TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.3 DETECTION/Serology

Section 2.3.6
Enzyme-Linked
Immunosorbent Assays:
Double Antibody “Sandwich”
(DAS-ELISA)

DAS-ELISA is a very sensitive plant virus identification technique. It has

the following advantages:

• Higher sensitivity than other serological methods for the

identification of most viruses, including PLRV (potato leaf roll virus)
and PVA (potato virus A), which are found only in low
concentrations in infected plants.

• Lower requirements of antiserum than other serological methods.

 Although this technique comprises numerous steps, it can be easily
adapted as part of a seed improvement program for rapid and reliable
detection of most viruses. It is the only serological method for detecting
PLRV in tubers or foliage.

Method

The principle underlying the modified double-antibody ELISA technique

(Clark and Adams 1977) is as follows:

1. Plate coating sensitizing. The virus-specific antibodies

(gammaglobulin: lgG) are adsorbed to the surface of each well in
the microtiter plate. The excess, non-adsorbed gammaglobulin is
then rinsed. A specific antibody is required for each virus tested.

2. Adding test samples. The sample to be tested is added to the

plate. If the plate contains particles of the specific virus (antigen)
for the antibodies coated during the previous stage, the particles
will adhere to the antibodies and will not be eliminated during
rinsing.

3. Adding the enzyme-gammaglobulin conjugate (lgG– AP). These

antibodies adhere only to the virus particles. Excess conjugate is
eliminated by rinsing.

4. Adding the substrate. Finally, the substrate is added to the plate.

The intensity of the yellow color that develops is proportional to the
quantity of virus in the sample.

1. Coating

The first step in this modified ELISA technique is to coat the wells of the
microtiter plate with a virus-specific antibody. Proceed as follows:

a) Prepare the adsorption buffer:

1.59 g Na2CO3
2.93 g NaHCO3
add distilled water up to 1
liter of solution
0.2g NaN3*

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 Figure 1. Steps in the DAS-ELISA test

b) Dilute the specific antibody in the absorption buffer to the adequate

dilution.

c) Add 200 l of IgG solution to each well of the microtiter plate.

d) Incubate for 2 to 4 hours at 37oC (all the wells in the plate may be
used).

e) After incubation, empty plate and wash with PBS-Tween. The

composition of PBS-Tween is as follows:
8.0 g NaCl
0.2 g KH2PO4
2.9 g Na2HPO4 .12H2O
0.2 g KCl
0.2 g NaN3
0.5 ml Tween 20
Add distilled water to make 1 liter
total volume

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 f) Wash the plate. Fill the wells with PBS-Tween and leave for 3
minutes. Empty the plate. Repeat 3 times. Washing eliminates
non-adhering IgG in the wells. The plates can be stored at –20oC
for long periods for routine application of this technique. They can
be prepared in advance.

2. Adding samples (antigen)

The test samples can be extracted by maceration of plant tissue and use
of a buffer solution to obtain a 1/10 to 1/20 dilution.

a) To sample maceration, add fresh extraction buffer (PBS-Tween +

2% PVP-40) in the bag.

Extraction buffer

2% PVP– 40
2% Egg albumin or non-fat milk
Add PBS-Tween

b) Including both healthy and infected samples as a control,

macerate the samples separately by rolling a test tube or a similar
object over each plastic sample bag. The sap may also be
extracted from the samples with a juice extractor.

c) Fill the wells with 200 µl of diluted samples. Use a clean pipette for
each sample. Normally, one well is enough for each sample.

d) Incubate the plate at 4°C for 18 hours (overnight) or at 37°C for 4

to 6 hours.

e) Wash the plate as described above. Repeat 3 times or until the

wells are totally clean of plant sap.

3. Adding the conjugate (enzyme-gammaglobulin IgG-AP)

Although other enzymes can be conjugated to the antibody molecules,

alkaline phosphatase has given the best results. To add the conjugate,
proceed as follows:

Conjugate buffer

2% PVP-40
0.2% egg albumin or non-fat milk
Add PBS-Tween

a) Dilute the enzymatic conjugate in PBS-Tween containing 2% PVP-

40 and 0.2% egg albumin or milk powder. (For the most

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 appropriate dilution, please refer to the chapter on antiserum
purification and conjugation).

b) Add 200 µl (0.2 ml) of diluted enzyme-gammaglobulin conjugate

(lgG–AP) to each well (fill wells along the sides with buffer solution
if they were not used for samples).

c) Incubate at 37oC for 3 to 6 hours.

d) Wash the plates as described. Repeat 3 times. Excess enzymatic

conjugate not adhering to virus particles will wash away.

4. Adding the substrate

Only the wells containing the enzyme will react at this stage.

a) Prepare the substrate buffer solution.

97 ml diethanolamine
800 ml H20
Adjust pH to 9.8
Add distilled water to make 1 liter

The buffer solution should be prepared just before use. It cannot be

stored.

b) Dissolve the enzyme substrate in buffer solution to a concentration

of 0.6 to 0.8 mg/ml.

Use powder or pelletized enzyme substrate. Using pellets of a

predetermined concentration is more convenient in order to avoid
repeated weighing of substrate.

c) Add 150 l of the substrate solution in each well.

d) Incubate the plate at room temperature until the reaction can be

observed (usually 30 to 60 minutes). In the wells containing
infected samples, the substrate solution will turn yellow.

The intensity of the yellow color indicates the amount of virus in the
sample. The stronger the color, the higher the amount of the virus.
Transparent wells indicate healthy samples. Healthy controls and
buffer solution wells should remain transparent.

Results are recorded in the sample list. Readings can be made visually
(qualitative), or with a photometer (quantitative) at 405 nm wavelength.

Recommended Literature
Clark, M.F. and A.N. Adams. 1977. Characteristics of microplate method
of Enzyme Linked Immunosorbent Assay for the detection of plant
viruses. Journal of General Virology 34:475–483.
Mathews, R.E.F. 1957. Plant virus serology. Cambridge University Press,
Church Army Press, Cowley, Oxford.

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