Enzyme-linked

Immunosorbent Assay
ELISA
Mehwish Hamid

Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is a

biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in
a sample.
 INTRODUCTION

Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay

(EIA), is a biochemical technique used mainly in immunology to detect the presence of an
antibody or an antigen in a 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. In
simple terms, in ELISA, an unknown amount of antigen is affixed to a surface, and then a
specific antibody is applied over the surface so that it can bind to the antigen. This antibody
is linked to an enzyme, and in the final step a substance is added that the enzyme can
convert to some detectable signal, most commonly a colour change in a chemical substrate.

ELISA methods are fundamental tools in the pharmaceutical industry with applications in
drug discovery, animal studies, and clinical trials. Traditional ELISA typically involves
chromogenic reporters and substrates that produce some kind of observable color change
to indicate the presence of antigen or analyte. Newer ELISA-like techniques utilize
fluorogenic, electrochemiluminescent, and real-time PCR reporters to create quantifiable
signals. These new reporters can have various advantages including higher sensitivities
and multiplexing.[1][2] In technical terms, newer assays of this type are not strictly ELISAs,
as they are not "enzyme-linked" but are instead linked to some non-enzymatic reporter.
However, given that the general principles in these assays are largely similar, they are often
grouped in the same category as ELISAs.

It is a useful and powerful method in estimating ng/ml to pg/ml ordered materials in the
solution, such as serum, urine, sperm and culture supernatant (Savige, 1998). ELISA has
been widely used in the life science researches (Ma, 1994; 2004).

HISTORY

Before the development of the ELISA, the only option for conducting an immunoassay was
radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. In
radioimmunoassay, the radioactivity provides the signal, which indicates whether a
specific antigen or antibody is present in the sample. Radioimmunoassay was first
described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.

Because radioactivity poses a potential health threat, a safer alternative was sought. A
suitable alternative to radioimmunoassay would substitute a non-radioactive signal in
place of the radioactive signal. When enzymes (such as peroxidase) react with appropriate
substrates (such as ABTS or 3,3’,5,5’-Tetramethylbenzidine), a change in color occurs,
which is used as a signal. However, the signal has to be associated with the presence of
antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody.
This linking process was independently developed by Stratis Avrameas and G.B. Pierce.
Since it is necessary to remove any unbound antibody or antigen by washing, the antibody
or antigen has to be fixed to the surface of the container; i.e., the immunosorbent has to be
prepared. A technique to accomplish this was published by Wide and Jerker Porath in 1966.

In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton
Schuurs and Bauke van Weemen in The Netherlands independently published papers that
synthesized this knowledge into methods to perform EIA/ELISA.

BASIC PRINCIPLE

The basic principle of an ELISA is to 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 Ags or Abs in a sample, depending on how the test is designed.

It can be further explained in terms of following steps:

1. Antibody is immobilized on micro-plate wells

2. Competition between drug in forensic sample and enzyme labeled drug for antibody
binding sites
3. The unbound material is washed out
4. Chromogenic substrate added to develop color
5. Resulting color is read in a spectrophotometer
 GENERAL METHODOLOGY

ELISA methods are routinely conducted in a standard 8-row by 12-column 96-well microplate
format that is readily automated and capable of high throughput quantification of analyte
concentrations. This general format is currently being extended to plates having 16 or more rows
and 24 or more columns, producing microplates with 384 or more wells.

Each well of the microplate is treated in a specific manner to produce a response (for many ELISA
methods, the typical response is a color change of the solution in the well, which is measured by a
spectrophotometer)

The bottom of each well is coated with a protein to which will bind the antibody you want to
measure. Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum
with antibodies (called primary antibodies). The serum is incubated in a well, and each well
contains a different serum (see figure below). A positive control serum and a negative control
serum would be included among the 96 samples being tested.

After some time, the serum is removed and weakly adherent antibodies are washed off with a series
of buffer rinses. To detect the bound antibodies, a secondary antibody is added to each well. The
secondary antibody would bind to all human antibodies and is typically produced in a rodent.
Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These
enzymes can metabolize colorless substrates (sometimes called chromagens) into colored products.
After an incubation period, the secondary antibody solution is removed and loosely adherent ones
are washed off as before. The final step is the addition the enzyme substrate and the production of
colored product in wells with secondary antibodies bound.

When the enzyme reaction is complete, the entire plate is placed into a plate reader and the optical
density (i.e. the amount of colored product) is determined for each well. The amount of color
produced is proportional to the amount of primary antibody bound to the proteins on the bottom of
the wells.
 ENZYME IMMUNOASSAY (EIA)

ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g., a
hormone or drug) in a fluid such as serum or urine. It is also known as enzyme immunoassay or
EIA.

The molecule is detected by antibodies that have been made against it; that is, for which it is the
antigen. Monoclonal antibodies are often used.

The test requires:

 The antibodies fixed to a solid surface, such as the inner surface of a test tube;
 A preparation of the same antibodies coupled to an enzyme — one (e.g., β-galactosidase)
that produces a colored product from a colorless substrate.

Performing the Test

1. The tubes are filled with the antigen solution (e.g.,

urine) to be assayed. Any antigen molecules present
bind to the immobilized antibody molecules.
2. The antibody-enzyme conjugate is added to the
reaction mixture. The antibody part of the conjugate
binds to any antigen molecules that were bound
previously, creating an antibody-antigen-antibody
"sandwich".
3. After washing away any unbound conjugate, the
substrate solution is added.
4. After a set interval, the reaction is stopped (e.g., by
adding 1 M NaOH) and the concentration of colored
product formed is measured in a spectrophotometer.
The intensity of color is proportional to the
concentration of bound antigen.
 TYPES OF ELISA

"INDIRECT" ELISA

The steps of "indirect" ELISA follows the mechanism below:-

1. A buffered solution of the antigen to be tested for is added to each well of a microtiter plate,
where it is given time to adhere to the plastic through charge interactions.
2. A solution of non-reacting protein, such as bovine serum albumin or casein, is added to
block any plastic surface in the well that remains uncoated by the antigen.
3. Next the primary antibody is added, which binds specifically to the test antigen that is
coating the well. This primary antibody could also be in the serum of a donor to be tested
for reactivity towards the antigen.
4. Afterwards, a secondary antibody is added, which will bind the primary antibody. This
secondary antibody often has an enzyme attached to it, which has a negligible effect on the
binding properties of the antibody.
5. A substrate for this enzyme is then added. Often, this substrate changes color upon reaction
with the enzyme. The color change shows that secondary antibody has bound to primary
antibody, which strongly implies that the donor has had an immune reaction to the test
antigen. This can be helpful in a clinical setting, and in R&D.
6. The higher the concentration of the primary antibody that was present in the serum, the
stronger the color change. Often a spectrometer is used to give quantitative values for color
strength.

The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the
enzyme molecules will produce many signal molecules. Within common-sense limitations, the
enzyme can go on producing color indefinitely, but the more primary antibody is present in the
donor serum the more secondary antibody + enzyme will bind, and the faster color will develop. A
major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-
specific; when serum is used as the source of test antigen, all proteins in the sample may stick to the
microtiter plate well, so small concentrations of analyte in serum must compete with other serum
proteins when binding to the well surface. The sandwich or direct ELISA provides a solution to this
problem, by using a "capture" antibody specific for the test antigen to pull it out of the serum's
molecular mixture.

ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple
positive or negative result (yes or no) for a sample. The cutoff between positive and negative is
determined by the analyst and may be statistical. Two or three times the standard deviation (error
inherent in a test) is often used to distinguish positive from negative samples. In quantitative ELISA,
the optical density (OD) of the sample is compared to a standard curve, which is typically a serial
dilution of a known-concentration solution of the target molecule. For example, if a test sample
returns an OD of 1.0, the point on the standard curve that gave OD = 1.0 must be of the same analyte
concentration as your sample.
 SANDWICH ELISA

A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample
antigen. The steps are as follows:

1. Prepare a surface to which a known quantity of capture antibody is bound.

2. Block any nonspecific binding sites on the surface.
3. Apply the antigen-containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply enzyme linked primary antibodies as detection antibodies that also bind
specifically to the antigen.
6. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
7. Apply a chemical that is converted by the enzyme into a color or fluorescent or
electrochemical signal.
8. Measure the absorbency or fluorescence or electrochemical signal (e.g., current) of
the plate wells to determine the presence and quantity of antigen.

The image to the right includes the use of a secondary antibody conjugated to an enzyme,
though, in the technical sense, this is not necessary if the primary antibody is conjugated to
an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process
of creating enzyme-linked antibodies for every antigen one might want to detect. By using
an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-
linked antibody can be used in a variety of situations. Without the first layer of "capture"
antibody, any proteins in the sample (including serum proteins) may competitively adsorb
to the plate surface, lowering the quantity of antigen immobilized. Use of the purified
specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen
from complicated mixtures before the measurement, simplifying the assay, and increasing
the specificity and the sensitivity of the assay.

A descriptive animation of the application of sandwich ELISA to home pregnancy testing

can be found here.

(1) Plate is coated with a capture

antibody
(2) sample is added, and any antigen
present binds to capture antibody
(3) detecting antibody is added, and
binds to antigen
(4) enzyme-linked secondary antibody
is added, and binds to detecting
antibody
(5) substrate is added, and is converted
by enzyme to detectable form.
 COMPETITIVE ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat
different than the first two examples:

1. Unlabeled antibody is incubated in the presence of its antigen (Sample).

2. These bound antibody/antigen complexes are then added to an antigen-coated well.
3. The plate is washed, so that unbound antibody is removed. (The more antigen in the
sample, the less antibody will be able to bind to the antigen in the well, hence
"competition.")
4. The secondary antibody, specific to the primary antibody is added. This second antibody is
coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the sample antigen concentration, the weaker the
eventual signal. 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.

(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-
linked antibody. The labeled antigen competes for primary antibody binding sites with
your sample antigen (unlabeled). The more antigen in the sample the less labeled antigen is
retained in the well and the weaker the signal).

It is common that the antigen is not first positioned in the well.

MULTIPLE AND PORTABLE ELISA (M&P ELISA)(ELISA REVERSE IN PUBLISHED PAPERS)

A new technique (EP 1 499 894 B1 in EPO Bulletin 25.02.209 N. 2009/09; USPTO 7510687
in USPTO Bulletin 31.03.2009; ZL 03810029.0 in SIPO PRC Bulletin 08.04.2009) 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.

The advantages of this technique are as follows:

1. 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
2. The sample volume can be increased to improve the test sensitivity in clinical
(saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples
3. 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.

INTER-ASSAY VARIABILITY AND BIAS

Assay methods typically exhibit experimental variability at intra- and inter-assay levels.
Both types of variation can arise from several sources. Because dilutions of material are
often made serially, there can be considerable correlation among a dilution series, which
results in serial dilution error, a source of intra-assay variation (Racine- Poon, Weihs, and
Smith 1991). Research continues on the effects of serial dilution error and methods of
dealing with it (Giltinan and Davidian 1994). The microplates themselves can exhibit
reproducible row and column patterns. These patterns result in considerable intraassay
variability when not identified and dealt with appropriately (Jones et al. 1995).

The microplate can also be a source of inter-assay variability when different plate types,
manufacturers, and lots result in variation in the observed optical densities. Additional
sources of inter-assay variation include experimental conditions under which the assay is
conducted, such as various environmental conditions (for example, incubation temperature
and incubation time), analysts, and biological reactants. These factors can enter the process
as fixed and/or random effects, increasing the variability of the optical density in either a
systematic or random fashion, respectively. These fixed and random patterns in optical
density affect model fitting by increasing the intra- and/or inter-assay variability or bias in
the model parameter estimates.

Increases in the variability or bias of parameter estimates produces greater variability in

the estimated analyte concentration. This variability and bias negatively impact drug
discovery, pharmacokenetic studies, drug stability, and clinical trials. For example,
increased variability could increase the sample size necessary to obtain acceptable
precision or lead to the wrong conclusions because of a significant undetected bias.

Therefore, variability and bias effects impact the system at several different levels: (1) the
raw optical density readings, (2) the estimation of model parameters, and (3) the
estimation of analyte concentration. The identification and elimination of these effects are
difficult because they arise from numerous sources and impact the bioassay process at
many levels. Several methods have been suggested to detect, control, or eliminate these
effects (Bunch, Rooke, and Harrison 1990; Buonaccorsi 1986; Lansky 1997; Larholt and
Sampson 1995; Plikaytis et al. 1994; Rodbard 1974; Rocke and Jones 1997; Sittampalam, et
al. 1996).
 APPLICATIONS

ELISA results using S-OIV A neuraminidase antibody at 1 μg/ml to probe the immunogenic and the
corresponding seasonal influenza A neuraminidase peptides at 50, 10, 2, and 0 ng/ml.

Because the ELISA can be performed to evaluate either the presence of antigen or the presence of
antibody in a sample, it is a useful tool for determining serum antibody concentrations (such as
with the HIV test[3] or West Nile Virus). It has also found applications in the food industry in
detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs. ELISA can also
be used in toxicology as a rapid presumptive screen for certain classes of drugs.

The ELISA was the first screening test widely used for HIV because of its high sensitivity. In an
ELISA, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens are
attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The
plate is then washed to remove all other components of the serum. A specially prepared "secondary
antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by
another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus, the
plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A
substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or
fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is
determining the "cut-off" point between a positive and a negative result.

A cut-off point may be determined by comparing it with a known standard. If an ELISA test is used
for drug screening at workplace, a cut-off concentration, 50 ng/mL, for example, is established, and
a sample that contains the standard concentration of analyte will be prepared. Unknowns that
generate a signal that is stronger than the known sample are "positive." Those that generate weaker
signal are "negative."

Literally hundreds of ELISA kits are manufactured for

 research
 human and veterinary diagnosis

Some examples:

1. 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)
 HTLV-1 and -2 (presence of antibodies)
2. measuring hormone levels
 HCG (as a test for pregnancy)
 LH (determining the time of ovulation)
 TSH, T3 and T4 (for thyroid function)
 hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes
3. detecting infections
 sexually-transmitted agents like HIV, syphilis, and chlamydia
 hepatitis B and C
  Toxoplasma gondii
4. detecting allergens in food and house dust
5. measuring "rheumatoid factors" and other autoantibodies in autoimmune diseases
like lupus erythematosus
6. measuring toxins in contaminated food
7. detecting illicit drugs, e.g.,
 cocaine
 opiates
 Δ-9-tetrahydrocannabinol, the active ingredient in marijuana
 REFERENCES

1. Exploring Enzyme-Linked Immunosorbent Assay (ELISA) Data with the SAS®

Analyst Application by Stewart L. Fossceco, Knoll Pharmaceutical Company,
Whippany, NJ. and Nathan A. Curtis, SAS Institute Inc., Cary, NC.

2. Enzyme-linked Immunosorbent Assay (ELISA) By Mary Lea Killian; USDA APHIS

VS, National Veterinary Services Laboratories, Ames, Iowa

3. Agriyork 400 (http://www.agriyork.co.uk/article.asp?ItemID=562)

4. ELISA (Enzyme-Linked Immunosorbent Assay) by Wright et al., 1987 & Wright et

al, 1996

5. Application of ELISA Technique by Hongbao Ma , Kuan-Jiunn Shieh , Sheau-Long

Lee