A SEMINAR ON

RIA AND ELISA

1

RIA AND ELISA

CONTENTS

INTRODUCTION DEFINATION HISTORY PRINCIPLE OF ELISA TYPES OF ELISA -

INDIRECT ELISA SANDWICH ELISA COMPETITIVE ELISA CHEMILUMINESCENCE ELISPOT ELISA

APPLICATIONS OF ELISA

ELISA

RIA

INTRODUCTION HISTORY PRINCIPLE OF RIA APPLICATIONS OF RIA ADVANTAGES OF RIA DISADVANTAGES OF RIA SUMMARY CONCLUSION REFERENCES

2

IMMUNOASSAY-

RIA AND ELISA

Assay employing antibodies for qualitative semiqualitative and quantitative analysis are broadly termed as immunoassay.

In other words immunoassays are methods utilizing immunological reactions for the analysis of the biological molecules.Example- dertermination of blood group of bacterial agglutination tests.

The methods generally used are follows-

3

DIRECT METHODS Precipitation Neutrilization Agglutination Compliment fixation test

INDIRECT METHODS Enzyme

immunoassay Immuno

fluorescence Radio

immunoassays

INTRODUCTION

ELISA also known as an enzyme linked immunosorbent assay is a biochemical Techniques used mainly in immunology to detected the Presence Of an antibody or an antigen in a sample.

What is antigen or antibody –

ANTIGEN- A substrate which activates the recipients immune system by generating antibodies an or activates specific immune cells.

RIA AND ELISA

Fig.-1- structure of antigen 4

ANTIBODY – Proteins produced in response to any substance or organism(antigen) edicting an immune response. it is also known as immunoglobulin Ig, which specifically binds the antigen.

RIA AND ELISA

Fig-2- structure of antibodies 5

Antigen – antibody reaction -

An antibody combines specifically with the corresponding antigen or hapten in a manner which is very similar to the binding of a enzyme to it is substrate and involvin hydrophobic and ionic interaction. The bonding between antigen and antibody. However involves no subsequent chemical reaction and it is stability depends upon the complementry shape of the antigen and antibody.

ANTIGEN + ANTIBODY Ag –Ab COMPLEX

Fig.-3- Antigen – Antibody reaction

RIA AND ELISA

6

General features of Antigen-Antibody reaction-

RIA AND ELISA

The reaction is specific, an Antigen combining only with it’s homologous Antibody and vice versa.

The specificity, however is not absolute and cross- reactions may occur due to antigenic similarity or relatedness.

There is no denaturation of the antigen or the antibody during the reaction.

Antibodies to the surface antigens of infectious agents are generally protective.

The combination is firm but reversible. The firmness of the union is influenced by the affinity and avidity of the reaction.

It is the function of the closeness of fit between an epitope and the antigen combining regions of it’s antibody.

Both antigen and antibodies participates in the formation of agglutinates or precipitates.

7

Enzyme-linked immunosorbent assay, commonly known as ELISA (or EIA), is similar in principle to RIA but depends on an enzyme rather than a radioactive label. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate.A number of enzymes have been employed for ELISA,

including - Alkaline phosphatase, Horseradish peroxidase, and B-galactosidase.

These assay approach the sensitivity of RIAs and have the advantage of being safer and less costly.

DEFI NATION

ELISA-

RIA AND ELISA

8

HISTORY

This techniques given by - “Engval and Perlmanu” – 1971

This techniques is highly versatile, senstital,simple Antigen of Antibody hazards.

RIA AND ELISA

9

PRINCIPLE

The principle of ELISA is as follows – one of the immunoreagent is immoblised through adsorption on the solid phase support (usually polyvinyl chloride or polystyrene) in such a way that there is loss to it’s activity.

The second immunoreagent is linked to an enzyme in a way that there is no loss either to immuno reactivity or to the enzyme activity.

After incubation and subsequent’s washing a chromogenic Substrate of the enzyme is supplied . If the two immuno reagents have bound to each other, colour will developed because of the presence of the linked enzyme, if not there will not be any Colour.

COLOUR LESS SUBSTRATE

PRODUCT COLOURSOLID PHASE ABSORBED

ANTIGEN

REACTING ANTIBODY

ANTI-ANTIBODYENZYME COJUGATE

Fig-4-Principle of ELISA

RIA AND ELISA

10

RIA AND ELISA

Fig-5-ELISA the general procedure

11

PROCEDURE

TYPES

OF

ELISA

A number of variations of ELISA have been developed, allowing qualitative detection or quantitative measurement of either antigen or antibody.

Each type of ELISA can be used qualitatively to detect the presence of antibody or anentig.

TYPES OF ELISA-

INDIRECT ELISA SANDWICH ELISA COMPETITIVE ELISA CHEMILUMINESCENCE ELISA ELISPOT ELISA

RIA AND ELISA

12

INDIRECT ELISA

Antibody can be detected or quantitatively determined with an indirect ELISA . Serum or some other sample containing primary antibody (Ab1) is added to an antigen-coated microtiter well . After any free Ab1 is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme- conjugated secondary antibody (Ab2). Any free Ab2 then is washed away, and a substrate for the enzyme is added. The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers.

RIA AND ELISA

Fig-6-Indirect ELISA 13

In this technique, the antibody (rather than the antigen) is immobilized on a microtiter well. A sample containing antigen is added and allowed to react with the immobilized antibody.After the well is washed, a second enzyme-linked antibody specific for a different epitope on the Antigen is added and allowed to react with the bound antigen.After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured.

RIA AND ELISA

SAND WICH ELISA

Fig- 7-Sandwich ELISA 14

In this technique, antibody is first incubated in solution with a sample containing antigen.The antigen-antibody mixture is then added to an antigen coated microtiter well. The more antigen present in the sample,the less free antibody will be available to bind to the antigen-coated well. In the commpetitive assay, however, the higher the concentration of antigen in the original sample, the lower the absorbance.

RIA AND ELISA

COMPETITIVE

ELISA Fig-8- Competitive ELISA 15

Measurement of light produced by chemiluminescence during certain chemical reactions provides a convenient and highly sensitive alternative to absorbance measurements in ELISA assays. In versions of the ELISA using chemiluminescence, a luxogenic (light-generating) substrate takes the place of the chromogenic substrate in conventional ELISA reactions.

For example - oxidation of the compound luminol by H2O2 and the enzyme horseradish peroxidase (HRP) produces light:

Ab-HRP+ Ag →Ab-HRP-Ag luminol+H2O2 light

The advantage of chemiluminescence assays over chromogenic ones is enhanced sensitivity.

RIA AND ELISA

CHEMILUMINESCENCE 16

In this approach, the plates are coated with the antigen (capture antigen) recognized by the antibody of interest or with the antibody (capture antibody) specific for the antigen whose production is being assayed. producing a ring of antigen-antibody complexes around each cell that is producing the molecule of interest.

The plate is then washed and an enzyme- linked antibody specific for the secreted antigen or specific for the species (e.g., goat anti-rabbit) of the secreted antibody is added and allowed to bind.

Subsequent development of the assay by addition of a suitable chromogenic or chemiluminescence-producing substrate reveals the position of each antibody- or antigen- producing cell as a point of colour or light.

RIA AND ELISA

ELISPOT

ELISA Fig-9-ELISPOT

assasy17

RIA AND ELISA

APPLICATION

ELISA used in the detection and quantitation of several antigen as well as antibodies.

ELISA method to determine antigliadin antibodies in children with coeliac disease.

It is useful tool for determining serum Antibody consentration (such as with the HIV test or virus).

It is also found opposite in detecting potential food allergens such as milk, peanuts, walnuts, almond, and eggs.

Indirect ELISA method to detect the presence of serum antibody against HIV. The causative agent of AIDS.

ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drug.

18

19

Radioimmunoassay(RIA)-

INTERODUCTION

Radioimmunoassay is one of the most important techniques in the clinical biochemical fields for the quantitative analysis of harmones, and drugs .

It combines the specificity of the immune reaction with the sensitivity of the radioisotope techniques.

Alternative names used for RIA include saturation analysis, displacement analysis and competitive radioassay.

The most commonly used labels are radioisotope and enzymes.

A variety of tests have been devised for the measurement of antigen and antibodies using such lebelled reactants.

The term binder- ligand – assay has been used for these reaction.

RIA AND ELISA

20

RIA AND ELISA

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. Berson and Rosalyn Yalow, to determine levels of insulin–anti-insulin complexes in diabetics. Although their technique encountered some skepticism, it soon proved its value for measuring hormone serum proteins, drugs, and vitamins at concentrations of 0.001 micrograms per milliliter or less.

In 1977, some years after Berson’s death, the significance of the technique was acknowledged by the award of a Nobel Prize to Yalow.

HISTORY

RIA AND ELISA

21

The principle of RIA involves competitive binding of radiolabelled antigen and unlabeled antigen to a high-affinity antibody.

.

PRINCIPLE

The labeled antigen is mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody. The antibody does not distinguish labeled from** unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody.

As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites. The decrease in the amount of radiolabelled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample. Example – 4 Ag* + 4 Ab 4Ag* Ab 4 Ag + 4Ag* + 4 Ab 2 Ag* Ab + 2 Ag Ab +2 Ag* +2 Ag 12 Ag + 4 Ag* + 4 Ab Ag* + Ab +3 Ag Ab + 3 Ag* + 9 Ag (sample) (labelled Ag) (coated Ab)

RIA AND ELISA

22

(a) Microtiter wells are coated with a constant amount of antibody specific for HBsAg, the surface antigen on hepatitis B virions. A serum sample and [125I]HBsAg are then added. After incubation, the supernatant is removed and the radioactivity of the antigen-antibody complexes is measured. (b) A standard curve is obtained by adding increasing concentrations of unlabeled HBsAg to a fixed quantity of [125I]HBsAg and specific antibody.

Fig- 10- A solid phase RIA to detected hapatites B - virus in blood sample

Cont.

METHODS

RIA AND ELISA

23

APPLICATIONS

RIA has significance in diagnostic biochemistry. RIA comes in handy for estimation of T3 and T4 separately.

RIA can help to differentiate the basic biochemical lesion in endocariniology whether the increased level of a as such or the tropic harmones.

An apparent increase of the thyroid harmones is observed in pregnancy or in persons talking oral contraceptives due to the increased level of a the serum thyroxin - bonding globulin.

This techniques offers safety to the patient in the use of drugs if there is only a narrow margin between the therapeutic and toxic dosage. This is applied during digitalisation in the in the management of congestive heart failure.

RIA AND ELISA

24

Estimation of peptids steroids harmone, vitamins, drugs, antibodies nuclic acids, structural proteins harmone receptor proteins.

Radioimmunoassay has tremendous application in the dignosis of harmonal disorders, cancers and therapeutic monitoring of drugs, besides being useful in biomedical research.

Cont.

APPLICATIONS

This techniques is also useful in diagnosing insulinomas, sex harmone sensitive tumors etc. and this facilitates proper treatment of the disease

RIA AND ELISA

25

ADVANTAGES

The ability to assay any compound that is immunogenic, available is pure form, and can be ratio labelled.

It’s high sensitivity some compounds may be detected at a level of pg cm-3 .

It’s high specificity.

It’s precision which is comparable to that of other physico- chemical techniques and for better then bioassay.

It’s case of automation so that a minimum of manual handling and data processing is necessary. This allow a large number of sample to be processed at minimal cost.

RIA AND ELISA

26

DISADVANTAGES

The relatively high cost of equipment and reagents. gamma scintillation counters are expensive to buy and maintain, and radioiodine is not a cheap reagent.

The short shelf-life of reagents that half lives of 125I and 131I are 60 days respectively necessitating relatively frequent labelling of antisera.

The rediological hazards of using radioiodine particularly during the labelling of antisera, which must be respected fairly regularly. staff should have regular thyroid scans and be rested it the lavel of radioactivity increases significantly.

Assays usually takes days rather than hours.

RIA AND ELISA

27

SUMMARY

Antigen-antibody interactions depend on four types of noncovalent interactions: hydrogen bonds, ionic bonds, hydrophobic interactions, and vander waals interactions.

The interaction of a soluble antigen and precipitating antibody in a liquid or gel medium forms an Ag-Ab precipitate.

The interaction between a particulate antigen and agglutinating antibody (agglutinin) produces visible clumping, or agglutination that forms the basis of simple, rapid, and sensitive immunoassays.

Radioimmunoassay (RIA) is a highly sensitive and quantitative procedure that utilizes radioactively labeled antigen or antibody.

The enzyme-linked immunosorbent assay (ELISA) depends on an enzyme-substrate reaction that generates a colored reaction product.

RIA AND ELISA

28

CONCLUSION

Immunoassay are methods utilizing immunological reaction for the analysis of biological molecules.

ELISA also known as an enzyme assay is a biochemical techniques used mainly in immunology to detected the presence of an antibody or an antigen in a sample.

The most sensitive techniques for detecting antigen orantibody is radioimmunoassay (RIA).

29

RIA AND ELISA

REFERENCES

S.V.S. RANA

AVINASH UPADHYAYKAKOLI UPADHYAYNIRMALENDU NATH

JAINS KUBY

ANANTHANARAYANAND PANEKER’S

INTERNET SOURCES CLASS NOTES-

2010-11

2007

2007

2006

2011

BIOTECHNIQUES THEORY AND PRACTICE2nd EditionRASTOGI PUBLICATION

BIOPHYSICALCHEMISTRY PRINCILEAND TECHNIQUES4th EditionHIMALAYA PUBLICATION

IMMUNOLOGY6th EditionW.H.FREEMAN COMPANYNEWYORK

MICROBIOLOGY7th Edition

30

THANKYOU