Enzyme Linked Immunosorbent Assay (ELISA):

Enzyme Linked Immunosorbent Assay (ELISA) By S.Abishegam II-Msc

INTRODUCTION:
INTRODUCTION ELISA, or enzyme-linked immunosorbent assay, are quantitative immunological
procedures in which the Ag-Ab reaction is monitored by enzyme measurements. It is a powerful
method for detection of small concentration of substance such as proteins hormones or metabolites
It is useful & powerful method in estimating ng/mL to pg/mL ordered materials in the solution
PRINCIPLE:
PRINCIPLE 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.
HISTORY:
HISTORY Before the development of the ELISA,RIA technique is used by radioactively-labeled
antigens or antibodies it identify the specific antigen or antibody is present in the sample. It was
developed by Rosalyn Sussman Yalow and Solomon Berson published in 1960.It is a poses a
potential health threat . so they introduced non-radioactive signal in place of the radioactive signal.
By using these technique in 1971, it was developed by Peter Perlmann and Eva Engvall . For their
invention they received the German scientific award of the "Biochemische Analytik" in 1976
TYPES:
TYPES
COMPETITIVE ELISA :
COMPETITIVE ELISA Used to determine small molecule antigens.(T3,T4,progesterone etc.)
antibody coated microwell serum antigen and labelled antigen added together--competition.
ADVANTAGES:
ADVANTAGES The major advantage of a competitive ELISA is the ability to use crude or impure
samples and still selectively bind any antigen that may be present.
INDIRECT ELISA:
INDIRECT ELISA The indirect, two-step method uses a labeled secondary antibody for detection.
First, a primary antibody is incubated with the antigen. This is followed by incubation with a
labeled secondary antibody that recognizes the primary antibody.
Advantages of indirect detection Advantages :
Advantages of indirect detection Advantages Wide variety of labeled secondary antibodies are
available commercially. Versatile, since many primary antibodies can be made in one species and
the same labeled secondary antibody can be used for detection. Immunoreactivity of the primary
antibody is not affected by labeling.
DISADVANTAGES:
DISADVANTAGES Cross-reactivity might occur with the secondary antibody, resulting in
nonspecific signal. An extra incubation step is required in the procedure.
SANDWICH ELISA:
SANDWICH ELISA Plate is coated with a capture antibody Sample is added, and any antigen
present binds to capture antibody Detecting antibody is added, and binds to antigen Enzyme-linked
secondary antibody is added, and binds to detecting antibody Substrate is added, and is converted
by enzyme to detectable form.
ADVANTAGES:
ADVANTAGES High specificity, since two antibodies are used the antigen/analyte is specifically
captured and detected Suitable for complex samples, since the antigen does not require purification
prior to measurement Flexibility and sensitivity, since both direct and indirect detection methods
can be used
ELISA Reverse method & device (ELISA-R m&d ):
ELISA Reverse method & device (ELISA-R m&d ) A new technique uses a solid phase made up of
an immunosorbent polystyrene rod with 8-12 protruding ogives . The entire device is immersed in a
test tube containing the collected sample and the following steps (washing, incubation in conjugate
and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard
microplates pre-filled with reagents.
ADVANTAGES:
ADVANTAGES The ogives can each be sensitized to a different reagent, allowing the simultaneous
detection of different antibodies and/or different antigens for multi-target assays; The sample
volume can be increased to improve the test sensitivity in clinical (blood, saliva, urine), food (bulk
milk, pooled eggs) and environmental (water) samples; One ogive is left unsensitized to measure
the non-specific reactions of the sample; The use of laboratory supplies for dispensing sample
aliquots, washing solution and reagents in microwells is not required, facilitating the development
of ready-to-use lab-kits and on-site kits.
ELISPOT:
ELISPOT The Enzyme-linked immunosorbent spot ( ELISPOT ) assay is a common method for
monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in
1983 . It is used to detect cytokines secreted from different cells.
PowerPoint Presentation:
SDASD
Enzymes Used in Elisa :
Enzymes Used in Elisa Horseradish peroxidase (most commonly used) Alkaline Phosphatase B-
galactosidase Lactoperoxidase Tetra Methyl benzidine
SUBSTRATES:
SUBSTRATES 1. PNPP ( p -Nitrophenyl Phosphate, Disodium Salt); When alkaline phosphatase
and PNPP are reacted, a yellow water-soluble reaction product is formed. This reaction product
absorbs light at 405nm. Safer to use — contains a non-mercury, non-azide preservative
OPD (o-Phenylenediamine ):
OPD (o-Phenylenediamine ) OPD is a water-soluble substrate for horseradish peroxidase (HRP) .
produces a yellow-orange product. detectable at 492nm by ELISA plate readers
TMB (3,3′,5,5′-tetramethylbenzidine ):
TMB (3,3′,5,5′-tetramethylbenzidine ) TMP produce blue colour when it react with horseradish
peroxidase (Amax = 370nm and 652nm) that changes to yellow (Amax = 450nm) upon addition of
a sulfuric or phosphoric acid stop solution. Slow, fast and ultrasensitive varieties of the most
popular substrate for ELISA
ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt):
ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) It is a water-
soluble HRP substrate that yields a green end product upon reaction with peroxidase. The green
product has two absorbance peaks, 410nm and 650nm. ABTS is less sensitive than OPD and TMB
in ELISA applications. It is less readily oxidized, and its color development is slower
(approximately 20 minutes).
Available kits in practice:
Available kits in practice Hepatitis A ( HAV) Hepatitis B surface Ag ELISA – anti HBs – detecting
antibodies hepatitis B antigen ELISA HBc – detecting hepatitis c antigen. ELISA HBe – detecting
HB e antigen in sample
PowerPoint Presentation:
Hepatitis D and E detecting kits HIV detecting kits Rubella ELISA Toxoplasma –ELISA CMV-
ELISA HSV IgG herpes Epstein Barr virus detection
ELISA PLATE:
ELISA PLATE 96 well plate Plate Made from clear polystyrene, is high grade for optic transmission
on which protein can be adsorbed (bind) easily C Special buffer used that will notUsually done
overnight at 4 denature Ab and maximize binding
APPLICATIONS:
APPLICATIONS Detecting infections such as sexually-transmitted agents like HIV, syphilis and
chlamydia hepatitis B and C Toxoplasma gondii Detecting allergens in food and house dust
Measuring"rheumatoid factors" and other autoantibody in autoimmune diseases like Lupus
erythematosus Measuring toxins in contaminated food
Screening donated blood for evidence of viral contamination by :
Screening donated blood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-
HIV antibodies) hepatitis C (presence of antibodies) hepatitis B (testing for both antibodies and a
viral antigen) Measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of
ovulation) TSH, T3 and T4 (for thyroid function)
Cont…:
Cont… ELISA is used to detect many bacterial and viral antigens, including human
immunodeficiency virus (HIV), malaria, cholera, measles, and mumps The ELISA has been used as
a diagnostic tool in medicine and plant pathology, as well as a quality control check in various
industries

Virtual Immunology Lab Glossary

Aliquot - a portion of a solution

Antibody - a type of protein called an immunoglobulin found in the blood that is produced by
immune cells in response to the presence of a foreign particle (antigen). Antibodies are specific to
each different type of foreign particle.

 Primary : first antibody used in an immunoassay to detect the foreign particle; in this case,
we are testing to see if the serum from the patients contains primary antibodies to SLE.
 Secondary antibody: the second antibody used in an immunoassay that detects the primary
antibody. Note, this antibody must be made in a different species (rabbit, donkey, horse)
than the primary antibody, in order to recognize the primary antibody as "foreign". In this
case, we are using HRP-tagged rabbit anti-human antibodies as our secondary antibody.

Antibody-Enzyme Conjugate - antibodies (usually secondary antibodies) are "tagged" or joined to

enzymes through a chemical process. Now the antibody is carrying an enzyme, so wherever the
secondary antibody binds, the enzyme is present also.

Antigen - any substance (usually a protein or carbohydrate) that induces the production of
antibodies by immune cells. Usually is a substance that is "foreign" to the host.

Antigenic Site - the part of the antigen that is recognized by the antibody

Assay - an examination or test

Centrifuge - an instrument used to spin tubes to separate solutions into different phases (liquid or
solid). In this example, the red blood cells and other blood components are "spun out" of the blood,
creating two layers in the tube: components below and serum above.

ELISA - Enzyme-Linked Immunosorbent Assay; this is an assay that uses an enzyme linked to an
antibody. In this experiment, a colorless substrate is turned into a colored product by the bound
enzyme. The amount of activity of this enzyme (as determined by detection of the amount of
colored product) is used as a measurement of the amount of bound antibody.

Enzyme - a protein that acts as a agent or catalyst to induce chemical changes in other substances.
An enzyme can be used repeatedly, because it is unchanged by the process.

Eppendorf® pipette - an instrument in the lab used to dispense small volumes of liquids
(milliliters or microliters usually)

HRP - horseradish peroxidase; an enzyme used to stimulate the conversion of the colorless
substrate into a colored product in this exercise

Humoral Immunity - this refers to the resistance that results from the presence of the specific
antibody within the serum

Nanometers (nm) - a unit of measurement, in this case, one-billionth of a meter (10-9 m)

Pipettor - a piece of equipment used to transfer a specified volume of a liquid

Sera/Serum - a clear watery fluid obtained after removing blood cells and other components from
blood by centrifugation that will contain antibodies

Serial Dilutions - when the samples are diluted in a specific, systematic manner (1:2; 1:10; 1:100)
to determine the concentration of a sample or the sensitivity of an assay

Spectrophotometer - an instrument for measuring the intensity of light of a specific wavelength

transmitted by a substance. In this example, it will determine the amount of a reaction product
which is absorbing the light in a solution.

Substrate - any substance on which an enzyme can act. In this example, HRP (the enzyme) will
interact with a substrate called ABTS (2,2'-azinobis-3-ethylbenzothiazoleine-6-sulfonic acid) to
produce a yellow solution.

Systemic Lupus Erythematosus (SLE) - Lupus is believed to be an autoimmune disease. The

cause of lupus has yet to be determined. The immune system is your body's defense mechanism
against foreign particles called antigens. Sometimes the immune system loses the ability to
distinguish between its own body components and antigens. Instead of fighting antigens, the
antibodies mistakenly fight the body's own cells. This is referred to as an autoimmune response
(auto means self) and the body mistakenly makes autoantibodies. A systemic disease is one in which
several different parts of the body may be affected. In systemic lupus, these include the skin,
kidneys, nervous system, lungs, heart and/ or blood-forming organs.

Titer - the concentration of a substance in a solution. For instance, the amount of a specific
antibody in the serum.