Ag-Ab reactions

Immunoelectrophoresis

5.Electroimmunodiffusion development of
precipitin lines can be speeded up by
electrically driving Ag and Ab
Counter immuno electro phoresis

AGGLUTINATION REACTIONS: when a

particulate antigen is combined with its
antibody in the presence of electrolytes
at suitable temp. and pressure the
particles are clumped or agglutinated
It is more sensitive to detect Abs. than
precipitation
Incomplete or monovalent abs do not cause
agglutination ,though they combine with
the ags, they inhibit agglutination by
complete abs added subsequently.

Applications:
1. Slide agglutination ex. Identification of many
bacterial isolates.
2.Tube agglutination : standard method for
quantitative measurement of abs
Ex. For diagnosis of enteric fever, Brucellosis,
Typhus fevers etc.
For Brucellosis several serial dilutions should
be kept to prevent false negatives by
prozone phenomenon
presence of blocking abs hider agglutination

Detection of blocking antibodies:

1. By doing the test in 5%hypertonic saline
or albumin saline
2. Heterophile agglutination tests:1.
Weil- Felix test
2.Streptococcal MG test
3.Paul-Bunnel test
4. cold agglutin test
Coombs test: Antiglobulin test for
detection of anti Rh abs that donot
agglutinate Rh positive abs in saline

1. Sera containing incomplete anti Rh abs

are mixed with Rh positive erythrocytes
the antibody globulin coats the surface of
the erythrocytes, though they are not
agglutinated
2. Such erythrocytes are washed to
remove all unattached proteins
3. Then the test is treated with rabbit
antiserum against human globulin
4. The coated cells are agglutinated

Direct antiglobulin test: one stage

1.Invivo sensitization of Rh positive red cells
with incomplete abs in Rh positive infant
2.Then the cells are washed and treated
with antiglobulin
Indirect test: two stage reaction
1. Sensitization of red cells with incomplete
abs in vitro
2. Then after washing, antiglobulin is
added to detect anti Rh antibodies
3. Ex. Rh typing and sero diagnosis of
Brucelllosis

Passive agglutination tests: very sensitive,

convenient. But false positives are common
Soluble ags are passively adsorbed on to or
chemically coupled to erythrocytes or other inert
particles-latex or bentonite
Poly saccharide ags-by simple mixing with cells
Protein ags tanned red cells are used
Coagglutination test:Cowan 1 strain of staph
aureus is usedas carrier particle to coat abs
Rose Waaler test: passive haemagglutination
test

2.bacteriophages- plaque inhibition tests

3. Toxin neutralization tests
Ex. Schick test for dophtheria
Eleks gel precipitation test
ASO test neutralizes hemolytic activity of
steptococcal hemolysin
Opsonization : opsonin was a heat stable
substance present in normal sera which facilitates
phagocytosis
Later a specific ab is called opsonin
Opsonin

normal factor
In serum
Specific ab

RA factor auto antibody which act as ab.

to gammaglobulin in rheumatoid arthritis
Latex particles 0.8 to 1 mm are used to
adsorb several types of ags.
Latex agglutination tests: ASO,CRP ,RA
factor, HCG and many other ags
Reverse passive agglutinaton test:
Ab is adsorbed on to inert particles to
detec or estimate ags.
Neutralization tests:
neutralizaion abs1. neutralization of
viruses

Opsonization : is a process by which a

particulate ag (bacteria) becomes more
susceptible to phagocytosis by combining
with opsonin.
Opsonic index: ratio of phagocytic
activity of patients blood for a given
bacterium to the phagocytic activity of
blood from a normal individual
by incubating citrated blood with bacterial
suspension of both test and control at 37 oc
for 15 min.
Estimation of average number bacteria
phagocytosed by -PNL

 Complement is system of serum proteins

present normally in serum
It aids the antibodies in lysing
organisms,promoting phagocytosis and
immune adherence
Complement dependent tests are:
1.CFT( versatile and sensitive)
2.Immune adherence test
3.Immobilization test
4.Cytolytic test

Complement fixation test

1.ag+test serum(contains ab.) complement
+complement
fixed
+hemolytic system
no hemolysis
positive CF test
2.ag+test serum (no ab.)
complement
+complement
not fixed
+hemolytic system
hemolysis
negative CF test

Indirect complement fixation test:

This test is done to sera that cannot fix guinea
pig compliment Ex.Avian sera
Test is done in duplicate
After the first step ,standard antiserum known to
fix compliment is added to one set
Hemolysis indicates positive test
In a positive test ,if serum contains ab would be
used up.

Conglutinating complement adsorption test:

A method for sera that cannot fix
compliment
Sheep erythrocytes sensitized with bovine
serum indicator system
Conglutinin present in bovine serum acts
as ab to compliment
Conglutinin causes agglutination of of
sensitized sheep erythrocytes,if these are
combined with compliment

Labelled antibody assays :by using

abs labelled to specific markers for
detecting ags or haptens.
Specific markers: 1. Fluorescent dyes-IF
2.Radio isotopes-RIA

(binder-

ligand)

3. Enzymes- EIAs
Immunoflorescence(Coons):is the property
of absorbing light rays of particular wave
length and emitting rays with different
wave length

 Immunofluorescence is a technique
allowing the visualization of a specific
protein or antigen in cells or tissue
sections by binding a specific antibody
chemically conjugated with a fluorescent
dye such as fluorescein isothiocyanate
(FITC).
There are two major types of
immunofluorescence staining methods:
fluorescent microscope. Dyes: Fluoresein
isothiocyanate, lissamine rhodamine

 Directmmunofluorescence staining in which the

primary antibody is labeled with fluorescence
dye,
Indirect immunofluorescence staining in which
a secondary antibody labeled with fluorochrome
is used to recognize a primary antibody.
Immunofluorescence staining can be performed
on cells fixed on slides and tissue sections.
Immunofluorescence stained samples are
examined under a fluorescence microscope or
confocal microscope.

DIRECT IF

INDIRECT IF

RIA:Radioisotopes are used as labels in agab assays

Based on the competitive binding of unlabelled
ag and radiolabeled ag for ab in the given
system
After incubation, amount of free radio labelled ag
is directly proportional to quantity of unlabelled
ag in the mixture
Extremely sensitive, but costly, radiation hazard
and short shelf life of reagents limit its use

Standard dose response curve should be prepared the ratio

of bound :total labels (B:T) plotted against the analyte conc.
RIA is a competitive binding assay, fixed amounts of ab. and
radiolabelled ag react in the presence of unlabelled ag.
The labelled and unlabelled ags. compete for limited binding
sites on the ab.
The competition is determined by the level of unlabelled(test)
ag present in the reacting system
The ag is separated into free and bound fractions and their
radioactivity is measured
The conc. of test ag is measured from the ratio of bound and
total antigen labels,using a standard dose response curve

EIAs
Two types:EMIT(homologous), ELISA(heterologous)
ELISA: ags or abs are labelled with enzymes
Use of immunosorbent material specific forone of the
components of the reaction
Immunosorbent materials used: paticulate,solid phase,
microwells membranes or discs of polyacrilamide,paper
or plastic
Enzymes used are alkaline phosphatase,horse radish
peroxidase,galactosidase etc.
Chromogenic substances- paranitrophenyl ,Ophenylene-diamine dihydrochloride specific to enzymes
are added

Classical ELISA and modifications like rapid

ELISA,cylinder ELISA cassette ELISA,
sandwich ELISA
E/rapid
competitive ELISA ,Capture ELISA and
immunometric tests are more specific

WESTERN BLOT TEST

It is an immuno electro blot
It is of equal sensitivity of ELISA with much
greater specificity
It is of three steps:1. separation of ag.
Components by polyacrilamide gel
electropherosis
2.Blotting of electrophoresed ligand
fractions on nitrocellulose membrane
3.Detection of ag-ab complex by EIA or
RIA

 Separation of ag components: ags or

proteins on the test organism suspension
disrupted &seperated by electrophoresis
The electrophoretically separated proteins
bands transferred to nitro cellulose
membrane or nylon.they adhere by means
of nonpolar interactions
Then incubated with test serum

 After washing anti human ab enzyme

conjugate is added,then substrate
Reactoin read by spectrophotometer
Applications:It is useful in detecting abs in
microbes with many cross reacting abs
Ex:HIV, T.pallidium,B.burgdoferi, HSV1&2,
Ricketteciae,leismaniae,BSE
HIV:hiv proteins separated are
HIV-1: Envelope proteins gp
160,gp120,gp41 .HIV-2 env.gp36

Typical Data
Negative: No bands
present
Positive: Bands at
either p31 OR p24
AND bands present at
either gp160 OR gp120
Indeterminate: Bands
present, but pattern
does not meet criteria
for positivity

 Chemiluminiscence assay uses

chemiluminiscent compounds that emit
energy in the form of a light during ag-ab
reactions. Ex. Drug sensitivity testing of
My.tuberculosis
Immunelectron microscope: Ag- Ab
reactions are visualized using EM
Immunoproxidase and immunoferritin
Immunochromatography:

IMMUNOELECTRON
MICROSOPE

How
Immunochromatography
Works
Add
Sample

HIV
IgG
antibodies Antibodies

Conjugate

Colloidal
gold
conjugated
to HIV
antigen

Test
Line

HIV antigen

Control
line

AntiIgG/gold
antibodies

12

Tests Based on
Immunochromatography

Lateral Flow Devices

Determine

Hema- Strip
Ora Quick
Unigold

Control
HIV Antigen

Sample pad
Specimen Flow

13