ELISA

DR. Arvind Kulkarni

OBJECTIVES OF IMMUNODIAGNOSISOBJECTIVES OF IMMUNODIAGNOSIS

SPEED OF DIAGNOSIS

SIMPLICITY OF THE TESTS

COST EFFECTIVENESS

CONVENIENCE OF ITS USE

ACCURACY

CRITERIA FOR SELECTIONCRITERIA FOR SELECTION

APPLICATION AT LABORATORY OR FIELDAPPLICATION AT LABORATORY OR FIELD INFRASTRUCTUREINFRASTRUCTURE EXPERTISEEXPERTISE SENSITIVITYSENSITIVITY SPECIFICITYSPECIFICITY PRODUCTIVITYPRODUCTIVITY EASE OF PERFORMANCEEASE OF PERFORMANCE FIELD FRIENDLINESSFIELD FRIENDLINESS AUTOMATIONAUTOMATION COST EFFECTIVENESSCOST EFFECTIVENESS

ENZYME IMMUNOASSAYENZYME IMMUNOASSAY

COMMONLY KNOWN AS ENZYME LINKED

IMMUNOSORBENT ASSAY (ELISA)

USES AN ENZYME IN PLACE OF FLUORESCENT DYES OR RADIO ISOTOPES

HAS BECOME TEST OF CHOICE DUE TO ITS SIMPLE PROTOCOL

ELISAELISA PRINCIPLE

IT ALSO MAKES USE OF ANTIGEN-ANTIBODY

COMPLEX.

THE ANTIBODY IS CONJUGATED WITH AN ENZYME.

THE FORMATION OF THE ANTIGEN-ANTIBODY

COMPLEX IS VISUALIZED BY THE PROPERTY OF

FORMATION OF A COLOURED COMPLEX BY THE

ENZYME WHEN THE SUBSTRATE IS ADDED.

ELISAELISA

ENZYMES USED

HORSERADISH PEROXIDASE (HRPO) The most common enzyme used in the

assay.

ALKALINE PHOSPHATASE PENCILLINASE.

ELISAELISAThe Basic Principle

IMMOBILISATION OF EITHER Ag / Ab.IMMOBILISATION OF EITHER Ag / Ab.

ADD THE TEST Ab. / AgADD THE TEST Ab. / Ag

ADD THE ENZYME CONJUGATEADD THE ENZYME CONJUGATE

ADD THE SUBSTRATEADD THE SUBSTRATE

READ THE COLOUR REACTIONREAD THE COLOUR REACTION

ELISAELISA

MAJOR TYPES

DIRECTDIRECT

INDIRECTINDIRECT

SANDWICHSANDWICH

COMPETITIONCOMPETITION

Direct ELISADirect ELISA

PRINCIPLEAntigen attached to the solid phase is reacted directly with an enzyme

labeled antiserum.

DISADVANTAGEThe sera raised against different

antigens have all to be labeled. Poor assay if used to detect antigen from ‘crude’ samples.

Indirect ELISAIndirect ELISA PRINCIPLE

SIMILAR TO DIRECT ELISA EXCEPT THAT THE SECOND

ANTIBODY IS CONJUGATED WITH THE ENZYME.

ADVANTAGES

ONLY A SINGLE ANTI-SPECIES ENZYME CONJUGATE IS

NEEDED TO TITRATE ANTISERA FROM MANY ANIMALS OF

A SINGLE SPECIES.

SANDWICH ELISASANDWICH ELISA

TWO TYPES

DIRECT SANDWICH ELISADIRECT SANDWICH ELISA

INDIRECT SANDWICH ELISAINDIRECT SANDWICH ELISA

DIRECT SANDWICH ELISADIRECT SANDWICH ELISA PRINCIPLE

THIS IS SIMILAR TO DIRECT ELISA EXCEPT THAT THE AB. IS ATTACHED TO THE SOLID PHASE AT A CONSTANT DILUTION.

ANTIGEN IS THEN ADDED.

BOUND ANTIGEN IS THEN DETECTED BY THE ADDITION OF ENZYME LABELED AB. SPECIFIC FOR THE CAPTURED ANTIGEN.

INDIRECT SANDWICH ELISAINDIRECT SANDWICH ELISA

PRINCIPLESIMILAR TO DIRECT SANDWICH

EXCEPT THAT THE SECOND AB. IS PRODUCED IN A DIFFERENT SPECIES FROM THE TRAPPING AB.

ADVANTAGE MANY SECOND ANTIBODIES MAY BE

TITRATED WITH A SINGLE CONJUGATE.

COMPETITION ELISA

FOUR VARIETIES OF COMPETITION ELISA

DIRECT ANTIBODY COMPETITIONDIRECT ANTIBODY COMPETITION DIRECT ANTIGEN COMPETITIONDIRECT ANTIGEN COMPETITION INDIRECT ANTIBODY INDIRECT ANTIBODY

COMPETITIONCOMPETITION INDIRECT ANTIGEN INDIRECT ANTIGEN

COMPETITIONCOMPETITION

DIRECT ANTIBODY COMPETITIONADSORPTION OF ANTIGEN TO THE SOLID PHASE.

SPECIFIC AB. LABELED WITH ENZYME IS ADDED.

THE LABELED AB. IS MIXED WITH ANOTHER AB.(COMPETING AB.) WHICH IS ABLE TO REACT WITH THE SOLID PHASE BOUND ANTIGEN.

THE COMPETING AB. ADDED AS A DILUTION RANGE, THUS REPLACES ALL OR SOME OF THE PRE-TITRATED CONJUGATED AB.

DEVELOPMENT OF THE COLOUR BY ADDING SUBSTRATEDECREASE IN THE COLOUR EXPECTED.

DIRECT ANTIGEN COMPETITION

THIS IS SIMILAR TO ANTIBODY COMPETITION EXCEPT THAT THE COMPETING SUBSTANCE FOR THE PRE-TITRATED CONJUGATED ANTIBODY IS ANTIGEN.

INDIRECT ANTIBODY COMPETITIONINDIRECT ANTIBODY COMPETITION

Essentially the same as the indirect ELISA except that a competing Ab. is added to the solid phase antigen either before or simultaneously with pre-titrated specific Ab..

The competing Ab. must be from a different The competing Ab. must be from a different

species from the pre-titrated Ab. since the anti-species from the pre-titrated Ab. since the anti-species conjugate must not react with both. species conjugate must not react with both.

If the competing Ab. is able to bind to the If the competing Ab. is able to bind to the antigen then it prevents the pre-titrated Ab. antigen then it prevents the pre-titrated Ab. reacting and this is observed as a decrease in reacting and this is observed as a decrease in the expected colour as compared to controls the expected colour as compared to controls without competitor.without competitor.

INDIRECT ANTIGEN COMPETITION

Similar to indirect ELISA where Ab. is Similar to indirect ELISA where Ab. is pre-titrated against the solid phase pre-titrated against the solid phase bound antigen by the use of anti-species bound antigen by the use of anti-species conjugate, which is challenged by the conjugate, which is challenged by the addition of dilution ranges of antigen in addition of dilution ranges of antigen in the liquid phase. the liquid phase.

The competition is reflected by a The competition is reflected by a decrease in the expected colour decrease in the expected colour obtained without competitor.obtained without competitor.

COMPARATIVE SENSITIVITY OF VARIOUS IMMUNOASSAYS*

ASSAY SENSITIVITY(g ANTIBODY N/ml)

Precipitation reaction in fluids 3 -20

Immunodiffusion 3 – 20

Immunoelectrophoresis 3 – 20

Passive agglutination 0.001 – 0.01

Immunofluorescence 1.0

Agglutination inhibition 0.001 – 0.01

Radioimmuno assay 0.0001 – 0.001

ELISA 0.0001-0.001

* Source : Immunology by J. Kuby ( 1992)