research Techniques made simple
Enzyme Immunoassay and Enzyme-
Linked Immunosorbent Assay
Stephanie D. Gan1 and Kruti R. Patel2
Journal of Investigative Dermatology (2013) 133, e12. doi:10.1038/jid.2013.287
INTRODUCTION
Enzyme immunoassay (EIA) and enzyme-linked immunosor-bent WHAT ENZYME-LINKED
assay (ELISA) are both widely used as diagnostic tools in IMMUNOSORBENT ASSAY (ELISA) DOES
medicine and as quality control measures in various industries;
• ELISA is a biochemical assay that uses antibodies
they are also used as analytical tools in biomedical research for
and an enzyme-mediated color change to detect
the detection and quantification of specific antigens or anti-bodies
the presence of either antigen (proteins, peptides,
in a given sample. These two procedures share similar basic
hormones, etc.) or antibody in a given sample.
principles and are derived from the radioimmunoassay (RIA). RIA
was first described by Berson and Yalow (Yalow and Berson, • Both “indirect” and “sandwich” ELISAs allow
1960), for which Yalow was awarded the Nobel Prize in 1977, to detection of antigen or antibody at very low
measure endogenous plasma insulin. RIA was then developed concentrations.
into a novel technique to detect and measure bio-logical • The competitive method detects compositional
molecules present in very small quantities, paving the way for the differences in complex antigen mixtures with high
analysis and detection of countless other biologi-cal molecules, sensitivity, even when the specific detecting
including hormones, peptides, and proteins. Because of the antibody is present in relatively small amounts.
safety concern regarding its use of radioactivity, RIA assays were • Multiple and portable ELISA is a ready-to-use,
modified by replacing the radioisotope with an enzyme, thus low-cost lab kit that is ideal for large population
creating the modern-day EIA and ELISA. screening in low-resource settings.
GENERAL PRINCIPLES
EIA/ELISA uses the basic immunology concept of an antigen LIMITATIONS
binding to its specific antibody, which allows detection of very • The enzyme-mediated color change will react
small quantities of antigens such as proteins, peptides, hor- indefinitely. Over a sufficiently long period of time,
mones, or antibody in a fluid sample. EIA and ELISA utilize the color strength will inaccurately reflect the amount
enzyme-labeled antigens and antibodies to detect the bio-logical of primary antibody present, yielding false-positive
molecules, the most commonly used enzymes being alkaline results.
phosphatase (EC 3.1.3.1) and glucose oxidase (E.C. 1.1.3.4).
• To detect a given antibody or antigen, a known
The antigen in fluid phase is immobilized, usually in 96-well
microtiter plates. The antigen is allowed to bind to a specific
reciprocal antigen or antibody must be generated.
antibody, which is itself subsequently detected by a secondary, • Nonspecific binding of the antibody or antigen to
enzyme-coupled antibody. A chromogenic sub-strate for the the plate will lead to a falsely high-positive result.
enzyme yields a visible color change or fluores-cence, indicating
the presence of antigen. Quantitative or qualitative measures can
be assessed based on such colorimet-ric reading. Fluorogenic
substrates have higher sensitivity and can accurately measure the ELISA assay is the direct or indirect detection of antigen by
levels of antigen concentrations in the sample. The general adhering or immobilizing the antigen or antigen-specific cap-ture
procedure for ELISA is outlined in Figure 1. antibody, respectively, directly onto the well surface. For sensitive
Various types of ELISAs have been employed with modifica- and robust measurements, the antigen can be spe-cifically
tion to the basic steps described in Figure 1. The key step in selected out from a sample of mixed antigens via a
Department of Dermatology, Department of Medicine, Boston University School of Medicine and Boston Medical Center, Boston, Massachusetts,
1
USA and 2Program in Molecular and Translational Medicine, Department of Medicine, Boston University School of Medicine and Boston Medical
Center, Boston, Massachusetts, USA
Correspondence: Stephanie D. Gan, 609 Albany Street, J-201, Boston, Massachusetts 02118, USA. E-mail: Stephanie.gan@bmc.org
© 2013 The Society for Investigative Dermatology www.jidonline.org 1
research Techniques made simple
to the specific test antigen to select it out of the serum, as
illustrated in the sandwich technique below.
Sandwich ELISA
The sandwich technique is used to identify a specific sample anti-
gen. The well surface is prepared with a known quantity of bound
antibody to capture the desired antigen. After nonspecific binding
sites are blocked using bovine serum albumin, the antigen-con-
taining sample is applied to the plate. A specific primary antibody
is then added that “sandwiches” the antigen. Enzyme-linked sec-
ondary antibodies are applied that bind to the primary antibody.
Unbound antibody–enzyme conjugates are washed off. Substrate
is added and is enzymatically converted to a color that can be
later quantified. Canady et al. (2013) analyzed patient sera using
the sandwich method to detect enhanced keratinocyte growth
factor (KGF) levels in the sera of keloid and scleroderma patients
compared to healthy controls to quantify human KGF (Figure 3).
Figure 1. Enzyme-linked immunosorbent assay (ELISA) technique One advantage of using a purified specific antibody to
used to detect an antigen in a given sample. The antigen (in liquid capture antigen is that it eliminates the need to purify the
phase) is added to the wells, where it adheres to the walls. Primary antigen from a mixture of other antigens, thus simplifying
antibody binds specifically to the antigen. An enzyme-linked secondary
the assay and increas-ing its specificity and sensitivity.
antibody is added that reacts with a chromogen, producing a color
change to quantitatively or qualitatively detect the antigen.
Competitive ELISA
The key event of competitive ELISA is the process of competi-
“capture” antibody. The antigen is thus “sandwiched” between tive reaction between the sample antigen and antigen bound
such capture antibody and a detection antibody. If the anti- to the wells of a microtiter plate with the primary antibody.
gen to be measured is small in size or has only one epitope First, the primary antibody is incubated with the sample anti-
for antibody binding, a competitive method is used in which gen and the resulting antibody–antigen complexes are added
either the antigen is labeled and competes for the unlabeled to wells that have been coated with the same antigen. After
antigen–antibody complex formation or the antibody is an incubation period, any unbound antibody is washed off.
labeled and competes for the bound antigen and antigen in The more antigen in the sample, the more primary antibody
the sample. Each of these modified techniques of ELISA can will be bound to the sample antigen. Therefore, there will be a
be used for a qualitative and quantitative purpose. smaller amount of primary antibody available to bind to the
antigen coated on the well. Secondary antibody conjugated to
TYPES OF ELISA an enzyme is added, followed by a substrate to elicit a chro-
Indirect ELISA mogenic or fluorescent signal. Absence of color indicates the
A sample that must be analyzed for a specific antigen is adhered presence of antigen in the sample.
to the wells of a microtiter plate, followed by a solution of nonre- The main advantage of competition ELISA is its high sensitivity to
acting protein such as bovine serum albumin to block any areas compositional differences in complex antigen mixtures, even when
of the wells not coated with the antigen. The primary antibody, the specific detecting antibody is present in relatively small amounts
which binds specifically to the antigen, is then added, followed by (Dobrovolskaia et al., 2006). This method can be used to determine
an enzyme-conjugated secondary antibody. A substrate for the the potency of U.S. standardized allergen extracts (Dobrovolskaia et
enzyme is introduced to quantify the primary antibody through a al., 2006) and to measure the total antibodies to the capsular
color change. The concentration of primary antibody present in polysaccharide of Haemophilus influenzae type
the serum directly correlates with the intensity of the color. One
application of the indirect ELISA method is demon-strated by
Haapakoski et al. (2013), who investigated the role of Toll-like
receptor activation during cutaneous allergen sen-sitization using
ovalbumin (OVA) in the modulation of allergic asthma. In one
experiment, dermal exposure to Toll-like receptor ligands
(lipopolysaccharide, Pam3Cys, P(I:C)) was demonstrated to
downregulate OVA-specific IgE antibodies in serum, as mea-
sured by the indirect ELISA technique (Figure 2).
A main disadvantage of indirect ELISA is that the meth-od
Figure 2. Indirect enzyme-linked immunosorbent assay (ELISA).
of antigen immobilization is not specific. When serum is used
Dermal exposure to Toll-like receptor ligands (lipopolysaccharide, Pam 3Cys,
as the test antigen, all proteins in the sample may adhere to P(I:C)) was demonstrated to downregulate ovalbumin-specific IgE antibodies
the wells of a microtiter plate. This limitation, however, can be in serum, as measured by the indirect ELISA technique. Reprinted
overcome using a capture antibody unique from Haapakoski et al. (2013).
2 Journal of Investigative Dermatology (2013), Volume 133 © 2013 The Society for Investigative Dermatology
research Techniques made simple
REFERENCES
Balsam J, Ossandon M, Bruck HA et al. (2013) Low-cost technologies for medical
diagnostics in low-resource settings. Expert Opin Med Diagn 7:243–55
Canady J, Arndt S, Karrer S et al. (2013) Increased KGF expression
promotes fibroblast activation in a double paracrine manner resulting in
cutaneous fibrosis. J Invest Dermatol 133:647–57
Dobrovolskaia E, Gam A, Slater JE (2006) Competition enzyme-linked
immunosorbant assay (ELISA) can be a sensitive method for the
specific detection of small quantities of allergen in a complex mixture.
Clin Exp Allergy 36:525–30
Figure 3. Sandwich enzyme-linked immunosorbent assay (ELISA). The Haapakoski R, Karisola P, Fyhrquist N et al. (2013) Toll-like receptor activation
“sandwich” method was used to detect enhanced keratinocyte growth factor during cutaneous allergen sensitization blocks development of asthma
(KGF) levels in the sera of keloid and scleroderma patients compared to through IFN-gamma-dependent mechanisms. J Invest Dermatol 133:964–72
healthy controls to quantify human KGF. Reprinted from Canady et al. (2013). Mariani M, Luzzi E, Proietti D et al. (1998) A competitive enzyme-linked
immunosorbent assay for measuring the levels of serum antibody to
Haemophilus influenzae type b. Clin Diagn Lab Immunol 5:667–74
b in human sera from vaccinated subjects (Mariani et al., 1998). Yalow RS, Berson SA (1960) Immunoassay of endogenous plasma insulin
in man. J Clin Invest 39:1157–75
Competitive ELISA is often used to detect HIV antibodies in the
sera of patients. The HIV antigen is coated on the surface of the
microtiter plate wells, and two specific antibodies are applied: one
conjugated with enzyme and the other of the sera of the patient.
Cumulative competition occurs between the two anti-bodies for QUESTIONS
the same antigen. If antibodies are present in the sera, then the This article has been approved for 1 hour of Category 1 CME Credit. To take the quiz,
antigen–antibody reaction occurs, leaving behind very low with or without CME credit, follow the link under the "CME CREDIT" header.
amounts of antigen available for binding with the enzyme-labeled
antibody. Most of the unbound enzyme-labeled anti-bodies are 1. Which of the following molecule(s) can be
washed off, producing minimal to no color change. Absence of detected by ELISA?
color is indicative of an HIV-positive sample. A. Proteins.
Multiple and portable ELISA B. Hormones.
Multiple and portable ELISA is a new technique that uses a mul- C. Antibodies.
ticatcher device with 8 or 12 immunosorbent protruding pins on a
D. All of the above.
central stick that can be immersed in a collected sample. The
washings and incubation with enzyme-conjugated antigens and 2. What does a weak color signal in competitive
chromogens are performed by dipping the pins in prefilled ELISA represent?
microwells with reagents. The main advantage of these ready-to-
use lab kits is that they are relatively inexpensive, can be used A. More antigen in the sample.
for large population screening, and do not require skilled per- B. Less antigen in the sample.
sonnel or laboratory equipment, making them an ideal tool for
low-resource settings (Balsam et al., 2013). Clinical applications C. Less antigen retained on the well.
include point-of-care detection of infectious diseases, bacterial D. Both a and c.
toxins, oncologic markers, and drug screening.
3. Which of the following is immobilized on
SUMMARY the microtiter well in sandwich ELISA?
EIA/ELISA is a powerful method not only for general biomedi-
A. Detection antibody.
cal research but also as a diagnostic tool. It allows detection
of all types of biological molecules at very low concentrations B. Sample.
and quantities. Although it has its limitations, EIA/ELISA
C. Capture antibody.
remains an important tool in both clinical and basic research,
as well as in clinical diagnostics. D. Secondary antibody conjugated to an enzyme.
CME Credit 4. What is a major advantage of ELISA in comparison
This article has been approved for 1 hour of Category 1 CME Credit. To
take the online quiz, follow the link below:
to other biological quantification techniques?
http://www.classmarker.com/online-test/start/?quiz=yxk51dc7bff36258 A. Detection of a molecule at a low concentration.
CONFLICT OF INTEREST B. Inexpensive.
The authors state no conflict of interest.
C. Low specificity.
SUPPLEMENTARY MATERIAL
A PowerPoint slide presentation appropriate for journal club or other
D. Easily available.
teaching exercises are available at http://dx.doi.org/10.1038/jid.2013.287.
© 2013 The Society for Investigative Dermatology www.jidonline.org 3
