Radio used as flourochrome to give yellow green flouresence

                                                 
Radio immune assay

Principle

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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