ELISA

DR. Arvind Kulkarni

OBJECTIVES OF IMMUNODIAGNOSIS

 SPEED OF DIAGNOSIS

 SIMPLICITY OF THE TESTS

 COST EFFECTIVENESS

 CONVENIENCE OF ITS USE

 ACCURACY
CRITERIA FOR SELECTION
 APPLICATION AT LABORATORY OR FIELD
 INFRASTRUCTURE
 EXPERTISE
 SENSITIVITY
 SPECIFICITY
 PRODUCTIVITY
 EASE OF PERFORMANCE
 FIELD FRIENDLINESS
 AUTOMATION
 COST EFFECTIVENESS
 ENZYME 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
 ELISA

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.
 ELISA
ENZYMES USED

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

 ALKALINE PHOSPHATASE
 PENCILLINASE.
 ELISA
The Basic Principle
IMMOBILISATION OF EITHER Ag / Ab.

ADD THE TEST Ab. / Ag

ADD THE ENZYME CONJUGATE

ADD THE SUBSTRATE

READ THE COLOUR REACTION
 ELISA

MAJOR TYPES
 DIRECT
 INDIRECT
 SANDWICH
 COMPETITION
 Direct ELISA

 PRINCIPLE
Antigen attached to the solid phase is
reacted directly with an enzyme
labeled antiserum.

 DISADVANTAGE
The sera raised against different
antigens have all to be labeled.
Poor assay if used to detect antigen
from ‘crude’ samples.
 Indirect 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 ELISA
TWO TYPES

 DIRECT SANDWICH ELISA

 INDIRECT SANDWICH ELISA

DIRECT 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 ELISA
 PRINCIPLE
SIMILAR 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 COMPETITION

 DIRECT ANTIGEN COMPETITION
 INDIRECT ANTIBODY COMPETITION
 INDIRECT ANTIGEN COMPETITION
DIRECT ANTIBODY COMPETITION
ADSORPTION 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

SUBSTRATE
DECREASE 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 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

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

If the competing Ab. is able to bind to the

antigen then it prevents the pre-titrated Ab.
reacting and this is observed as a decrease in
the expected colour as compared to controls
without competitor.
INDIRECT ANTIGEN COMPETITION

Similar to indirect ELISA where Ab. is

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

The competition is reflected by a

decrease in the expected colour
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)