TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.3 DETECTION/Serology

Section 2.3.7
Enzyme-Linked
Immunosorbent Assays on
Nitrocellulose Membranes
(NCM-ELISA)

NCM-ELISA is an immuno-enzymatic test that uses nitrocellulose

membranes instead of the polystyrene microtitration plates as a support
for the reagents used in the serological reaction. The test is as sensitive
as the direct double antibody sandwich ELISA (DAS-ELISA), easier to
perform, and can be completed in a shorter period of time. It also has
another very important advantage: the samples can be dotted onto the
nitrocellulose membrane and stored for several weeks before continuing
with the test; or they can be sent to another laboratory for development.
 All the steps are done at room temperature. The test consists of:

• Blotting a very small amount of the test sample (2 to 30 ul) onto

the membrane.

• Blocking areas not utilized by the samples.

• Reacting the virus particles with specific antibodies (IgG).

• Detecting the virus specific antibodies with enzyme-labeled

antibodies by means of an appropriate substrate (see figure).

Figure 1. Steps in the NCM-ELISA test.

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 The intensity of the coloration, which is proportional to the virus
concentration, is stable for a long period. The membrane can be easily
stored.

As in DAS-ELISA, several viruses can be detected at the same time on

one membrane by using a mixture of antibodies that are specific for the
virus to be tested. This is especially useful for seed production programs
where it is generally important to learn only if the sample is infected with
virus(es) or not.

Materials and Methods

1. Preparation of buffers and solutions

a) TBS pH 7.5 (2000 ml)

4.84 g TRIS (0.02M)

58.44 g NaCl (0.5M)
0.40 g NaN3 (0.01%)*

*Optional. Use only if the buffer solution is stored for several days. Sodium azide is a toxic
substance. Handle with care.

Dissolve in 1990-ml distilled water and adjust pH to 7.5 with HCI. Add
distilled water to 2000-ml final volume.

b) Extraction solution

For potato plant samples: (100 ml)

DIECA 0.01 M
EDTA 0.01 M
100 ml of TBS

For sweetpotato plant samples: (100 ml)

0.2 g Na2SO3 (0.2%)
100 ml of TBS

c Blocking solution (25 ml)

5 g milk powder
0.5 ml Triton X-100
25 ml TBS

Dissolve milk powder in a small volume of TBS and complete to 25 ml

with TBS. Add Triton X-100 and mix well. Prepare fresh blocking solution
for each test.

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 d) Antibody solution (50 ml)
1 g milk powder
50 ml TBS

Dissolve milk powder in a small quantity of TBS. Complete to 50

ml with TBS.

e) T-TBS washing solution (2000 ml)

2000 ml TBS
1 ml Tween-20 (0.05%)

f) Substrate buffer pH 9.5 (500 ml)

6.05 g (0.1M) TRIS base

2.92 g (0.1M) NaCl
0.51 g (5m M) MgCl2 6H20
0.05 g (0.01%) NaN3

Dissolve TRIS, NaCl, and MgCl2.6H2.O in distilled water. Adjust pH

to 9.5. Add distilled water to 500-ml final volume.

g) NBT* and BCIP* stock solutions

NBT stock solution
40 mg NBT*
2 ml N, N-dimethyl formamide (DMF) 70%

BCIP stock solution

20 mg BCIP*
2 ml N, N-dimethyl formamide (DMF)

*NBT and BCIP are highly toxic substances. Wear gloves and handle them carefully.

Mix each stock solution well. Store away from light at 4oC.

h) Developing solution (to be prepared immediately before use).

25 ml substrate buffer
100 µl NBT stock solution
100 µl BCIP stock solution

Dissolve NBT stock solution in 25-ml substrate buffer. Once

dissolved, add BCIP stock solution drop by drop, mixing constantly
in vortex.

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 2. Sample preparation

a) Collect and identify samples in separate plastic bags (this must be

done on the same day the test is performed).

b) Extraction

• Prepare extraction buffer solution for either potato or

sweetpotato samples.

• Cut three approximately 1-cm disks from each potato leaf,

or one disk for sweetpotato samples.

• Place disks from one sample in a plastic bag and add 1 ml

extraction buffer solution (per each three or one leaf disk
of potato or sweetpotato respectively).

• Macerate tissue using a test tube or a wooden rod.

• Let the bag stand for 30 to 45 minutes at 4oC.

3. Application of samples on nitrocellulose membrane

Always use gloves and forceps when handling the nitrocellulose

membranes. Fingerprints may interfere with the reactions.

a) Cut nitrocellulose membranes to desired size. Draw 0.9 x 0.9-cm

squares using a pencil. For identification, number the membrane
at bottom left corner.

b) Pre-wet the membrane by gently immersing it in TBS for 5 minutes

over a piece of Watman filter paper a little larger than the
membrane.

c) Stack 2 dry filter papers on the work surface and lay the pre-wet
membrane adhered to the wet filter paper over them. This avoids
the formation of bubbles. Allow the liquid on the surface of the
membrane to be absorbed (1–2 minutes).

d) Using a clean Pasteur pipette, carefully place one drop of

supernatant (without plant tissue) from each bag on the center of
each nitrocellulose membrane square. (Do not touch the
membrane with the tip of the pipette while dotting the sample.)
Use a clean pipette for each sample.

e) Transfer the membrane to a dry filter paper and let it dry for 30
minutes at room temperature, or for 15 minutes at 37oC.

f) If desired, put the membrane between two dry filter papers when
dry. Protect the membrane with cardboard for mailing or storing
until ready to continue with the test.

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 4. Development of the serological test

All incubation and washing steps are done at room temperature. If a

shaker is available, incubations are done with gentle agitation at 50 rpm
and washing at 100 rpm.

a) Add 25 ml blocking buffer solution to a petri dish and immerse the

membrane at 45o angle. Avoid the formation of air bubbles.
Incubate for 60 minutes. In the meantime, prepare the virus-
specific antibody solution (or the mixture of antibodies). Add
adequate IgG dilution to make 25 ml of antibody buffer solution
(antibody solution 1).

b) Discard the blocking solution and then add the mixture of antibody
solution 1 to the petri disk. Cover with lid and seal with parafilm or
adhesive tape to prevent evaporation. Incubate overnight.

c) Discard antibody solution 1 and remove the unbound antibodies by

washing the membrane with constant agitation in 30 ml of T-TBS
four times for 3 minutes each time. In the meantime, prepare
antibody solution 2, add an adequate dilution of the enzyme-
conjugated anti-lgG (antibody 2) to 25 ml of antibody solution. Put
it in a clean petri dish.

d) Remove the membrane from the last wash and place it in the petri
dish containing antibody solution 2. Incubate for 1 hour.

e) Discard antibody solution 2 and remove the unbound antibodies by

washing the membrane with constant agitation in 30 ml of T-TBS,
four times for 3 minutes each time. During final washing, prepare
the developing solution in a dark flask or cover carefully. Wearing
gloves, add 100 l of NBT stock solution to 25 ml of substrate
solution. Mix well, then add 100 l of BCIP stock solution, drop by
drop. Mix well and pour into a clean petri dish.

f) Remove the membrane from the last wash and place it in the petri
dish containing the developing solution. Be careful to completely
immerse the membrane in the solution. Wait for 15 to 30 minutes
for the reaction. Stop the reaction by discarding the developing
solution and washing the membrane with tap water three times for
5 minutes each time.

g) Let membrane dry on filter paper and store between two dry filter
papers.

Note: Occasionally, chlorophyll pigments or other coloring substances in

the samples may darken or mask positive reactions. To see the
reactions, eliminate these compounds by immersing the membrane in a
bleach (2 to 5%) or calcium hypochlorite (2%) solution for 20 minutes.
Wash in distilled water and dry.

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 Recommended Literature
Hawks, R. E., E. Niday and J. Gordon. 1982. A Dot-immunobinding assay
for monoclonal and other antibodies. Analytical Biochemistry
119:142–147.
Hibi, T. and Y. Saito. 1985. A dot-immunobinding assay for the detection
of tobacco mosaic virus in infected tissues. Journal General Virology
66:1191–1194.
Lizárraga, C. and E.N. Fernández-Northcote. 1989. Detection of potato
viruses X and Y in sap extracts by a modified indirect enzyme-linked
immunosorbent assay on nitrocellulose membranes (NCM-ELISA).
Plant Diseases 73:11–14.

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