In the Laboratory

Enzyme-Linked Antibodies: A Laboratory Introduction

to the ELISA
Gretchen L. Anderson and Leo A. McNellis
Department of Chemistry, Indiana University South Bend, South Bend, IN 46634

ELISAs (enzyme-linked immuno-sorbent assays) have Many antibodies used in ELISAs are commercially avail-
revolutionized diagnostic procedures in medicine, agriculture, able as enzyme-linked antibodies (also called enzyme-linked
and home health-care management. Enzyme-linked antibodies antibody conjugates). Antibodies are produced by injecting
are used routinely to detect minute quantities of viruses (e.g., an animal (e.g., a rabbit) with the antigen of interest. Within
hepatitis virus), hormones (e.g., human chorionic gonado- several weeks, the animal produces many copies of an anti-
tropin as evidence of pregnancy), and other antibodies (e.g., body that specifically recognizes and binds the antigen. The
antibodies to the AIDS virus as an indication of infection). antibodies are identified, purified, and covalently bound to an
The use of ELISAs has spread from clinical applications to enzyme. This enzyme-linked antibody now acts as a specific
layman use in home pregnancy tests, golf-course turf man- probe to detect the presence of the antigen in a sample. A
agement, and diagnosis of crop disease. different antibody or enzyme-linked antibody preparation is
ELISAs provide an ideal laboratory exercise for elementary purchased for each antigen to be tested.
biochemistry students such as health science and nursing This laboratory exercise demonstrates the effectiveness
majors, students enrolled in chemistry survey courses, or high of ELISAs by using enzyme-linked antibodies to detect biotin
school students. The technique is current, conceptually in the form of biotinylated albumin. This choice of antigen
straightforward, and rapid enough to be completed in a typical is advantageous in terms of availability and cost of reagents
2-hour laboratory class. It provides students with a clear (enzyme-conjugated antibiotin antibodies are among the least
example of practical, contemporary uses of basic chemical expensive) and avoidance of potentially hazardous biological
principles. It introduces them to the microscale quantities specimens. Because the volumes of samples are very small
typical of ELISAs and integrates the use of enzymes, anti- (usually less than 100 µL), the assay can be performed in com-
bodies, and the specificity of protein binding. mercially available small plastic (polyethylene) wells similar
The procedure outlined below demonstrates the impres- to a tiny plastic muffin tin. For ill-understood reasons, large
sive sensitivity of the ELISA through the detection of femto- molecules, particularly proteins, bind tightly to the inside
molar (10᎑15 M) quantities of biotin, a water-soluble vitamin. surface of the wells, but small molecules such as biotin do
The advantages of this choice of antigen are the low cost not bind efficiently. The efficiency of the ELISA is therefore
($1.00 or less per student), minimal equipment needs, mod- improved by attaching the biotin to a large protein such
erate amount of instructor or technician preparation time, as albumin. Commercially available biotinylated albumin is
and nonpathogenicity of the samples. used here to insure that biotin remains bound to the surface
of the ELISA wells. Peroxidase-linked antibiotin antibody is
Background also commercially available. It catalyzes the reduction of color-
less TMB (3,3′,5,5′-tetramethylbenzidine) dye in the presence
In vivo, antibiodies (or immunoglobulins) constitute a of hydrogen peroxide to yield a colored product:
family of proteins that specifically bind to antigens recognized
peroxidase
as foreign to the host. The antigen may be a polypeptide or a TMBox + H2O2 → TMBred + O2 + 2H+
portion of a protein, a carbohydrate, or virtually any small (colorless) (colored)
molecule. Formation of the antigen–antibody complex is an
essential step in the immune response to eliminate molecules, Typical ELISA Procedure
viruses, or cells that are not part of the host organism. Anti-
bodies bind noncovalently to target antigens by virtue of their A typical ELISA procedure consists of four basic steps
complementary 3-dimensional structures. A specific antibody (Figure 1 shows these steps as related to the assay for
will bind only to its target, even in complex mixtures. biotinylated albumin described here):
The ELISA takes advantage of the specificity of antibodies 1. Bind the antigen to the ELISA well and wash away excess.
to detect small amounts of a particular antigen in a complex Samples used in ELISA diagnostic procedures are gen-
mixture such as blood or extracts of plant tissue (1–9). One erally complex mixtures consisting of a few molecules
way to detect the presence of a specific antibody–antigen of the antigen of interest in a matrix of extraneous
complex is to measure the activity of an enzyme covalently proteins, lipids, carbohydrates, etc. Some of these “con-
attached to the antibody. The enzyme chosen (usually per- taminating” molecules will bind to the ELISA well,
but will not be detected later. Any molecules that do
oxidase or alkaline phosphatase) is one that catalyzes a color not bind to the well are removed with a buffered rinse.
conversion in a dye when appropriate substrates are added.
2. Block exposed sites on the wells with albumin and wash
Since each enzyme molecule catalyzes the formation of thou- away excess. The enzyme-linked antibody (to be added
sands of colored dye molecules, and since the absorbance of in a subsequent step) will bind nonspecifically to the
the dye is proportional to the amount of antibody–antigen ELISA well if any of the well surface is exposed. To
complex present in the sample, the ELISA can detect prevent this, a large excess of an inexpensive protein
picomolar or femtomolar (10᎑12 and 10᎑15 M, respectively) (such as albumin) is added to the well. Excess albumin
concentrations of a particular antigen in a mixture. is removed from the well with a buffered rinse.

JChemEd.chem.wisc.edu • Vol. 75 No. 10 October 1998 • Journal of Chemical Education 1275

In the Laboratory

3. Bind enzyme-linked antibody to available antigens. 1:2000 dilution of antibiotin peroxidase conjugate
When the appropriate enzyme-linked antibody is (Sigma) or according to recommendations of vendor
added to the well, it binds only to its specific antigen. TMB (3,3′,5,5′-tetramethylbenzidine-HCl) tablet
After washing to remove unbound antibody, the (Sigma)
amount of enzyme-linked antibody remaining in the
well is proportional to the amount of antigen in the 30% hydrogen peroxide
well. At this point, the enzyme-linked antibody is un- Strip of 8 polyvinyl ELISA wells (ELISA plates, available
detectable because it is colorless and present in minute from scientific warehouses as 96-well plates, can easily
quantities. be cut into 8-well strips)
4. Add enzyme substrate to detect presence of antigen. The Humid box (a plastic sandwich box containing a damp
amount of enzyme bound to the ELISA well can be paper towel works well)
determined by its activity with a suitable substrate. If
the enzyme-catalyzed reaction results in a color change Pipettors capable of measuring 100 µL and 2 µL (drops
of a dye, the presence of the enzyme can be determined and droppers can also be used)
visually and its concentration estimated by the intensity Pasteur pipet attached to an aspirator for removing liq-
of the color produced or quantified by spectroscopy. uid from ELISA wells
Since enzymes typically catalyze the specific reaction Distilled or deionized water should be used to make all
of hundreds of molecules of substrate, the detection reagents.
power of the ELISA is greatly amplified.
Experimental Procedure
Materials
Bind the antigen to the ELISA well and wash away excess:
PBS buffer (phosphate buffered saline): 8.0 g NaCl, 1.44 1. Add 100 µL (or one drop from an eyedropper) of each
g Na2HPO4, 0.24 g KH2PO4 per liter, pH 7.2 dilution of biotinylated BSA to an ELISA well, and
0.05 M phosphate citrate buffer pH 5.0: 51 mL 0.2 M 100 µL of 3% BSA to one well to serve as a negative
Na2HPO4 added to 49 mL 0.1 M citric acid and brought control.
to 500 mL with water 2. Incubate 15 min at room temperature in a humid box.
3% BSA (bovine serum albumin) (Sigma) in water 3. Remove the contents of the wells by aspiration. This
Serial dilutions of biotinylated BSA (Sigma) including is most easily accomplished using a Pasteur pipet at-
1:100, 1:500, 1:1000, 1:5000, 1:10,000 and 1:50,000 tached to an aspirator. To avoid contamination be-
dilutions tween wells, rinse the pipet by aspirating water before

Figure 1. General ELISA protocol. Antigens in a com-

plex mixture bind nonspecifically to the sides of a poly-
ethylene well. After removal of any unbound antigens,
albumin is used to coat the remaining exposed sur-
face of the well, and excess albumin is removed. An
enzyme-linked antibody specific for the antigen of
interest is added and the excess is removed. When
the enzyme substrate is added, a color change is ob-
served, which is proportional to the amount of antigen
of interest in the original sample.

Legend: antigen of interest

albumin

enzyme-linked antibody

1276 Journal of Chemical Education • Vol. 75 No. 10 October 1998 • JChemEd.chem.wisc.edu

 In the Laboratory

continuing to the next well. Avoid scraping the sides for pregnancy, and design an ELISA experiment that would
or bottom of the well. To wash the wells, completely detect the presence of HIV antibodies in serum.
fill each well with PBS; remove the wash by aspira- Variations of the basic ELISA are used in hospitals, clinics,
tion. Repeat twice. and agricultural and research laboratories for many purposes.
Block the wells with albumin and wash away excess: For example, the basic ELISA can be used in histochemistry
1. Completely fill each well with 3% BSA. Incubate 15 to determine the presence and location of compounds within
min at room temperature in a humid box. a cell using a dye that precipitates when reduced. The presence
2. Aspirate to remove the BSA solution and then wash of particular antibodies, such as antibodies to the AIDS virus,
the wells three times with PBS as before. can be detected and quantified using a competitive ELISA in
which enzyme-linked antibodies compete with patient anti-
Bind enzyme-linked antibody to available antigens: bodies for HIV antigens on a solid support. Therapeutic and
1. Add 100 µL of antibiotin-peroxidase conjugate solu- nontherapeutic drugs, infectious agents, hormones, and numer-
tion to each well. ous biological compounds are among the types of materials
2. Incubate 15 min at room temperature in a humid box. detected and quantified by ELISAs.
3. Meanwhile, prepare the enzyme substrate (the substrate ELISAs are often used to quantify antigens by choosing
must be made within 10 min of use). Dissolve one appropriate dilutions and comparing absorbances in these
TMB tablet in 1.0 mL of phosphate-citrate buffer. ELISA wells to those of standards in other wells of the same
When the tablet is dissolved, dilute the TMB with 9.0 ELISA plate. Because of the small volumes and large number
mL of phosphate-citrate buffer and add 2 µL of 30%
hydrogen peroxide.
of wells in an ELISA plate, absorbances are usually measured
directly in the well via a microplate reader. Reproducible re-
4. Wash the ELISA wells three times with PBS.
sults for quantitation require technical skill, precise timing,
Add enzyme substrate and observe color change: and/or automated procedures; for this reason the qualitative
1. Add 100 µL of prepared enzyme substrate to each well. ELISA as outlined here is probably more appropriate for
The intensity of the blue color is proportional to the freshman-level and high school courses. However, if students
amount of biotin (and biotinylated BSA) in the sample are familiar with Beer’s law, they can easily appreciate how the
well. Simple visualization can be used to determine the qualitative ELISA shown here can be extrapolated to quan-
detection limits of the assay and the useful concentra- titative detection.
tion range of the antigen. This can serve as a powerful
demonstration of the detection power of ELISAs.
The ELISA experiment has several advantages: (i) it uti-
lizes state-of-the art diagnostic procedures used routinely in
2. If a microplate reader is available, the absorbance can
be obtained for each well. In this case, careful mea-
clinical diagnoses; (ii) it reinforces concepts learned in
surement of biotinylated albumin added to each well accompanying lectures such as the specificity of enzymes,
can serve as a standard curve and demonstrate the lin- enzyme assays to detect minute levels of compounds, and the
earity of the response through Beer’s law as well as the chemistry of peroxidase; and (iii) it introduces new concepts
usual concentration range of the antigen. If using a and procedures such as the specificity of antibodies and the
microplate reader, the enzyme reaction should be micro scale of assays often used in clinical situations.
stopped after 20 min by adding 100 µL of 3 M sulfu-
ric acid. The absorbance is read at 450 nm. Acknowledgment
Discussion The development and testing of this laboratory experi-
ment was funded in part by a Curriculum Development
The ELISA described here is simple, fast, and relatively Grant from Indiana University South Bend.
cost-effective. The experiment demonstrates the power of
antibodies and enzymes to detect the presence of small Literature Cited
amounts of a particular antigen in a mixture. ELISAs are often
used in this way to detect the presence of antigens (e.g., 1. Van Weeman, B. K.; Schuurs, A. FEBS Lett. 1971, 15, 232–236.
human chorionic gonadotropin in urine, the “pregnancy test”) 2. Engvall, E.; Perlmann, P. Immunochemistry 1971, 8, 871–874.
or of a particular virus in a biological sample to confirm a 3. Rubenstein, K. E.; Schneider, R. S.; Ullman, E. F. Biochem.
Biophys. Res. Commun. 1972, 47, 846–851.
diagnosis.
4. Monroe, D. Anal. Chem. 1984, 56, 920A–931A.
This laboratory exercise has been used successfully in our
5. Antibodies: A Laboratory Manual; Harlow, E.; Lane, D., Eds.; Cold
freshman chemistry classes for nursing and health science Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988.
majors. Technical difficulties are generally related to adding 6. Calbreath, D. F. Clinical Chemistry: A Fundamental Textbook;
reagents in the proper order to the proper wells. Aspiration Saunders: Philadelphia, 1992; pp 153–177.
of the excess reagents avoids cross contamination of wells and 7. Anderson, S. C.; Cockayne, S. Clinical Chemistry: Concepts and
is reproducible so long as care is taken to rinse the aspiration Applications; Saunders: Philadelphia, 1993; pp 92–106.
pipet between wells and to avoid scraping the wells. Follow- 8. Antibody Techniques; Malik, V. S.; Lillihoj, E. P., Eds.; Academic:
up questions incorporated in our lab reports ask the students San Diego, 1994.
to calculate the detection limit of biotin in their experiment, 9. Crowther, J. R. ELISA: Theory and Practice; Humana: Totow,
extrapolate the ELISA concept to develop a diagnostic test NJ, 1995.

JChemEd.chem.wisc.edu • Vol. 75 No. 10 October 1998 • Journal of Chemical Education 1277