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

Contents

ELISA

OVERVIEW

PRINCIPLE

PROTOCOL

DIFFERENT TYPES

MATERIAL REQUIRED

MERITS&DEMERITS

APPLICATIONS

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

• 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

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.

MATERIALS REQUIRED

• Test sample

• Antibody /Antigen

• Polystyrene microtiter plates

• Blocking buffer

• Washing buffer

• Substrate

• Enzyme

Enzymes Used in ELISA

Horseradish peroxidase(very

commonly used )

Alkaline phosphatase

Beta –galactosidase

Lacto peroxidase

Tetra Methyl Benzidine (substrate)

The enzyme substrate should be colourless

After degradation by the enzyme it should be strongly coloured

fluorescent

TYPES:

a)Indirect ELISA

b)Sandwich ELISA

c) Competitive ELISA

Sandwich ELISA direct method

2 Antibodies Required

Must Recognize Different Epitopes

1st Antibody Is Referred To As Capture Ab

2nd Antibody Detection Ab

2nd Antibody Is Biotinylated

Enzymes Commonly Used: HRP (Horse Radish

Peroxidase) And AKP (Alkaline Phosphatase)

Substrate is TMB (Chromogen)

The ELISA plate is coated with Antibody to detect

specific antigen

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

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

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.

• 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….

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).

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.

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

Limitations

To carry out the test antibody must be

available

Concentration may be unclear

Possibility of False positive

Possibility of False negative

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

CONTENTS

RADIO IMMUNOASSAY (RIA)

OVERVIEW

PRINCIPLE

MATERIAL REQUIRED

PROTOCOL

INSTRUMENTAION

MERITS&DEMERITS

APPLICATIONS

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

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

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

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 ]

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

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

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

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]

Conti…

Intensity of radioactivity is inversely correlated

with the conc of the antigens in the test sample

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

The results obtained by plotting a graph

antibody bound to labelled antigen Unlabelled antigen

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.

APPLICTIONS :

Used in the estimation of pharmaceutical

drugs like

Morphine

Clonazepam

Barbiturates

Neobentine

Flurazepam

And others

Insulin

Gastrin

Glucogon

Growth hormones

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

References :

A textbook of pharmaceutical analysis

Biotechnology and its applications in pharmacy