TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.3 DETECTION/Serology

Section 2.3.2
Antiserum Production

The choice of animals to be immunized for antiserum production is

determined by four basic considerations.

• The species from which the antigen is isolated.

• Whether polyclonal or monoclonal antibodies are needed.
• The amount of antigen available.
• The amount of antiserum needed.

Rabbits, mice, rats, hamsters, or guinea pigs are used according to the
researcher’s needs (see Table 1).
Table 1.

Animal Maximum volume of Possibility of production of Remarks

antiserum monoclonal antibodies
(ml obtainable)
Rabbit 500 No The best choice for the production of polyclonal
antibodies, when the antigen is a limit factor

Mouse 2 Yes Offers the best monoclonal antibody production

Rat 20 Yes A good choice for the production of monoclonal

antibodies

Hamster 20 No A good choice for the production of polyclonal

antibodies when the antigen is a limit factor.

Guinea pig 30 No Difficult to bleed

CIP employs rabbits in its polyclonal antisera production process, using

the following steps:

a. First immunization: Multiple intradermal (4 weeks before

production).

b. Bleeding: To test the effect of antiserum and titer.

c. Second immunization: Intramuscular (to supply the necessary

boost in antigen numbers).

Procedure

Step a): First immunization

1. Immunogen preparation

10 mM Phosphate buffer pH 7.4 0.5 ml

purified virus 100–200 µg
Freund’s adjuvant* 0.5 ml

*Complete or incomplete.

Mix the virus in 10 mM of Phosphate buffer, and add Freund's adjuvant.

Shake vigorously for 5 min using a tube shaker (vortex) until a uniform
and dense suspension is obtained. It is advisable to keep this on ice to
prevent degradation.

2. Inoculation

Shave an area on the rabbit's back. This has to be large enough to allow
40 or 50 inoculation sites for approximately 20 l of immunogen in each.

Use a 1 ml syringe and a 26 G / " needle. Inoculate intradermally on

both sides of the back using the backbone as a reference. Take
appropriate precautions to avoid infection in the inoculated area.

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 Step b): Bleeding

The fourth week after inoculation, the first bleeding can proceed in order
to test the titer (specificity) of the antibody produced. The titer is
determined by the microprecipitation technique described later.

Place the rabbit in an appropriate box to keep it still during the bleeding.
Locate the marginal vein in one of the ear, (if this is not visible, use a
lamp to warm the ear and swell the vein).

Shave the ear and make an angular incision of about 45° (never cut
transversally), making sure that the cut is deep enough to allow clean
bleeding. Collect the blood in a clean glass tube. A maximum of 30 ml of
blood may be collected in each session.

Stop the bleeding by pressing the cut with a sterile piece of cotton. Press
for about 10 to 20 min until the bleeding stops.

Step c) Second immunization

Once the titer has been obtained, it is convenient to give regular booster
immunizations in order to collect the maximum amount of antiserum.
Booster immunizations should be given every 6 weeks and the blood
collected 10 days after each booster.

Table 2. Main methods for rabbit immunization

Route Abbreviation Maximum volume Adjuvant Immunogen Remarks

requirement

Subcutaneous sc 800 µl per site yes/no Soluble or insoluble Easy to inoculate

Intramuscular Im 0.5 ml yes/no Soluble or insoluble Slow release

Intradermal Id 100 µl per site yes/no Soluble or insoluble Difficult to

inoculate slow
release

Intravenous iv 1 ml no Soluble, ionic and non- Ineffective for

ionic* primary
immunizations

Intraperitoneal Ip Not advisable in Detergents

rabbits

*Less than 0.2% salts or 0.3 M urea.

Microprecipitation Test

This test is used to determine the titer and specificity of the crude
antibody.

a) Materials:

Petri dishes


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 Lidded plastic boxes


W ax pencil, ruler


Appropriate-sized test tubes



1-ml pipettes


Syringe


Antiserum


Healthy and infected plant leaves



Low speed centrifuge



Vortex


Saline solution (NaCl 0.85%)



b) Sample preparation:

Weigh the samples (should be slightly more than 1 g each); dilute 1/1
(w/v) in saline solution; and macerate carefully with a roller or test tube.

Collect the macerate in a tube, spin at 5000 rpm for 5 min, and pour the
supernatant into a test tube.

Prepare battery of 12 test tubes which contain 1ml of saline solution each
and label them according to the following dilutions: 1/2, 1/4, 1/8, 1/16,
1/32, 1/64, 1/128, 1/256, 1/512, 1/1,024, 1/2,048, and 1/4,096.

Add 1ml supernatant to the first tube and mix thoroughly. Transfer 1 ml
from this tube to the second and so on until all twelfth tubes are used.

c) Antiserum preparation

Prepare a battery of 12 tubes with 1 ml saline solution; label as in b)

above.

Add 1 ml antiserum to the first tube, mix well, and transfer 1 ml from this
tube to the second; continue until you finish the dilutions.

d) Procedure test

Using a wax pencil, draw perpendicular lines on a petri dish to make a

grid design. Write in the schematic diagram as shown below.

With a syringe, dispense one drop of each dilution of both the antiserum
and antigen in the center of each square so that each antiserum dilution
will be tested against each dilution of the clean and infected sample. Be
sure to clean the syringe 10 times with water between each sample.

Place the dishes in a rotating shaker at 100 rpm for 15 min. Incubate the
dishes in a wet chamber or in a large container with a lid lined with wet
paper.

Take the first reading after 2 hours. Watch the reactions in the dark room
by means of a stereoscope with a side lamp. The presence of
precipitation shows a positive reaction.

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• Sec 2.3.2 – 99 • Page 4 - INTERNATIONAL POTATO CENTER
 Record the data from the readings and read again after 24 hours.

Ab ½ ¼ 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 1/2048

Ag
½
¼
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
1/2048

Recommended Literature
Mathews, R.E.F. 1957. Plant Virus Serology, Cambridge University
Press, Church Army Press. Cowley, Oxford.
Nowotny, 1969. Basic Exercises in immunochemistry. A Laboratory
Manual. Springer-Verlag. Berlin, Germany.
Harlow, E. and D. Lane. 1988. Antibodies: a laboratory manual. Cold
Spring Harbor Laboratory. New York, USA.

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