Radio immune assay
Principle
It involves
binding of antigen and antibody
Antigen
(labelled mostly with gamma rays)
Procedure
Antigen is
mixed with antibodies
Increasing
amount of antigens is added ( one is labelled and other one is unknown)
These
antigens compete with each other for binding sites which are on antibody
molecule
Isolation of
free and bound antigen take place
Antigen
which is radiolabeled is counted by precipitation with antiserum and free antigen
is left in supernatant.
Uses
RIA used in
assay drugs like morphine
Vitamin
analysis e.g. folic acid
ELISA
principle
For
detection of antigen and antibody
Enzymes
cause conversion of substrate (colorless) to a product (colored) indicating
antigen antibody binding presence
Procedure
1st
step is coating in which antigen and antibody are coated on plate
2nd
step is blocking in which blocking agent is used to cover all unbound sites
present on plates
In 3rd
step detection is done in which enzyme conjugated Ab to target Ab ( antigen to
target Ag)
Addition of
substrateto produce signal which is recorded (enzyme substrate reaction)
Uses
Dag Nala
disease
Direct flouresent antibody technique
Principle
Used for
detection of antigen in samples e.g. viral
Used as
direct test because flouresent dye is used with antibody
FITC
(fluorescein isothyocyanate) which is used as flourochrome to give yellow green
flouresence
Procedure
Glass slide
fixed with antigen is taken
Incubation
is done with Ab(linked with FITC)
Wash to
remove unbound Ab
Dark field
illumination is observed under microscope with light source
Particles
which are Ab labelled bound show flouresence
Results
Flouresence
presence. Positive
If not
present . negative
Uses
For
identification of viruses from infected animals with rabies
Serum neutralization test
Principle
Used for
quantification of titer of neatralising antibody of viruses
Procedure
Antibodies
solutions are diluted and mixed with viral suspension
Incubation
is done allowing the antibody to react with virus
Western blot
Principle
Also called
immunoblotting used for detection of proteins
Immunochromatographic
based principles are used where proteins are isolated as their mol w8
Separated
proteins are then transfered on membrane (nitrocellulose) and are detected by
using Ab(primary) , substrate and enzyme labellled Ab
Procedure
SDS page is
used for separation of proteins
Proteins are
then transferred on PVDF membrane
Membrane
blocking is done with proteins which are neutral
Incubation
of protein with Ab is done
Blot is
incubated with chemiluminesent HRP substrate and film exposed
Uses
For lyme
disease ,creutzfeldt Jacob disease
Hemagglutination
inhibition test
Principle
Used for
measurement of specific Ag which is soluble
It is called
HI because soluble antigen has ability to block red blood cells which are
coated with Ab
Procedure
In this test
fixed amount of RBCs( coated with antigen) is mixed with Ab to Ag which is also
fixed
If antigen
is present in the sample it vwill compete antigen coated on RBCs for binding
Abs thereby causing inhibition of agglutination of RBCs
Results
Agglutination
. test is positive and vice versa
Complement fixation test
Principle
Serum is
heated and measured amount of complement and Ag is added in this heated serum
Procedure
Patient
serum is first mixed with known Ag
If Abs are
present in the serum complement is fixed by the formation of AG-Ab comlex
Then sheep
RBCs and hemolysin are added
Lysis of
sheep cells indicate that there are no Abs
Result
Abs are
present result is positive and vice versa
PCR
Principle
A chain
reaction in which small fragments of DNA sction are identified which are then
used as a template for producing primers that start reaction.
Procedure
1st
step is initialization in which temperature is increased to 97 degree celcius
DNA
polymerase is activated
2nd
step is denaturation in which temp is 94 to 96 degree centigrade for half minute which cause breakage of
hydrogen bond b/w complementary basec producing single stranded DNa
3rd
step is annealing in which temp is 50 to 65 deg cent for 20 t0 40 secnd which
cause annealing of promer to single stranded DnA template
In 4th
step which is extension taq polymerase has its optimum activity temp is 75 to
78 deg centigrade. Primers are extended towards each other so that DnA segment
is copied b/w two primers
