PRESENTED BYPRESENTED BY

S . VISWANTH REDDYS . VISWANTH REDDYM.Pharmacy 1M.Pharmacy 1ststYear(pharmacology)Year(pharmacology)

Gokaraju rangaraju college of pharmacyGokaraju rangaraju college of pharmacy

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ENZYME LINKED IMMUNOSORBENT ASSAY

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CONTENTSCONTENTS

Introduction to ELISAIntroduction to ELISAPrinciple & procedurePrinciple & procedureMaterials neededMaterials neededTypes of ELISATypes of ELISAAdvantages & disadvantages of ELISAAdvantages & disadvantages of ELISAApplicationsApplications

ELISAELISA

RIARIAIntroduction to RIAIntroduction to RIAPrinciple & procedurePrinciple & procedureMaterials neededMaterials neededAdvantages & disadvantages of RIAAdvantages & disadvantages of RIAInstrumentationInstrumentationApplicationsApplications

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INTRODUCTION TO ELISAINTRODUCTION 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.

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Why known Why known as ......?as ......? EEnzyme nzyme LLinked inked IImmunommunossorbent orbent AAssayssay

1 .Antigen of interest is absorbed on to plastic

surface (‘sorbent’).

2. Antigen is recognised by specific antibody

(‘immuno’).

3. This antibody is recognised by second antibody

(‘immuno’) which has enzyme attached (‘enzyme-

linked’).

4. Substrate reacts with enzyme to produce product,

usually coloured . 5

BASIC PRINCIPLE OF ELISABASIC PRINCIPLE OF ELISAUse 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

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Materials NeededMaterials NeededTesting sampleAntibody (1st, 2nd) / AntigenPolystyrene microtiter plateBlocking bufferWashing bufferSubstrateEnzyme

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ANTIGEN (Ag)ANTIGEN (Ag)Any molecule that induces production of

antibodies when introduced in the body of an animal is called antigen.

OR any “thing”, foreign to the immune

system. e.g. bacteria, viruses, (or their parts), pollen, etc.

Protein moleculeCarbohydrate molecule.MicroorganismsAllergens.Viruses Etc.

SYMBOL FOR ANTIGEN

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ANTIBODY ( Ab)Antibody: proteins produced by

the immune system which help defend against antigens

SYMBOL FOR ANTIBODY

Y10

Antibodies (ImmunoglobulinsAntibodies (Immunoglobulins))

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Specimen Sample For ELISASpecimen Sample For ELISASERUM

CSF

SPUTUM

URINE

SEMEN

SUPERNATANT OF CULUTRE

STOOL12

Enzymes Used in ElisaEnzymes Used in ElisaHorseradish peroxidase (most

commonly used)Alkaline Phosphataseβ-galactosidaseLactoperoxidaseTetra Methyl benzidine In case of peroxidase, the substrate hydrogen peroxide is

converted into water and o2 in the presence of electron donors . (like diaminobenzidine or 4-chloronaphthol which themselves oxidized in the reaction).

Oxidation of diaminobenzidine produces dark brown color while that of 4-chlorornaphthol yields purple color which is the basis of ELISA

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ENZYME SUBSTRATEENZYME SUBSTRATEInitially the substrate should be colorlessAfter degradation by the enzyme it

should be strongly colored or fluorescent.

ENZYME SUBSTRATE CHROMOGEN STOPPING

Alkaline Phosphatase

p-NPP p-NPP+ diethandamine+MgCl2

1 M NaOH

Horse radish Peroxidase

H2O2 Tetramethylbenzidine + Phosphate – Citrate buffer

1 M H2SO4

Horse radish Peroxidase

H2O2 O – Phenylenediamine + HCl

1 M HCl

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Substrate

Primary antibody

Enzyme

Secondary antibody

Different antigens in sample

Coloured product

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Basic Basic Steps Steps Of Of EEnzyme-nzyme-LLinkedinked IImmunommunossorbantorbantAAssayssay

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TYPES OF ELISATYPES OF ELISA

◦Indirect elisa

◦Sandwhich elisa

◦Competetive elisa

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Indirect elisaIndirect elisa

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Sandwich elisaSandwich elisaAntigens such as tumor markers, hormones and

serum proteins may be determined

Antigen in the sample binds with the capture antibody on the microwell and becomes immobilized.

The antibody of the enzyme conjugate binds with the immobilized antigen to form a sandwich of antibody-antigen-antibody/enzyme bound to the microwell.

Enzyme reaction product is directly proportional to concentration of standard or analytical antigen 19

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ELISA SANDWICH FORMATELISA SANDWICH FORMAT

Y Y Y

Y Y Y

Y

YY

Y Y Y

Y Y Y

2nd antibody with enzyme

Antibody/Antigen

Antibody

Y Y YY Y Y

enzyme produces colour

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Competitive ElisaCompetitive Elisa Used to determine small molecule antigens.(T3,T4,progesterone etc.) antibody coated microwell serum antigen and labelled antigen added together--competition. antibody-antigen-enzyme complex bound is inversely related to the concentration

of antigen present in the sample. The bound enzyme conjugate reacts with the chromogenic substrate added to

produce a color reaction (blue to yellow color). . Increased serum antigen results in reduced binding of the antigen-enzyme

conjugate with the capture antibody producing less enzyme activity and color (yellow) formation

Substrate product concentration is inversely proportional to the concentration of standard or test antigen added

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Comparison between Indirect Sandwich & Competitive ELISA

to detect Ab (HIV, HCV)

to detect Ag ( Tumor Markers, Hormones )

to detect Ag ( Free Testosterone)

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ResultsResults

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Importance of incubation step:-Importance of incubation step:-During the test performance incubation

time and mentioned temperature is must required For the proper binding between antigen and antibody and also binding with conjugate and color development of substrate.

Importance of Washing :- For the removal of any unbound Antibody/Antigen proper washing and taping is required other wise we get the incorrect result.

So incubation & washing is much important for good results.

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Elisa PlateElisa Plate

Microtitre wellsGenerally 96

wellsMarked on one

side alphabeticallyNumerically on

the other sideComes with the

kit

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Using a clean Pipette , add 100 µL of diluted serum sample (Dilute the sera to be tested 1:100 in the sample diluents) to each well. Incubate 1 hour at 37°C.

TEST PERFORMANCETEST PERFORMANCE

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Measures the absorbance at 450nm With the help of ELISA READER.

Calculate the absorbance for each sample and reference.

We used Ascent Software for Calculation of the result

ELISA PLATE READY FOR READING

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Advantages of ELISAAdvantages of ELISA

Reagents are relatively cheap & have a long shelf life

ELISA is highly specific and sensitiveNo 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.

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Disadvantages of ELISADisadvantages 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

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LimitationsLimitations

•Results may not be absolute

•Antibody must be available

•Concentration may be unclear

•False positive possible

•False negative possible

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APPLICATIONS OF ELISAAPPLICATIONS OF ELISA

detection of Mycobacterium antibodies in tuberculosis

detection of rotavirus in feces

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

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APPLICATIONS OF ELISAAPPLICATIONS OF ELISA

1- Hormones 7- Vaccine Quality Control

2- Proteins 8- FOR GMO (Genetically modified organism)

3- Infectious Agent ( Viral, Bacterial, Parasitic, Fungal )

9- For Rapid Test

4- Drug Markers 10- IgG, IgM, IgA

5- Tumor Markers 11- In New Born Screening

6- Serum Proteins 12- In Clinical Research

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Sensitivity of various immunoassays

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Equipments for performing the ELISA test

PipettesIncubator

ELISA reader

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ELISA READERELISA READER

THERMOLAB SYSTEM (USA)

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RadioimmunoassaRadioimmunoassay y

]

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RadioimmunoassayRadioimmunoassay (RIA) is a very sensitive (RIA) is a very sensitive  in vitro assay assay technique used to measure concentrations  technique used to measure concentrations of antigens (for example, hormone levels in theblood) by use of antigens (for example, hormone levels in theblood) by use of antibodies.of antibodies.

The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy

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.

INTRODUCTIONINTRODUCTION

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To perform a radioimmunoassay, a known quantity of To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. gamma-radioactive isotopes of iodine attached to tyrosine.

This radiolabeled antigen is then mixed with a known This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two amount of antibody for that antigen, and as a result, the two specifically bind to one another. specifically bind to one another.

Then, a sample of serum from a patient containing an Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. unknown quantity of that same antigen is added.

This causes the unlabeled (or "cold") antigen from the serum This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for to compete with the radiolabeled antigen ("hot") for antibody binding sites. As theconcentration of "cold" antigen antibody binding sites. As theconcentration of "cold" antigen is increased, more of it binds to the antibody, displacing the is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. the supernatant is measured using a gamma counter.

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Principle of Principle of RadioimmunoassayRadioimmunoassayPrinciple: Uses an immune reaction

[Antigen – Antibody reaction] to estimate a ligand

Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ab*

◦ Unbound Ag* and Ag washed out ◦ Radioactivity of bound residue measured◦ Ligand conc is inversely related to

radioactivity

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

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Advantages & Advantages & Disadvantages of RIADisadvantages of RIA

Advantages◦Highly specific: Immune reactions are

specific◦High sensitivity : Immune reactions are

sensitiveDisadvantages

◦Radiation hazards: Uses radiolabelled reagents

◦Requires specially trained persons◦Labs require special license to handle

radioactive material◦Requires special arrangements for

Requisition, storage of radioactive material radioactive waste disposal.

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Requirements for RIARequirements for RIA

1. Preparation & characterisation of the Antigen [Ligand to be analysed]

2. Radiolabelling of the Antigen3. Preparation of the Specific

Antibody4. Development of Assay

System

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Preparation & Preparation & Radiolabelling of the Radiolabelling of the Antigen Antigen

Antigens prepared by.. ◦Synthesis of the molecule ◦ Isolation from natural sources

Radiolabelling [Tagging procedure]◦ 3 H 14 C 125 I are used as radioactive

tags◦Antigens are tagged to 3 H 14 C 125

◦Tagging should NOT affect Antigenic specificity & Antigenic activity !

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Preparation of the Specific Preparation of the Specific AntibodyAntibody

Antigen injected intradermally into rabbits or guinea pigs antibody production

Antibodies recovered from the serumSome ligands are not Antigenic

◦ Hormones, Steroids, Drugs HAPTENS◦ Eg: Gastrin, Morphine, ◦ Haptens conjugated to albumin antigenic

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Development of the Assay Development of the Assay SystemSystem

A crucial step is separation of unbound antigens

This achieved by binding the antibodies to the microtitre well surface [Solid phase RIA]

Antigens bound to the fixed antibodies remain stuck to the inner surface

Decanting & washing the well removes unbound antigens

Other techniques of separation: Centrifugation

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Assay ProcedureAssay ProcedureAdd known amounts of the test sample +

labelled antigen into the microtitre wells Incubate allow the reaction to reach

completionDecant & wash contents of the well

removes all unbound antigensRadioactivity remaining in the Microtitre

wells measured by a Counter [GM counter , Scintillation counter etc]

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

Sensitive to very low conc of antigens 48

-Radio-isotopes,- Enzymes

FluorescentChemi-luminescent probesMetal tags

Antibodies: types of Antibodies: types of labellinglabelling

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RRadioadioiimmunommunoaassayssay(RIA(RIA))

Advantages◦ Flexibility◦ Sensitivity◦ Size

Disadvantages◦ Toxicity◦ Shelf life◦ Disposal costs

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Radioimmunoassay is widely-used because of its great sensitivity.

Using antibodies of high affinity, it is possible to detect a few picograms (10−12 g) of hormone in the tube.

The greater the specificity of the antiserum, the greater the specificity of the assay

Advantages

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The main drawbacks to radioimmunoassay are the expense and hazards of preparing and handling the radioactive antigen.

Both 125I or 131I emit gamma radiation that requires special counting equipment

The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4).

limitations

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INSTRUMENTATIONINSTRUMENTATION

Incubate tissue withradioactive ligand

Expose to filmor emulsion

Isotope will emitradiation (usually beta)

Radiation will hit silver grains in emulsion and expose them

Autoradiography54

INSTRUMENTATIONINSTRUMENTATION

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REFERENCESREFERENCES

 Engvall E, Perlman P (1971). "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G". Immunochemistry 8 (9): 871–4. Gofflot; El (2004). 

 Journal of Immunoassay and Immunochemistry 25 (3): 241–58. Retrieved 13 December 2012.

Kuhar M, Yamamura HI (Jul 1976). "Localization of cholinergic muscarinic receptors in rat brain by light microscopic radioautography". Brain Res. 110 (2): 229–43.

 E. Rutherford and H. Geiger (1908) "An electrical method of counting the number of α particles from radioactive substances," Proceedings of the Royal Society (London), Series A, vol. 81, no. 546,

 A Handbook of Radioactivity Measurements Procedures, 2nd edition: (Report No. 58), National Council on Radiation Protection and Measurements (NCRP) , 1985 ISBN 0-913392-71-5,pages 30-31

WWW.GOOGLE.COM/IMAGES

WWW.SLIDESHARE.NET

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