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Immunological techniques must view slides

2. Whatareimmunological
techniques
Immunological techniques are the wide varieties of
methods and specialized experimental protocols
devised by immunologists for inducing, measuring,
and characterizing immune responses.
These techniques are not restricted to the field
ofimmunology, but are widely applied by basic
scientists in many other biological disciplines

3. Immunologic techniques include:
ELISA
RIA
IMMUNOPRECIPITATION
WESTERN BLOT

4. Anenzyme-linked immunosorbentassay, also called
ELISA or EIA, is a test that detects and measures
antibodies in your blood. This test can be used to
determine if you have antibodies related to certain
infectious conditions. Antibodies are proteins that
your body produces in response to harmful substances
calledantigens.

5. An ELISA test may be used to
diagnose:
HIV, which causes AIDS
Lyme disease
pernicious anemia
Rocky Mountain spotted fever
rotavirus
squamouscell carcinoma
syphilis
toxoplasmosis
varicella-zoster virus, which
causeschickenpoxandshingles

6. Requirement
•Microtiterplate
•Antigen
•Antibody
•PBS Buffer
•BSA blocking protein

8. Types of ELISA
DIRECT ELISA
INDIRECT ELISA
SANDWICH ELISA

10. DirectELISA
Blood sample
is taken
Centrifugation
Plasma +
serum
Serum+PBS
BUFFER

11. STEP 1
PBSalong with serum is poured in the well of
microtiterplate.
As the serum contain antigen, when we pour sample in
the well antigen get attached with the well surface.
This step is called coating.

12. Blocking
This is the step in which we use non reactive protein
and pour it in the well.
This protein will cover or block the space in the well
other than where antigen is present.
Blocking is done because microtiterplate has ability
for binding antibodies and proteins.Sowe want to bind
antibody only with antigen not with surface.
Blocking agent is BSA protein.

13. Attachment of enzyme linked
antibody
Next step is the attachment of enzyme linked
antibody.
These antibodies are available in markets or we can
prepare them.
This enzyme linked antibody is specific to antigen,that
has enzyme HRP.
After attachment of antibody and antigen we will add
substrate specific to enzyme.

14. Detection
When enzyme and substrate combine color change in
the well that shows the presence of antigen.
Instensityof color shows severity of infection.
If color change does not take place it means there is no
infection and there is no antibody.

15. Analysis
We a analysethe test by ELISA reader.
ELISA plate is placed in reader.
This gives quantitative information and qualitative
information of antigen.

16. Direct
ELISA

18. Indirect ELISA
Used for antigen detection.
Disease specific microtiterplate is used.
In this specific plate antigen is already present and
surface is already blocked.
So,nextsample containing antibody is added.
This antibody will attach with antigen.Antibodyused
is called primary antibody.
Then we will use seconderyantibody that is also called
detection antibody that is enzyme linked.

19. Enzyme attach is HRP or alkaline phosphate.
Seconderyantibody bind with primary antibody as it is
specific to primary antibody.
After that specific substrate added and colorful
solution is obtained.
Then data analysis is performed.

21. Sandwich ELISA
First step is serum separation that contain antibodies.
If we have some sort of infection then we must have
antibodies that will produce in response to antigen.
PBS buffer is added to this sample.
We will add sample in the well of plate,antibodywill
attach to the surface.
Then blocking step is performed.
Here antibody is called capture antibody.

22. Then sample containing antigen is added.
After that enzyme linked antibody is added.
After that substrate is added reaction takes place and
color change occur.
Sandwich ELISA is 2 to 3 times more sensitive.

24. Radio immunoassay
One of the most sensitive techniques for detecting
antigen or antibody is radioimmunoassay (RIA).
The technique was first developed in 1960 by two
endocrinologists, S. A. Bersonand Rosalyn Yalow, to
determine levels of insulin–anti-insulin complexes in
diabetics.
• In 1977, some years after Berson’sdeath, the
significance of the technique was acknowledged by the
award of a Nobel Prize to Yalow.

25. Requirement
If we want to detect antigen serum A antigen then we
need 3 things
Anti a antibody
RadiolabeledA antigen
Unlabeled A antigen

26. Principle of RIA
Radioimmunoassay (RIA) involves the separation of a
protein (from a mixture) using the specificity of
antibody -antigen binding and quantitationusing
radioactivity.

27. Steps involved in RIA
In the basic method of Radioimmunoassay, we use the
target antigen which is labeled radioactively and
bound to its specific antibodies.
We will require a limited and known antibody to be
added in a specific amount inRadioimmunoassay.
A sample is then added in order to initiate a reaction
competitive in nature, of the labeled antigens from the
preparation, and the unlabeled antigens from the
sample, with the specific antibodies.

28. Steps involved RIA
Consequently, unlabeled antigen added to the sample
mixture will compete with radiolabeledantigen for the
limited supply of antibody.
Even a small amount of unlabeled antigen added to
the assay mixture of labeled antigen and antibody will
cause a decrease in the amount of radioactive antigen
bound, and this decrease will be proportional to the
amount of unlabeled antigen added.

29. The bound antigens are then separated from the
unbound ones, and the radioactivity of the free
antigen remaining in the supernatant is measured
using a gamma counter.

31. Applications of RIA
The test can be used to determine very small
quantities (e.g. nanogram) of antigens and antibodies
in the serum.
The test is used for quantitationof hormones, drugs,
HBsAg, and other viral antigens.
Analyze nanomolarand picomolarconcentrations of
hormones in biological fluids

32. Immunoprecipitation
Immunoprecipitation is the technique of precipitating
a protein antigen out of solution using an antibody
that specifically binds to that particular protein. This
process can be used to isolate and concentrate a
particular protein from a sample containing many
thousands of different proteins.

33. Target antigens are usually immunoprecipitated from
complex solutions, such as cell lysates, the goal being
to isolate and eventually detect and measure a specific
protein (i.e., the antigen of the specific antibody).

34. StepsinvolvedinIP
First, lyseyour cells using some sort of lysisbuffer.
Next, add in an antibody that will bind your protein of
interest and form a protein-antibody complex. Then
drop in some resin which can bind the antibody.
These proteins are specifically designed to bind the
heavy chains of antibodies so they can easily pull out
your protein-antibody complexes.

35. Agarosebeads offer higher capacity per bead but
magnetic beads are MUCH easier to separate because
you can use a magnet to keep them in place.
Finally, spin everything down and remove the
supernatant. With the remaining bead-antibody-
protein conjugates, you can either denature everything
and run aSDS-PAGE western blot.

37. ApplicationofIP
Detect proteins of interest.
Study protein-protein interact.
Identify protein in protein complex.
Protein expression in specific tissues.

38. Westernblotting
Awestern blottingis a laboratory method used to
detect specific protein molecules from among a
mixture of proteins. This mixture can include all of the
proteins associated with a particular tissue or cell type
It not only used for proteins but also to find out
different chemical reactions inside the cell,protein-
protein interaction.

39. Principleofwesternblotting
Western blotting is an Immunoblotting technique
which rely on the specificity of binding between a
molecule of interest and a probe to allow detection of
the molecule of interest in a mixture of many other
similar molecules.
In Western blotting, the molecule of interest is a
protein and the probe is typically an antibody raised
against that particular protein.

40. Steps involved in western blotting
technique
Extraction of molecule
Separation
Tranfer
Probing
Analysis

41. Step 1
First of all we will treat the cell simply with detergent.it
will break down cell membrane.Cytosoliccomponents
come out.
Then we will do centrifugation that gives us
supernatant and pellet.Pelletcontain cell debris,
membrane , organelles while supernatant contain
protein components.
We will take supernatant that contain mixture of
proteins.

42. Separation of proteins
We will separate proteins on the basis of molecular
weight.Thisis done by SDS-PAGE electrophoresis.
SDS-PAGE stands for sodium dodicylsulphate
polyacralamidegel electrophoresis.
It uses gel acralamide.
This type of electrophoresis separate proteins on the
basis of molecular weight.

44. Transfer
On completion of protein separation by
polyacrylamidegel electrophoresis (PAGE), the next
step is to transfer the proteins from the gel to a solid
support membrane, usually made of a chemically inert
substance, such as nitrocellulose or PVDF. Blotting
makes it possible to detect the proteins on the
membrane using specific antibodies. The proteins
transferred from the gels are immobilized at their
respective relative migration positions at the time
when the electric current of the gel run was stopped.

46. Antibodyprobing
Once your protein samples are separated and
transferred onto a membrane, the protein of interest is
detected and localized using a specific antibody.
Usually, Western blotting protocols utilize an
unlabeled primary antibody directed against the target
protein and a species-specific, labeled secondary
antibody directed against the constant region of the
primary antibody.

47. The secondary antibody serves not only as a carrier of
the label, but is also a mechanism to amplify the
emitted signals, as many secondary antibodies can
theoretically bind simultaneously to the primary
antibody.
This is one of the most effective ways to maximize the
potential sensitivity of the assay. For this reason,
secondary antibodies are most often polyclonal and
can target epitopeson the framework regions of the
primary antibody; specificity is thus limited to species
and immunoglobulin isotype.

48. The signal emitted by the labeled secondary antibody
is then measured and is proportional to the quantity of
protein of interest present on the membrane.
With this highly specific immunodetectionprocess, it
is possible to reveal the presence of a very low quantity
of a specific protein in a complex sample.

49. Imaging
The last step in the Western blotting workflow before
data analysis is image capture. Enhanced
chemiluminescence(ECL) is based on the reaction
between an added luminolsubstrate and horseradish
peroxidase(HRP)-labeled antibodies. In the presence
of HRP, hydrogen peroxide catalyses the oxidation of
luminol, a reaction that results in the emission of light.

50. The light signal can then be detected on X-ray film or
by digital imaging with a charge-coupled device (CCD)
camera-based imager. When using fluorescence
detection, a fluorophoreis conjugated to the primary
or secondary antibody. Light is emitted by the
fluorophoreafter excitation via a specific wavelength
of light. A photomultiplier tube (PMT) or a CCD can
be used to collect and convert the emitted light to an
electrical signal. The electrical signal is then digitized
for image display and analysis.

51. Analysis
Detection of signals, using either X-ray film, scanners,
or a charge-coupled device (CCD) camera-based
imager, results in one or more visible protein bands on
the membrane image. The molecular weight of the
protein can be estimated by comparison with marker
proteins and the amount of protein can be determined
as this is related to band intensity (within the limits of
the detection system.