TIPS, TRICKS & A Quick & Simple Single-Antibody
TECHNIQUES Immunoassay for the Student
Laboratory
• PAUL L. GUY
ABSTRACT
In this exercise students use a simplified version of an immunoassay that relies on
the nonspecific binding of proteins to nitrocellulose followed by the specific binding
of antibodies to antigen. This antigen–antibody interaction is detected when a
chromogenic substrate, catalyzed by the enzyme conjugated to the antibody,
produces a color change. The immunoblot is less expensive and faster to perform
than plastic-plate-based assays. I have used this assay for over 10 years in
undergraduate courses: in ecology to estimate disease incidence; in botany to
explore immunological techniques; and in biotechnology as an exercise in product
development.
Key Words: Antibody; antigen; disease incidence; enzyme; immunoassay;
immunoblot; nitrocellulose.
Introduction
Enzyme-linked immunoassays are commonly used in medical, vet-
erinary, and plant research and diagnosis because of their high sen-
sitivity, robustness, and relatively low cost when many samples need
to be tested (Tijssen, 1985). Typically, immunoassays are used to
test organisms for infection by viruses, bacteria, or fungi; however,
antibodies can be produced to detect a wide range of proteins and
some other molecules. So there are immunoassays to detect influ-
enza (Landry, 2009), canine distemper (von Messling et al., 1999),
and barley yellow dwarf viruses (Guy, 1988); immunoassays to test
whether you are pregnant; and immunoassays to detect GMOs
(Stave, 2002) or gluten (Wu et al., 2012) in your crops. These assays
utilize an enzyme (often alkaline phosphatase) chemically linked to
an antibody that binds to specific parts of target antigens (usually
proteins). This specific binding is detected by incubating the conju-
gated antibody–antigen complex with a substrate that, when cata-
lyzed by the enzyme, produces a color change that can be analyzed
The American Biology Teacher, Vol. 81, No. 3, pp. 197–199, ISSN 0002-7685, electronic ISSN 1938-4211. © 2019 National Association of Biology Teachers. All rights
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THE AMERICAN BIOLOGY TEACHER A QUICK & SIMPLE SINGLE-ANTIBODY IMMUNOASSAY FOR THE STUDENT LABORATORY
visually or with a spectrophotometer (Tijssen, 1985; Graddon &
Randles, 1986).
Materials
A range of ornamental, field-crop, and lawn species can be used in this
exercise. White clover (Trifolium repens) is a reliable species to use, as
it is widespread in pastures and lawns and is a common weed. Virus
incidence is usually high in white clover, and individual plants may
be infected with more than one virus. Conjugated antibodies to alfalfa
mosaic virus, clover yellow vein virus, and white clover mosaic virus
(three viruses that commonly infect clover; McLaughlin et al., 1996)
are available from Agdia (Elkhart, Indiana, USA) or DSMZ (Braunsch-
weig, Germany). They also provide positive and negative controls.
The chemicals listed below are available from Sigma.
Procedure
For each leaf sap sample, place 0.1 g of clover leaves in a mortar
with 0.9 mL of phosphate buffered saline (PBS: 8 g NaCl, 0.2 g
KH2PO4, 2.9 g Na2HPO4.12H2O, 0.2 g KCl in 1 L deionized
water pH 7.4) and grind thoroughly with a pestle. Place a piece
(20 × 25 mm) of nitrocellulose membrane (Bio Rad Trans-Blot
membrane) on a paper towel. Pipette drops (about 1–2 µL) of leaf
sap onto the membrane and allow to air dry for 5 minutes. For one
student’s layout, see Figure 1.
Half fill a Petri dish with blocking buffer (PBS, 0.05% Tween
20 [v/v], 5% [w/v] skim milk powder), dip the membrane (use for-
ceps!) into the Petri dish edge-first, then submerge. Agitate briefly
every 5 minutes for a total of 30 minutes.
Remove the membrane from the dish and blot lightly by resting
on a paper towel. Tip blocking buffer out of the dish and dry the dish
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Figure 1. An example of a student’s immunoblot.
with a paper towel. Place membranes in the dish (sample side up).
Pipette ~0.5 mL of alkaline phosphatase conjugated antibody, using
the manufacturer’s recommended dilution (in PBS-Tween, 0.5% skim
milk powder), onto the membrane to form a “liquid pillow.” Incubate
for 1 hour.
Using a torn piece of paper towel, gently soak the conjugate
solution off the face of the membrane and then flood the dish with
deionized water. Agitate for 1 minute.
Transfer the membranes to a new Petri dish containing AP 7.5
buffer (15.75 g Tris-HCl, 5.85g NaCl, 0.4g MgCl2 in 1 L deion-
ized water pH 7.5 with 0.05% [v/v] Triton X-100). Agitate for
5 minutes.
Remove membranes to fresh paper towel while you replace AP
7.5 with AP 9.5 buffer (12.1 g Tris base, 5.85 g NaCl, 1 g MgCl2 in
1 L deionized water pH 9.5). Agitate the membranes briefly in the
new buffer and allow to equilibrate for 5 minutes.
Replace with fresh AP 9.5 and continue to wash for another
5 minutes.
During the conjugated-antibody incubation step, dissolve 5 mg of
BCIP (5-Bromo-4-chloro-3-indolyl-phosphate) in 100 µL dimethyl-
formamide and combine with 10 mg NBT (nitro-blue tetrazolium)
dissolved in 30 mL AP 9.5. This is the color development solution.
Remove the membrane from the dish and blot lightly on paper
towel. Tip AP 9.5 buffer out of the dish and dry with a paper towel.
Place membranes in the dish (sample side up). Pipette ~1 mL of
color development solution onto each membrane.
After 15 minutes, record color development of individual spots.
Samples containing virus turn purple; virus-free samples remain
green.
When color has developed, flood the dish with AP 7.5 and agi-
tate for 2 minutes. Tip off AP 7.5 and flood with deionized water.
Remove and dry the immunoblot (keeps indefinitely).
Procedure Variations
Samples can be applied to the membrane in other ways. Tooth-
picks are a cheap and effective way of applying the sap extracts
to the membrane. Sap from herbaceous species such as daffodil
or faba bean can be applied directly by pressing freshly cut stems
to the membrane. Increase the number of field samples applied by
not duplicating samples on the membrane. Students can test 20
samples per membrane and can compare virus incidence between
different sites. This encourages student exploration: they can
compare their lawn or favorite sports ground with other students’
samples. Students then discuss which statistical analyses are suit-
able for determining whether the differences they observe are
significant.
198 THE AMERICAN BIOLOGY TEACHER
Figure 2. How the immunoblot works.
Two or three membranes can be processed in one Petri dish,
thereby reducing plastic and buffer consumption.
A densitometer can be used to measure the intensity of color
development as a proxy for measuring relative virus concentration.
A simpler way to do this is to cover the dish after the color devel-
opment solution has been applied and score the sample spots as
positive or negative every 2 minutes. Students will get the sense
that the spots develop at different rates. You can then encourage
them to think about and discuss what this means.
Assessment
Students are required to write lab reports demonstrating their under-
standing of the immunological/biochemical principles involved in
producing their immunoblot and comparing and contrasting its util-
ity and sensitivity with that of standard ELISA assays (which they
can run in parallel). The learning objective is a deeper understanding
of the strengths and limitations of the different assays. For example,
student-directed research lets them discover that samples can bind
to nitrocellulose under a greater range of conditions than to plastic
surfaces. They are expected to demonstrate their understanding by
generating a graphic similar to Figure 2.
In a third-year biotechnology class, students are asked to regard
the immunoblot as a product they are developing, and they are
assessed on a brochure they produce (see Figure 3) outlining the
benefits of using their kit as well as describing how it works. This
encourages lateral thinking, and some students find it a challenge
not to produce a standard lab report. During the blocking-buffer
incubation step, the students are told they will be interviewing the
technician who set up the day’s lab. They discuss among themselves
and formulate questions they need to ask the technician to gauge the
amount of time and effort required to assemble each component of
the lab. During the conjugated-antibody incubation step, the stu-
dents interview their technician with the objective of gaining a
deeper understanding of how the lab was set up. They also use this
to develop the sales pitch they will use in their brochure.
VOLUME 81, NO. 3, MARCH 2019
Figure 3. Examples of students’ brochures.
Conclusion
Immunoassays are used extensively in the biological sciences. This
quick, simple, cost-effective lab exercise introduces students to the
important concepts of specific and nonspecific binding used in a vari-
ety of ways in different immunoassays (Tijssen, 1985). The dot-blot
assay is an inexpensive way to test field samples for the presence of
antigen and is a useful vehicle for students to explore product devel-
opment and marketing in biotechnology.
Acknowledgment
Thanks to Vickey Tomlinson for enduring many years of student
questioning.
References
Graddon, D.J. & Randles, J.W. (1986). Single antibody dot immunoassay:
a simple technique for rapid detection of a plant virus. Journal of
Virological Methods, 13, 63–69.
Guy, P.L. (1988). Pasture ecology of barley yellow dwarf viruses at
Sandford, Tasmania. Plant Pathology, 37, 546–550.
THE AMERICAN BIOLOGY TEACHER A QUICK & SIMPLE SINGLE-ANTIBODY IMMUNOASSAY FOR THE STUDENT LABORATORY
Landry, M.L. (2009). Developments in immunologic assays for respiratory
viruses. Clinics in Laboratory Medicine, 29, 635–647.
McLaughlin, M.R., Larsen, R.C., Trevathan, L.E., Eastman, C.E. & Hewings, A.D.
(1996). Virus diseases of American pasture and forage crops. In Pasture
and Forage Crop Pathology (pp. 289–302). Madison, WI: American
Society of Agronomy & Crop Science Society of America.
Stave, J.W. (2002). Protein immunoassay methods for detection of biotech
crops: applications, limitations, and practical considerations. Journal of
AOAC International, 85, 780–786.
Tijssen, P. (1985). Practice and Theory of Enzyme Immunoassays.
Amsterdam, The Netherlands: Elsevier.
von Messling, V., Harder, T.C., Moennig, V., Rautenberg, P., Nolte, I. &
Haas, L. (1999). Rapid and sensitive detection of immunoglobulin M
(IgM) and IgCG antibodies against canine distemper virus by a
new recombinant nucleocapsid protein-based enzyme-linked
immunosorbent assay. Journal of Clinical Microbiology, 37,
1049–1056.
Wu, M.J., McKay, S., Hegedus, E. & Chin, J. (2012). Proteolytic extraction
enhances specific detection of the novel ‘S’-type low molecular weight
glutenin subunit in wheat by monoclonal antibody. Journal of Cereal
Science, 55, 53–60.
PAUL L. GUY is an Associate Professor in the Department of Botany,
University of Otago, New Zealand; e-mail: paul.guy@otago.ac.nz.
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