Sandhu Premjeet et al.

/ Journal of Pharmacy Research 2011,4(12),4581-4583

Review Article Available online through
ISSN: 0974-6943 www.jpronline.info
Enzyme-Linked Immuno-Sorbent Assay (ELISA),
basics and it’s application : A comprehensive review

Sandhu Premjeet1,2*,Gupta Deepika 1, Bhardwaj Sudeep1,Jain Sonam 2, Kataria Sahil 1, Rathore Devashish 3, Kumar Sunil 1
College of Technical Education, Institute of Pharmaceutical Sciences and Drug Research, Sri Ganganagar, Rajasthan, INDIA
2
School of Pharmaceutical Sciences,RGPV University campus, Bhopal, M.P, INDIA
3
School of Pharmacy,DAVV, Indore, M.P, INDIA
Received on:20-08-2011; Revised on: 15-09-2011; Accepted on:10-11-2011

ABSTRACT
Enzyme immunoassay, a bioanalytical method incorporating an antigen–antibody reaction to capture the analyte of interest and an enzyme reporter system
to detect the captured analyte, is one of the most widely used immunoassay formats. The method is sometimes applied only qualitatively to indicate the
presence of an antigen in a matrix. However, in the more common quantitative implementation, a calibration curve is incorporated, from which the
concentration of the analyte in unknown samples is interpolated. Immunoassays have been widely applied in support of medical practice and drug development.
However, in recent years, there has been a decline in the application of immunoassays to the quantitation of low-molecular weight xenobiotics, primarily due
to the advent of liquid chromatography–mass spectrometry (LC–MS) methods, which have high sensitivity and specificity. Immunoassays remain the method
of choice for the quantitation of protein macromolecules and antibodies in complex matrices. Another major application of immunoassays is in the detection
and quantitation of biomarkers, which are evolving to be of pivotal importance in the evaluation of pharmacological, toxicological, and clinical activities of
candidate drugs1. It’s a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample2.

Key words: Enzyme, Immuno assays, biomarkers, antibody, antigen.

INTRODUCTION
Immunoassays generally vary in the type of critical antibody binding reagent used enzymes are horseradish peroxidase, alkaline phosphatase, p-nitrophenyl
or the detection and reporter systems used to monitor the end-point of the phosphatase ß galactosidase4. These enzymes have been used to link to the
binding reaction. Enzyme immunoassay formats fall broadly into two categories, antibody or antigen molecule as may be the case. When mixed with a suitable
namely heterogeneous and homogeneous. In a heterogeneous assay, at least substrate each of these enzymes generates a colored reaction product 5. ELISA
one key reagent is immobilized on a solid surface and there is at least one procedure are popular primarily because they require little interpretive skill to
‘‘washing’’ step before the final detection step. In contrast, in a homogeneous read the results tend to be clearly positive or clearly negative6.
assay, all reagents are in solution together and there is no ‘‘washing’’ step prior
to signal generation and detection. Both categories of assay include formats HISTORY
described as competitive and non-competitive. In a competitive assay, there is Before the development of the EIA/ELISA, the only option for conducting an
direct competition between the labeled and the unlabeled antigen (analyte or immunoassay was radioimmunoassay, a technique using radioactively -labeled
ligand) in solution or, in some cases, between immobilized and soluble antigen antigens or antibodies. In radioimmunoassay, the radioactivity provides the
for a limited number of antibody binding sites. In non-competitive assays, signal which indicates whether a specific antigen or antibody is present in the
antibody binding sites to capture and detect the antigen are not limiting because sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman
the antigen is incubated with excess capture antibody and enzyme-labeled Yalow and Solomon Berson published in 1960.
detection antibody. An example of a competitive homogeneous assay format
is the enzyme-multiplied immunoassay (EMIT) system, in which enzyme- Because radioactivity poses a potential health threat, a safer alternative was
labeled antigen competes directly in solution with unlabeled antigen in the sought. A suitable alternative to radioimmunoassay would substitute a non-
biological sample (or calibration standard and quality control samples) for a radioactive signal in place of the radioactive signal. When enzymes (such as
limited number of antibody binding sites. The reaction endpoint is detected peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’-
and quantitated spectrophotometrically without any intervening wash steps. Tetramethylbenzidine), this causes a change in color, which is used as a signal.
Enzyme-linked immunosorbent assay (ELISA) is an example of a heterogenous However, the signal has to be associated with the presence of antibody or
noncompetitive immunoassay 3. antigen, which is why the enzyme has to be linked to an appropriate antibody.
This linking process was independently developed by Stratis Avrameas and
ELISA- (ENZYME LINKED IMMUNOSORBENT ASSAY) G.B. Pierce. Since it is necessary to remove any unbound antibody or antigen
Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) by washing, the antibody or antigen has to be fixed to the surface of the
have become household names for medical laboratories, manufacturers of in container, i.e. the immunosorbent has to be prepared. A technique to accomplish
vitro diagnostic products, regulatory bodies, external quality assessment and this was published by Wide and Jerker Porath in 1966 2.
proficiency-testing organizations (Rudolf M). It is a biochemical technique
used mainly in immunology to detect the presence of an antibody or an antigen In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden,
in a sample2. In principle, the method is similar to that of the RIA which and Anton Schuurs and Bauke van Weemen in The Netherlands, independently
involves competitive binding, it does not involve in use of any radio-labelled published papers which synthesized this knowledge into methods to perform
material and the sensitivity of the method is equally good 0.0001-0.001 µg/ml. EIA/ELISA.
The method is safe and cheaper as compared to RIA. Here the enzyme conjugated
to an antibody reacts with a colorless substrate to generate a colored reaction Numerous variants of ELISA
product. The color is measured spectrophotometrically. The most commonly A number of variation ELISA have been developed, allowing qualitative detection
or quantitative measurement of either antigen or antibody. Each type of
*Corresponding author. ELISA can be used qualitatively to detect the presence of antibody or antigen.
Premjeet Sandhu Alternatively, a standard curve based on known concentration of antibody or
Seth G. L. Bihani antigen is prepared, from which the unknown concentration of a sample can
S.D. College of Technical Education, be determined.
Institute of Pharmaceutical Sciences
and Drug Research, Sri Ganganagar, 1.Indirect ELISA :
Rajasthan, INDIA Antibody can be detected or quantitatively determined with an indirect ELISA.
Tel.: + 91-
E-mail:[email protected] Serum or some other sample containing primary antibody (Ab1) is added to an
antigen- coated microtiter well and allowed to react with the antigen attached
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 Sandhu Premjeet et al. / Journal of Pharmacy Research 2011,4(12),4581-4583
to the well. After any free Ab1 is washed away, the presence of antibody bound 2. The avidity of the first antibody for the antigen.
to the antigen is determined by adding an enzyme- conjugated secondary anti- 3. The avidity of the second antibody for the antigen.
isotype antibody (Ab2), which binds to the primary antibody. Any free Ab2 then 4. The specific activity of the second antibody.
is washed away, and a substrate for the enzyme is added. The amount of colored The amount of the capture antibody that is bound to the solid phase can be
reaction product that forms is measured by specialized spectrophotometric adjusted easily by dilution or concentration of the antibody solution. The
plate readers, which can measure the absorbance of all of the wells of a 96- well avidity of the antibodies for the antigen can only be altered by substitution
plate in seconds7. with other antibodies. The specific activity of the second antibody is determined
by the number and type of labeled moieties it contains9.
Indirect ELISA is the method of choice to detect the presence of serum
antibodies against human immunodeficiency virus (HIV), the causative agent 1.Competitive ELISA :-
of AIDS. For detection of anti- HIV-1 and anti- HIV-2 antibodies in the When two “matched pair” antibodies are not available for your target, another
patient serum. The well of the polystyrene micrititre plate are coated with option is the competitive ELISA. Another advantage to the competitive
purified HIV-1 and HIV-2 antigens or synthetic peptides representing ELISA is that non-purified primary antibodies may be used. Another variation
immunodominant epitopes of HIV-1 and HIV-2, which constitutes the solid for measuring amounts of antigen is competitive ELISA 9.
phase antigens. Diluted test serum or plasma sample is added to such a well and
incubated. If antibodies specific for HIV-1 and/or HIV-2 is/are present in the In this technique, antibody is first incubated in solution with a sample containing
test sample they will form stable complexes with antigens coated on the well. antigen. The antigen- antibody mixture is then added to an antigen-coated
Well is then washed and a conjugate of goat anti-human immunoglobin, which micrititer well. The more antigen present in the sample, the less free antibody
has been labelled with the enzyme horse-radish peroxidase, is added. If the will be available to bind to the antigen-coated well. Addition of an enzyme
antigen- antibody complex is present, the peroxidase conjugate will bind to conjugated secondary antibody (Ab2) specific for the isotype of the primary
the complex and remains in the well is removed by washing and the presence antibody can be used to determine the amount of primary antibody bound to
of enzyme immobilized on the complexes is shown by incubation in the the well in an indirect ELISA 7.
presence of enzyme substrate (ortho-phenylene-diamine dihydrochloride
solution). Incubation with enzyme substrate produces a yellow-orange colour
in the test well. If the sample contains no anti-HIV-1 and/or anti-HIV-2, then
the labeled antibody cannot be found and no colour develops. The absorbance
value of each well is read by an ELISA plate read at wavelength of 492±2 nm8.

Figure: 3 Competitive ELISA method

1.Reverse ELISA :
A new technique uses a solid phase made up of an immunosorbent polystyrene
rod with 4-12 protruding gives. The entire device is immersed in a test tube
Figure: 1 Indirect ELISA method containing the collected sample and the following steps (washing, incubation
2.Sandwich ELISA : in conjugate and incubation in chromogenous) are carried out by dipping the
One of the most useful of the immunoassays is the two antibody “sandwich” gives in microwells of standard microplates pre-filled with reagents.
ELISA. This assay is used to determine the antigen concentration in unknown
samples. This ELISA is fast and accurate, and if a purified antigen standard is The advantages of this technique are as follows:
available, the assay can determine the absolute amount of antigen in an unknown 1.The gives can each be sensitized to a different reagent, allowing the
sample. The sandwich ELISA requires two antibodies that bind to epitopes that simultaneous detection of different antibodies and different antigens for multi-
do not overlap on the antigen. This can be accomplished with either two target assays;
monoclonal antibodies that recognize discrete sites or one batch of affinity- 2.The sample volume can be increased to improve the test sensitivity in
purified polyclonal antibodies. Antigen can be detected or measured by a clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water)
sandwich ELISA. samples;
3.One give is left unsensitized to measure the non-specific reactions of the
In this technique, the antibody (rather than the antigen) is immobilized on a sample;
microtiter well. A sample containing antigen is added and allowed to react with 4.The use of laboratory supplies for dispensing sample aliquots, washing solution
the immobilized antibody. After the well is washed, a second enzyme- linked and reagents in microwells is not required, facilitating ready-to-use lab-kits
antibody specific for a different epitopes on the antigen is added and allowed and on-site kits2.
to react with the bound antigen. After any free second antibooby is removed
by washing, substrate is added, and the colored reaction product is measured7. FACTORS THAT INFLUENCE ASSAY PERFORMANCE
1.The greatest impact of the behavior of the assay is typically based on the
antibody pair itself. If the pair is a “good” match for the desired analyte, other
unwanted factors are greatly minimized. If the pair is a “poor” match with
lower affinity for the desired analyte, it may take a lot of time and creativity
to achieve acceptable parameters.
2.Non-specific binding can cause high background values and poor recovery
and linearity, which can often be controlled with IgG antibodies or serum.
3.False positive values can occur on Non-specific binding of samples to one or
both of the antibodies present which is often detected when performing spiked
recovery and linearity testing. In the ELISA format, these samples can typically
be controlled by the use of IgG antibodies and/or heterophilic blockers.
4.Hook Effect may give incorrect sample values for highly concentrated
samples and can usually be detected during linearity studies.

VALIDATION OF ELISA ASSAYS

1.Spiked recovery tests are performed using the blank, low standard, medium
Figure : 2 Sandwich ELISA method
standard and high standard points spiked into at least 8 different samples in
The Sensitivity of the Sandwich ELISA is Dependent on Four Factors: duplicate. This test should be repeated at least two times to determine if results
1. The number of molecules of the first antibody that are bound to the solid are reproducible and within acceptable criteria for the coefficient of variation
phase. (CV). Acceptable criteria are CVs of 80–120%.

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 Sandhu Premjeet et al. / Journal of Pharmacy Research 2011,4(12),4581-4583
2.Linearity tests are performed on at least 8 different samples in duplicate. At ELISA can also be used to determine the level of antibodies in faecal
least three dilution of the neat samples should be performed and more if content,specifically the direct method.
possible. This test should be repeated at least two times to assure that the
results are reproducible and acceptable (80–120% CVs). ELISA test are utilized to detect substances that have antigenic properties,
3.Intra-assay variation is tested by running 8 different samples (of varying primarily proteins (as opposed to small molecules and ions such as glucose and
concentration) in replicates of ten across the microtiter plate and determining potassium). Some of these include hormones, bacterial antigens and antibodies.
the % CVs of the samples. Acceptable criteria are typically CVs of 80–120%. ELISA methods are fundamental tools in the pharmaceutical industry with
4.Inter-assay variation is determined by evaluating at least 8 samples (of applications in drug discovery, animal studies, and clinical trials12.
varying concentration) in duplicate on at least three different microtiter
plates on different days using the same reagent lots. The % CVs are then There are variations of this test, but the most basic consists of an antibody
calculated. Acceptable criteria is typically CVs of 80–120%. attached to a solid surface. This antibody has affinity for (will latch on to) the
5.The microtiter plates coated with the capture antibody must also be validated substance of interest, for example, human chorionic gonadotropin (HGC), the
by running the blank, low, and high standard points in replicates of 32, on a commonly measured protein which indicates pregnancy. A mixture of purified
minimum of three plates, and the % CVs calculated. Acceptable criteria is HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are
typically CVs of =10%. added to the test system. If no HCG is present in the test sample, then only
6.The sensitivity of the assay is determined by evaluating the blank and the HCG with linked enzyme will bind. The more HCG which is present in the test
lowest standard point in replicates of at least 20. The following calculation is 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 13.

ELISA tests are generally highly sensitive and highly specific and less expensive
technique used in serology to detect antigens or antibodies14. They have the
used to determine the sensitivity of the assay: 1. Further specificity of the added advantages of not needing radioisotopes or a radiation-counting apparatus
assay is evaluated by running standards of several similar analytes of available ELISA assays are widely applied in clinical laboratory testing15.
species in the assay to determine if any similar analytes can be detected by the
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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 12.December 2011 4581-4583