09042013 – Ag-Ab interactions

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o immune-mediated hemolytic anemia.o
o Hemolytic disease of the newborn
(ABO, Rh)
o
o
identify antibodies present in a
patient's serum prior before blood
transfusion.
Blood transfusion preparation.
Antenatal antibody screening.
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Aggregation of “particulate antigens”
bound with agglutinins of more than
one specificity.
Example: coagglutination
staphylococcus aureus protein A for
serotyping strains of
Streptococcus pneumoniae,
o Example: Influenza virus
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o Heterophiles antibodies tests (Monospot test, Paul –
Bunnell test):
o agglutination of the horse RBCs by heterophile antibodies in
patient's serum for rapid diagnosis of EBV infectious
mononucleosis.
o Cold agglutnin test:
o Cold agglutinins are antibodies made by the immune system in
response to infection having the ability to agglutinate RBC in Low
temperature. E.g. Mycoplasma pneumoniae
o Weil felix test:
o it is agglutination test for the diagnosis of rickettsial infections.
o based on the presence of antigenic “cross-reaction”
between Rickettsia and certain serotypes of Proteus
o VDRL: screening for syphilis caused by treponema
pallidum.
o Widal test: standard tube agglutination test for
salmonella typhi.
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oIt is a reaction in which the Ab
interact with a free soluble Ag .
oAt the optimum concentration
,positive reaction form a precipitin
line
oThis is precipitation reaction to
test identity between two antigens
or between two antibodies
oThis is precipitation reaction to
test identity between two antigens
or between two antibodies
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Sample
concentration
Ag
Mg/ml
Sample
Diameter
Diameter /mm
oThis is precipitation reaction to
test presence of entire subclass of
immunoglobulin by applying
specific antiglobulin forming a
precipitation rings.
oThis is method to separate the serum by electrophoresis.
oCreate a trench containing specific anti serum.
oDiffusion of both serum and anti serum forming a precipitin lines
o technique used to detect
specific proteins in the given
sample.
o It uses ”gel
electrophoresis” to separate
proteins (or polypeptide) and
are then transferred to a
membrane.
o (nitrocellulose) where they
are stained with labled
antibodies specific to the
target protein.
Test Ag
Sample AB
Enzym linked Ab
Enzyme substrate
oAntibodies containing fluorescent dye react with serum
(either Ag or Specific Ab)..
oPositive reaction is shown under fluorescent microscope.
oTesting the presence of the
antitoxin in the serum.
oIf they are present the toxin
will be neutralized .RBCs not
lysed.
oIf there is no antitoxin the
toxin will lyse the RBCs
The toxin( hemolysin)
sample (serum) Ab
RBC lysed with the toxin
The toxin( hemolysin)
sample (serum) Ab
RBC lysed with the toxin
o Testing immunity of the patient against diphtheria.
o 0.2 ml of the diphtheria toxin in the test forearm and 0.2 ml of
inactivated diphtheria toxin (by heat ) in the control forearm.
oPreparing sensitized complement & two
systems:
oSystem1:
oContaing serum sample & prepared
antigen
oSystem 2 (indicator system):
ocontaining sensitized sheep RBCs
& Ab to sheep RBCs.
oIn +ve reaction the serum containing Ab
will react with the Ag the complement will
be consumed by system 1 (RBCs will not
be lysed)..
Test A g
Sam ple A B
com ple m ent
Sheep RB C
A b t o sheep RB C
oIn –ve reaction where there is no Ab in
the serum so the complement will
consumed by system 2 (RBCs will be
lysed).
Test Ag
Sample AB
complement
Sheep RBC
Ab to sheep RBC
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oThis a solid phase reaction through
which the serum antibody will be
captured to the wells using capture
Ag.
oAn Antihuman globulin conjugate
containing chromogenic enzyme will
react with the sample Ab.
oAdding enzyme substrate will
produce colour on the well.
oSo +ve test is manifested by
presenece of colour , in –ve test
there is no colour.
Ag
Sample AB
Enzym linked Ab
Enzyme substrate
oThis a solid phase reaction through
which the serum antibody will be
captured to the wells using capture
Ag.
oThe conjugate here is directed to the
Ag i.e. the sample and conjugate will
compete each other.
oAdding enzyme substrate will not
produce colour on the well.
oSo +ve reaction is manifested by no
colour , -ve reaction there is a colour.
Ag
Sample AB
Enzym linked Ab
Enzyme substrate
oThis a solid phase reaction through
which the serum antigen will be
captured to the wells using capture
Ab.
oThe conjugate here is directed to the
Ag i.e. the Ag will be sandwiched
between capture & test Abs
oAdding enzyme substrate will
produce colour on the well.
oSo +ve test is manifested by
presenece of colour , in –ve test there
is no colour.
Sample Ag
AB
Enzym linked Ab
Enzyme substrate
o is a biophysical laser based
technology employed in cell
counting, cell marker detection,
allowing synchronized analysis of
the physical
and chemical characteristics of up
to thousands of particles per
second.
o Example: used in the
diagnosis blood cancers, and CD4
cell counting in AIDS patients
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o rapid and easy.
o Antigen-antibody immune
complexes flow through a
region and encounter
antibody against them,
resulting in a visible coloured
line
o E.g Used in rapid diagnosis of
Malaria
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