This document provides information about various antibody-based techniques, including monoclonal and polyclonal antibodies, antigens, and several types of antigen-antibody reactions like radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Western blots, and precipitation reactions. It specifically describes RIA as a sensitive technique that uses radioactively labeled antigens and antibodies, ELISA as an assay that uses enzyme-labeled antibodies for detection, and Western blot as a method to detect specific proteins using labeled antibodies.

1. ANTIBODY BASED
TECHNIQUES
PRESENTED BY :
PRIYANKA PATHANIA
BTECH. BIOTECH (5TH
SEM)
12btb030

2. What are antibodies...??
An antibody (Ab) , also known as
immunoglobulin (Ig), is a large “Y”
shaped protein produced by plasma
cells that is used by the immune
system to identify and neutralize
foreign objects like bacteria and viruses.
Each antibody recognizes a specific
antigen unique to its target.

4. Antibodies (Immunoglobulins)

5.  Monoclonal antibodies :
(mAb) are antibodies that are identical
because they are produced by one type of
immune cells, all clones of a single parent cell.
 Polyclonal antibodies :
Are the antibodies that are derived from
different cell lines. They differ in amino acid
sequence.

6. What is an antigen...??
 An antigen (Ag), is a substance or any
molecule that induces an immune response in
the body.
 An antigen is often foreign or toxic to the body
for example bacteria or virus, which once in
the body, attracts and is bound to a respective
and specific antibody.

8. INTRODUCTION
 The antigens and the antibodies combine
specifically with each other. This interaction
between them is called Antigen-Antibody reaction.
 It may be abbreviated as Ag – Ab reaction.
 These reactions form the basis for detection of
infectious disease causing agents and also some
non-specific Ag’s like enzymes.

9. Types of Ag- Ab reactions...
 RIA – Radioimmuno Assay
 ELISA – Enzyme Linked Immuno Sorbent
Assay
 Western blots
 Precipitation Reactions

10. Radioimmunoassay (RIA)
 Involves the separation of a protein (from a
mixture) using the specificity of antibody -
antigen binding and quantify it using
radioactivity
 The technique was introduced in 1960 by Berson
and Yalow as an assay for the concentration of
insulin in plasma.

11. INTRODUCTION
 Radioimmunoassay (RIA) is a very
sensitive in vitro assay technique used to
measure concentrations of antigens (for
example, hormone levels in the blood) by use
of antibodies.
 The RAST test (radio allergosorbent test) is an
example of radioimmunoassay. It is used to
detect the causative allergen for an allergy

12.  To perform a radioimmunoassay, a known
quantity of an antigen is made radioactive,
frequently by labeling it with gamma-
radioactive isotopes of iodine attached to
tyrosine.
 This radiolabeled antigen is then mixed with a
known amount of antibody for that antigen, and
as a result, the two specifically bind to one
another.

13.  This causes the unlabeled (or "cold") antigen
from the serum to compete with the
radiolabeled antigen ("hot") for antibody
binding sites. As the concentration of "cold"
antigen is increased, more of it binds to the
antibody, displacing the radiolabeled variant,
and reducing the ratio of antibody-bound
radiolabeled antigen to free radiolabeled
antigen. 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.

15. Radioimmunoassay (RIA)

16. Advantages & Disadvantages of
RIA
 Advantages
 Highly specific: Immune reactions are specific
 High sensitivity : Immune reactions are sensitive
 Possible to detect picograms of Ag
 Sepharose beads used in RIA are reuseable

17.  Disadvantages
 Radiation hazards: Uses radio labelled
reagents
 Requires specially trained persons
 Labs require special license to handle
radioactive material
 Requires special arrangements for
storage of radioactive material
radioactive waste disposal.

18. ENZYME LINKED
IMMUNOSORBENT ASSAY
(ELISA)

19. INTRODUCTION TO ELISA
 ELISA, or enzyme-linked immunosorbent assay,
are quantitative immunological procedures in
which the Ag- Ab reaction is monitored by
enzyme measurements.
 The term ELISA was first used by Engvall &
Perlma in 1971.
 The ELISA test, or the enzyme immunoassay
(EIA), was the first screening test commonly
employed for HIV. It has a high sensitivity.

20. Elisa Plate
 Microtitre wells
 Generally, 96 wells
 Marked on one side
alphabetically
 Numerically on other
side
 Comes with the kit

21. BASIC PRINCIPLE OF ELISA
 Use an enzyme to detect the binding of
antigen (Ag) antibody (Ab).
 The enzyme converts a colorless substrate
(chromogen) to a colored product, indicating
the presence of Ag : Ab binding.
 An ELISA can be used to detect either the
presence of Antigens or antibodies in a
sample depending how the test is designed.
 ELISA was dveloped in 1970 and became
rapidly accepted

23. TYPES OF ELISA
• Indirect elisa
• Sandwich elisa
• Competetive elisa

24. Comparison between Indirect, Sandwich & Competitive
ELISA

25. Equipments for performing the
ELISA test

26. ELISA READER

27. Advantages of ELISA
 Reagents are relatively cheap & have a long
shelf life
 ELISA is highly specific and sensitive
 No radiation hazards occur during labelling or
disposal of waste.
 Easy to perform and quick procedures
 Equipment can be inexpensive and widely
available.
 ELISA can be used to a variety of infections.

28. Disadvantages of ELISA
 Measurement of enzyme activity can be more
complex than measurement of activity of some
type of radioisotopes.
 Enzyme activity may be affected by plasma
constituents.
 Kits are commercially available, but not cheap
 Very specific to a particular antigen. Won’t
recognize any other antigen
 False positives/negatives possible, especially
with mutated/altered antigen

29. APPLICATIONS OF ELISA
 detection of Mycobacterium antibodies in
tuberculosis
 detection of hepatitis B markers in serum
 detection of HIV antibodies in blood samples
 It has also found applications in
the food industry in detecting potential food
allergens, such
as milk, peanuts, walnuts, almonds, and eggs.

30. Western blot (Immunoblotting)
 Blots are techniques for transferring DNA ,
RNA and proteins onto a carrier so they can be
separated, and often follows the use of a gel
electrophoresis. The Southern blot is used for
transferring DNA, the Northern blot for RNA
and the western blot for PROTEIN.
 A technique for detecting specific proteins
separated by electrophoresis by use of labeled
antibodies.

31. Procedure
 In Western blotting, a protein mixture is
separated on a polyacrylamide slab gel
that has been treated with sodium dodecyl
sulfate, a dissociating agent.
 The protein bands are then transferred to
a nitrocellulose membrane by capillary
blotting.
 And the individual protein bands are
identified by flooding the blot with antigen-
specific

32. primary antibody, followed by incubation and
washing and finally addition of radiolabelled or
enzyme labelled secondary antibody specific
for the primary antibody.
 The antigen-antibody complexes (bands) that
form are visualised by autoradiography.

33. Western Blotting Procedure

34. Applications
 The confirmatory HIV test employs a western blot
to detect anti-HIV antibody in a
human serum sample. Proteins from known HIV-
infected cells are separated and blotted on a
membrane as above. Then, the serum to be
tested is applied in the primary antibody
incubation step; free antibody is washed away,
and a secondary anti-human antibody linked to an
enzyme signal is added. The stained bands then
indicate the proteins to which the patient's serum
contains antibody.
 Western blot can also be used as a confirmatory
test for Hepatitis B infection.