus - labeled with a radioactive atom or
enzyme) is added to the membrane.
 Formation of hydrogen bonds occurs between the
complementary bases.
 Unreacted probe is removed by washing and hybridization
is detected by an assay for the probe.

Southern hybridization for detection of
DNA fragments.

Northern hybridization for RNA
detection

Dot blot hybridization.
 The nucleic acid is
placed onto
nitrocellulose in an
apparatus that
focuses the
individual spots into
concentrated areas,
similar to a microtiter
plate

Application of Microarray Analysis:

 The development of microarrays has been
fueled by the application of robotic technology
to routine molecular biology, rather than by
any fundamental breakthrough.
 It used for Identification of specific viruses or
specific viral sequences as many as included
in the test.

DNA microarray
 (gene chip, DNA chip or biochip) is a collection of
DNA probes attached to a solid surface, such as
glass, plastic or silicon chip forming an array. Sample
DNA or RNA is extracted, RNA is reverse transcribed
to cDNA and the DNA or cDNA is labelled with
fluorescent labels. The labelled DNA is denatured
and hybridized with the probes on the array.
Unbound probes are washed away and the array is
visualized using confocal laser microscope scanner

Serological assays

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