Elisa & ria

1. ENZYME LINKED IMMUNO SORBENT
ASSAY(ELISA)&
RADIO IMMUNOASSAY (RIA)
P.SAHITHI REDDY
M.PHARMACY
H.T.NO:256213886024
Under the guidance of

Mr Utham prasad

2. Contents

ELISA

OVERVIEW
PRINCIPLE
PROTOCOL
DIFFERENT TYPES
MATERIAL REQUIRED
MERITS&DEMERITS
APPLICATIONS

3. OVERVIEW:

Enzyme Linked Immuno sorbent
Assay (ELISA)

Term Was Coined By Engvall and
Pearlmann in 1971
is a quantitative technique used in
immunology to detect the presence of
an antibody or an antigen in sample

4. •Antigen of interest is absorbed on to
surface sorbent
•Antigen is recognised only by the specific
antibody immuno
•This antibody is recognised by second
antibody (immuno) which has enzyme
attached (enzyme linked)
•Substrate reacts with enzyme to produce
product usually depicted by coloured

5. PRINCIPLE:

ELISA is based on specific interaction between Ag and
their corresponding Ab. One of the immuno reactant is
coated to a solid phase support such as polycarbonate,
polyvinyl polyacrylamide tubes or microwells.
To the respective antibody or antigen is added
To antigen-antibody complex so formed an enzyme
linked antibody is added. A colourless substance is
added to well. This substance is a substrate for the
enzyme which catalyses the conversion of colourless
substance to coloured product.

6. MATERIALS REQUIRED


•Test sample
•Antibody /Antigen
•Polystyrene microtiter plates
•Blocking buffer
•Washing buffer
•Substrate
•Enzyme

7. Enzymes Used in ELISA

Horseradish peroxidase(very
commonly used )
Alkaline phosphatase
Beta –galactosidase
Lacto peroxidase
Tetra Methyl Benzidine (substrate)

9. The enzyme substrate should be colourless
After degradation by the enzyme it should be strongly coloured
fluorescent

10. TYPES:

a)Indirect ELISA
b)Sandwich ELISA
c) Competitive ELISA

12. Sandwich ELISA direct method

2 Antibodies Required
Must Recognize Different Epitopes
1
st
Antibody Is Referred To As Capture Ab
2
nd
Antibody Detection Ab
2
nd
Antibody Is Biotinylated
Enzymes Commonly Used: HRP (Horse Radish
Peroxidase) And AKP (Alkaline Phosphatase)
Substrate is TMB (Chromogen)

13. The ELISA plate is coated with Antibody to detect
specific antigen

14. Prepare a surface to which a known quantity of capture
antibody is bound
Block any non specific biding sites on the surface
Apply the antigen – containing sample to the plate
Wash the plate ,so that unbound antigen is removed
Apply enzyme linked primary antibodies as detection
antibodies which also bind specifically to the antigen
Protocol for sandwich
ELISA

15. Wash the plate, so that the unbound antibody-
enzyme conjugates are removed
Apply a chemical which is converted by the enzyme
into a coloured product
Measure the absorbency of the plate wells to
determine the presence and quantity of antigen

17. Indirect ELISA:

•The protein antigen to be tested for is added to each
well of ELISA plate, where it is given time to adhere
to plastic through charge interactions
• A solution of non-reacting protein is added to block
any plastic surface in the well that remains uncoated
by the protein antigen
•Then the serum is added, which contains a mixture of
the serum antibodies, of unknown concentration,
some of which may bind specifically to the test
antigen that is coating the well.

18. •Afterwards, a secondary antibody is added, which will bind to
the antibody bound to the test antigen in the well. This
secondary antibody often has an enzyme attached to it
•A substrate for this enzyme is then added. Often, this
substrate changes colour upon reaction with the enzyme. The
colour change shows that secondary antibody has bound to
primary antibody, which strongly implies that the donor has
had an immune reaction to the test antigen.
•The higher the concentration of the primary antibody that was
present in the serum, the stronger the colour change. Often a
spectrometer is used to give quantitative values for colour
strength
•
Conti….

20. COMPETIVE ELISA

•The labelled antigen competes for primary antibody
binding sites with the sample antigen (unlabelled).
The more antigen in the sample, the less labelled
antigen is retained in the well and the weaker the
signal).

23. RESULTS

After reading the results the standard curve is drawn were the
concentration is plotted on the X-axis and the absorbance on
the Y-axis.

24. Advantages


•Reagents are relatively cheap& have a long shelf life
•Easy to perform and quick procedures
•No radiation hazards during handling
•Has wide usage to variety of infections

26. Limitations

To carry out the test antibody must be
available
Concentration may be unclear
Possibility of False positive
Possibility of False negative

27. Applications

The various sections devoted to the uses are

Detection of mycobacterium antibodies in tuberculosis
Detection of rotavirus in faeces
Detection of hepatitis B markers in serum
Detection of HIV antibodies in blood sample
Hormones
Proteins
Drug markers
Serum proteins
Vaccine quality control
Tumour markers

29. CONTENTS

RADIO IMMUNOASSAY (RIA)

OVERVIEW
PRINCIPLE
MATERIAL REQUIRED
PROTOCOL
INSTRUMENTAION
MERITS&DEMERITS
APPLICATIONS

30. Overview:

 Radio immuno assay was developed by
Berson&yalow(1956) for the quantitative
measurement of insulin in human plasma
RIA principles have found wide application in the
field of drug analysis kinetic studies, immuno
diagnosis
RIA is specific, sensitive &rapid

31. An immunoassay is a test that uses antibody and antigen
complex as means as a mean of generating a measureable
result

An antibody :antigen complex is known as a immune
complex.

Immunoassays are different from other types of laboratory
tests, such as colorimetric tests, because they use antibody:
antigen complexes to generate a signal that can be measured .

In contrast, most routine clinical chemistry tests utilize
chemical reaction between the reagent and sample to generate
a test result

32. Principle

RIA is a competitive binding assay.
The antibody & labelled antigen are always present as
limiting factors and the concentrations of unlabelled
antigens(sample) under examination is increased
continually

Uses an immune reaction
(Antigen –antibody reaction ) to estimate ligand

33. Unbound Ag* and Ag washed
Radioactivity of bound residue measured
Ligand conc is inversely related to radioactivity

[Ag: ligand to be measured ;Ag* radiolabelled ligand ]

35. Materials required

a)Preparation &characterisation of Antigen [Ligand to be
analysed ]
b)Radiolabelling of the Antigen
c)Preparation of the specific antibody
d)Development of Assay system

36. PREPARATION AND RADIOLABELLING
OF THE ANTIGEN

Antigens prepared by

synthesis of the molecule
isolation from natural sources

Radiolabelling [Tagging procedure ]

3H,14C,125I are used as radioactive tags
Antigens are tagged to 3H14c125I
tagging should NOT affect antigenic specificity and activity

37. PREAPRATION OF SPECIFIC
ANTIBODY

Antigen injected intradermal into rabbit
antibody production
Antibodies recovered from the serum
Some ligand are not antigenic
hormones ,steroids drugs HAPTENS
E.g.: castrin morphine

38. ASSAY Procedure

Add known amounts of the test sample+ labelled
antigen into the microtitre wells

 incubate – allow the reaction to reach completion
Decant and wash the contents of the well-
removes all unbound antigens
Radioactivity remaining in the mocrotitre wells
measured by a counter [GM counter, scintillation
counter]

39. Conti…


Intensity of radioactivity is inversely correlated
with the conc of the antigens in the test sample

40. INSTRUMENTATION:

CENTRIFUGE:

 swing bucket rotor : 1200-2500 rpm
Fixed angle head rotor :3500-4000 rpm

RADIOACTIVE counter

 gamma counter : which is used for a gamma energy
emitting isotopes. E.g. 125I.
Scintillation counter : it is used for beta energy emitting
isotopes.eg. Tritium 3H and 14C isotopes

42. The results obtained by plotting a graph
antibody bound to labelled antigen Unlabelled antigen

43. Merits and demerits

Merits :
highly specific : immune reactions are specific
high sensitivity : immune reaction are sensitive

Demerits:
radiation hazards :uses radiolabelled reagents
Requires specially trained personnel
Labs require special license to handle radioactive
material
Requires special arrangements for :requisition,
storage of radioactive material
radioactive waste disposal.

44. APPLICTIONS :

Used in the estimation of pharmaceutical
drugs like
Morphine
Clonazepam
Barbiturates
Neobentine
Flurazepam
And others
Insulin
Gastrin
Glucogon
Growth hormones

45. Limitations :

The limitations of the RIA are
Its expensive
Being hazardous Handling the radioactive material
The radio isotopes used 125I or 131I emit gamma
radiation these requires a special counting machine

46. References :

A textbook of pharmaceutical analysis
Biotechnology and its applications in pharmacy