ELISA

ENZYME-LINKED IMMUNO-
SORBENT ASSAY
 INTRODUCTION

• The enzyme-linked immuno-sorbent assay (ELISA). An ELISA test uses

components of the immune system and chemicals to detect immune responses
in the body. The ELISA test involves an enzyme (a protein that catalyzes a
biochemical reaction). It also involves an antibody or antigen (immunologic
molecules).
 INTRODUCTION

• ELISA is a popular format of a "wet-lab" type analytic biochemistry assay that

uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a
substance, usually an antigen, in a liquid sample or wet sample.

• The ELISA has been used as a diagnostic tool in medicine and plant pathology,
as well as a quality-control check in various industries.
 Use of an ELISA test

• ELISA tests are widely utilized to detect substances that have antigenic
properties, primarily proteins (as opposed to small molecules and ions such
as glucose and potassium). The substances detected by ELISA tests include
hormones, bacterial antigens and antibodies
 Principle of ELISA test

• There are variations of the ELISA test, but the most basic type consists of an
antibody attached to a solid surface. This antibody has affinity for the
substance of interest, E.g. human chorionic gonadotropin (HCG), the
commonly measured protein which indicates pregnancy. A mixture of purified
HCG linked to an enzyme and the test sample (blood, urine, etc) are added to
the test system. If no HCG is present in the test sample, then only HCG with
linked enzyme will bind. The more HCG which is present in the test sample, the
less enzyme linked HCG will bind. The substance the enzyme acts on is then
added, and the amount of product measured in some way, such as a change in
color of the solution.
 Principle of ELISA test
• As a wet lab analytic biochemistry assay, ELISA involves detection of an "analyte" (i.e. the specific
substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a
method that continues to use liquid reagents during the "analysis" (i.e. controlled sequence of
biochemical reactions that will generate a signal which can be easily quantified and interpreted as a
measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction
chamber or well needed to keep the reactants contained; It is opposite to "dry lab" that can use dry
strips - and even if the sample is liquid (e.g. a measured small drop), the final detection step in
"dry" analysis involves reading of a dried strip by methods such as reflectometry and does not need
a reaction containment chamber to prevent spillover or mixing between samples.
 Types of ELISA

Four kinds of ELISA :

1. Direct ELISA
2. Indirect ELISA
3. Competitive ELISA
4. Sandwich ELISA
 Direct ELISA

• Direct ELISAs involve attachment of the antigen to the solid phase, followed
by an enzyme-labeled antibody. This type of assay generally makes
measurement of crude samples difficult, since contaminating proteins
compete for plastic binding sites.
 Indirect ELISA

• Indirect ELISAs also involve attachment of the antigen to a solid phase, but
in this case, the primary antibody is not labeled. An enzyme-conjugated
secondary antibody, directed at the first antibody, is then added. This
format is used most often to detect specific antibodies in sera.
 Competitive ELISA

• The third type of ELISA is the Competition Assay, which involves the
simultaneous addition of 'competing' antibodies or proteins. The decrease in
signal of samples where the second antibody or protein is added gives a
highly specific result.
 Sandwich ELISA

• Sandwich ELISAs involve attachment of a capture antibody to a solid phase

support. Samples containing known or unknown antigen are then added in a
matrix or buffer that will minimize attachment to the solid phase. An
enzyme-labeled antibody is then added for detection.
 Sandwich ELISA
• The ELISA method is a benchmark for quantization of pathological antigens
and there are indeed many variations to this method. ELISAs are adaptable
to high-throughput screening because results are rapid, consistent and
relatively easy to analyze. The best results have been obtained with the
sandwich format, utilizing highly purified, pre matched capture and detector
antibodies. The resulting signal provides data which is very sensitive and
highly specific.
 Applications of ELISA
• ELISA Test Applications in Antibody Concentration Determination
• ELISA Test Applications in Monoclonal Antibody Screening
• ELISA Test Applications in Virus test (HIV, West Nile Virus, NDV)
• ELISA Test Applications in Home Pregnancy Test
• ELISA Test Applications in Food industry (detecting potential food allergens
such as milk, peanuts, walnuts, almonds and eggs)

• ELISA Test can be used to diagnostic diseases

THANKS