TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.3 DETECTION/Serology

Section 2.3.8
Serological Assays with
Enzymatic Conjugates:
Penicillinase Enzymatic
Conjugate
(PNC-DAS-ELISA)

The double antibody sandwich (DAS) variant of the enzyme-linked

immunosorbent assay (ELISA) has become a routine method for potato
virus detection.

This serological test uses enzyme-antibody complexes called enzymatic

conjugates. For simplicity of evaluation, the enzymes used in DAS-ELISA
should produce some kind of color reaction. At present, the most widely
used enzyme is alkaline phosphatase obtained from the intestines of
calves and from Escherichia coli.
 Many developing countries cannot include this rapid and efficient method
in their seed programs due to the high cost per sample. Most of the cost
originates in the enzyme and the enzymatic substrate used, i.e., p-
nitrophenyl phosphate.

CIP has tested DAS-ELISA for potato virus detection using penicillinase
(B-lactamase), which has a cost comparable to alkaline phosphatase.
However, this method uses a cheaper substrate: the iodine-starch-
penicillin complex.

DAS-ELISA using penicillinase (PNC-DAS-ELISA) is based on the

iodometric method, with the iodine-starch complex as the pH indicator.
This complex is formed through the arrangement of iodine atoms among
the matrix of the dissolved starch molecule. This blue complex is quite
stable.

When the penicillinase acts

on the penicillin, the
resulting penicillonic acid
acts on the iodine complex,
making it colorless.

Using this substrate, the

sensitivity of the penicillinase
test is equal to, and
frequently higher than, that
obtained with alkaline
phosphatase when tested
with viral purifications diluted
in the sap of healthy potato
plants.

This visual evaluation test is

a very economical option as
a routine technique.

Figure. Diagrammatic
representation
of the PNC-DAS-ELISA

Methods

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 1. Plate coating

a) Prepare coating buffer solution (carbonate buffer 0.05 M). Adjust

pH to 9.6.

1.59 g Na2CO3
2.93 g NaHCO3
0.2 g NaN3*

b) Dilute coating IgG in the buffer solution and mix well (IgG can be
diluted up to 1/1000).

c) Fill each well with 150 ml of diluted IgG. Place the plate in a plastic
bag and seal with adhesive tape.

d) Incubate for 3 hours at 37°C or at room temperature for 5 hours.

e) Eliminate lgG solution from the plate by shaking vigorously and

drying on paper towels.

2. Plate blocking

a) Prepare the blocking solution by dissolving non-fat milk powder or

egg albumin in phosphate buffer saline solution (pH 7.4).

8.0 g NaCl
0.2 g KH2PO4
2.9 g Na2HPO4 12H20
0.2 g KCl
0.2 g NaN3*
Add distilled water to make
1 liter final volume

b) Fill each well with 200 l blocking solution. Seal plate as before.

c) Incubate plate for 1 hour at 37oC.

d) Wash wells with washing buffer solution (PBS-Tween). Repeat 3

times for 3 minutes each time.

*Optional: use only if the buffer solution is stored for several days.

3. Adding samples

a) Place labeled samples in plastic bags (this may be done 1 or 2

days in advance if the samples can be stored at 4oC). Use infected
(positive) and healthy controls.

b) Prepare the maceration buffer solution: PBS-Tween with 2%

polyvinyl pyrrolidone (PVP-40), and 1% non-fat milk powder or egg
albumin.

c) Add buffer solution to samples. Dilution 1:5 (sample weight: buffer

volume).

P.V. • Sec 2.3.8 – 99 • Page 3 - INTERNATIONAL POTATO CENTER

 d) Macerate samples with a test tube or a wooden roller.

e) Fill wells with 150 l extracted sap (including positive and healthy
controls) following a predetermined scheme.

f) Cover the plate and incubate at 4oC overnight.

g) Wash the plate with PBS-Tween until all traces of sap have
disappeared from the wells. Repeat 3 times for 3 minutes each
time.

4. Adding the enzymatic conjugate

a) Prepare the conjugate buffer solution: PBS-Tween with 0.2% non-

fat milk powder or egg albumin.

b) Dilute the enzymatic conjugate in buffer solution.

c) Fill wells with 150 l diluted enzymatic conjugate.

d) Cover the plate and incubate at 37oC for 3 hours.

Note: Substrate preparation must begin 1 hour before the end of plate
incubation period with the enzymatic conjugate.

e) Wash the plate 3 times for 3 minutes each time.

5. Developing the plate

a) Prepare a 2% starch solution in 0.2 M phosphate buffer solution.

Adjust pH to 7.2 with 0.5 M HCl or 0.5 M NaOH.

b) Dissolve starch by heating. Allow to cool, stirring constantly to

prevent aggregation of starch.

c) Add 25 ml 0.2 M phosphate buffer solution pH 7.2 and 100 l

iodine stock solution (200 mg iodine and 5.32 g potassium iodide
in 10 ml distilled water) to 5 ml starch solution. Adjust pH to 7.2.
The solution is purple blue.

d) Add penicillin G to the substrate buffer solution (this must be done

during the last washing). Make sure the solution is completely cold.

e) Fill wells with 150 l substrate solution and wait for the reaction.
In positive reactions, the solution in the well becomes totally or
partially colorless, as compared with the healthy control, which
remains dark blue to purple.

Note: Reaction usually begins 10 minutes after placing the substrate in

wells.

Recommended Literature
Khurana, S.M.P. M.N. Singh and S. Kumar. 1989. Peroxidase and
penicillinase based–ELISA for detection of potato viruses. Annual

P.V. • Sec 2.3.8 – 99 • Page 4 - INTERNATIONAL POTATO CENTER

 Meeting of the Indian Phytopathological Society. New Delhi (28 Feb–
2 Mar.
Sudarshana, M.R. and D.V.R. Reddy. 1989. Penicillinase-based enzyme-
linked immunosorbent assay for the detection of plant viruses.
Journal of Virological Methods 26(1):45.

P.V. • Sec 2.3.8 – 99 • Page 5 - INTERNATIONAL POTATO CENTER