ELISA :

ELISA ELISA…… Enzyme-linked immunosorbent assay (or) Enzyme

immunoassay (EIA) It is a biochemical technique used mainly in immunology
to detect the presence of an antibody or an antigen in a sample.

Cont….. :
Cont….. ELISA, or enzyme-linked immunosorbent assay, are quantitative
immunological procedures in which the Ag- Ab reaction is monitored by
enzyme measurements. (or) An enzyme linked immunosorbent assay (ELISA)
is a test performed in an immunology laboratory to determine levels of protein
in a biological sample

Why known as ......? Enzyme Linked Immunosorbent Assay :

Why known as ......? Enzyme Linked Immunosorbent Assay 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.

HISTORY :
HISTORY Prior to the development of the EIA/ELISA, the only option for
conducting an immunoassay was radioimmunoassay, a technique using
radioactively-labeled antigens or antibodies. Radioimmunoassay was first
described in a paper by Rosalyn Sussman Yalow and Solomon Berson
published in 1960

Cont….. :
Cont….. The term ELISA was first used by Engvall & Perlma in 1971.

What is an ELISA test ? :

What is an ELISA test ? An enzyme-linked immunosorbent assay, or ELISA,
test is a type of medical diagnostic test used to detect whether a certain
antibody or antigen is present in a patient. It can be useful for a range of
different purposes relating to immunology, such as disease testing and virus
testing For example An ELISA HIV test may be administered to determine
whether a patient has been infected with HIV antibodies. ELISA tests are
sometimes also used in testing for illegal drug use. An ELISA test can also
help detect allergic reactions in food products like nuts or dairy items.

What is the use of an ELISA test? :

What is the use of an ELISA test? ELISA tests are widely utilized to detect
substances that have antigenic properties, primarily proteins The substances
detected by ELISA tests include hormones, bacterial antigens and antibodies.

How does an ELISA test work? :

How does an ELISA test work? 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 (will latch on to) the substance of interest For
example……… human chorionic gonadotropin (HCG), the commonly
measured protein which indicates pregnancy. A mixture of purified HCG
linked (coupled) 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.

BASIC PRINCIPLE OF ELISA :

BASIC PRINCIPLE OF ELISA Use 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.

Slide 13:
Substrate Different antigens in sample

ELISA Qualitative/Quantitative :
ELISA Qualitative/Quantitative Qualitative determines antigen or antibody is
present or absent Quantitative determines the quantity of the antibody Titer
The highest dilution of the specimen usually serum which gives a positive
reaction in the test

Common terms used in ELISA :

Common terms used in ELISA

Antigen :
Antigen A substance that when introduced into the body stimulates the
production of an antibody. An antigen is an organic compound…….. protein,
polysaccharide or glycolipid Antigens include Toxins, Bacteria, Foreign blood
cells, Microorganisms Allergens. Viruses Etc.

Antibodies :
Antibodies Antibodies (also known as immunoglobulins, abbreviated Ig) are
gamma globulin proteins that are found in blood or other bodily fluids of
vertebrates, and are used by the immune system to identify and neutralize
foreign objects, such as bacteria and viruses. (OR) proteins produced by the
immune system which help defend against antigens

Cont… :
Cont… Antibodies are produced by a kind of white blood cell called a plasma
cell. They are typically made of basic structural units—each with two large
heavy chains and two small light chains

Structure of Antibody :
Structure of Antibody

Enzyme substrate :
Enzyme substrate In biochemistry, a substrate is a molecule upon which an
enzyme acts. In the case of a single substrate, the substrate binds with the
enzyme active site, and an enzyme-substrate complex is formed Initially the
substrate should be colorless After degradation by the enzyme it should be
strongly colored or fluorescent.

Cont…. :
Cont…. Substrates are critical for the detection and visualization steps of an
ELISA. The choice of substrate depends on a number of factors, including the
degree of sensitivity required, different instrumentation and filter requirements,
safety issues, and choice of enzyme. Most immunoassays use
antibodies/proteins conjugated to enzymes in order to generate signal through
the catalytic properties of the enzyme.

Cont…. :
Cont…. Horseradish peroxidase (HRP) and alkaline phosphatase (AP). are
the two widely used enzymes employed in ELISA assay It is important to
consider the fact that some biological materials have high levels of
endogenous enzyme activity HRP has been shown historically to be more
sensitive than AP primarily due to its faster catalytic rate.

Enzyme substrate :
Enzyme substrate

Microtiter plate :
Microtiter plate A Microtiter plate (spelled Microtitre outside the United States
and Canada) or microplate is a flat plate with multiple "wells" used as small
test tubes. The microplate has become a standard tool in analytical research
and clinical diagnostic testing laboratories. A very common usage is in the
enzyme-linked immunosorbent assay (ELISA) and forms the basis of most
modern medical diagnostic testing in humans and animals

Materials Needed :
Materials Needed Testing sample Antibody (1st, 2nd) / Antigen Polystyrene
microtiter plate Blocking buffer Washing buffer Substrate Enzyme

Equipment for performing the ELISA test :

Equipment for performing the ELISA test

ELISA Plate 96 Wells :

ELISA Plate 96 Wells Made from clear polystyrene, these ELISA plate is high
grade for optic transmission. Arranged in the standard 8 x12 well
configuration, available as flat bottom wells. The ELISA plate fits all standard
optical readers. Marked on one side alphabetically Numerically on the other
side

Slide 30:
ELISA Plate 96 Wells

ELISA reader:
ELISA reader A micro ELISA reader includes a housing, a tray longitudinally
movably provided in the housing, a carrier transversally movably mounted on
the tray, a light source