TECHNIQUES IN PLANT VIROLOGYCIP Training Manual2.3 DETECTION/Serology

Section 2.3.2Antiserum Production

The choice of animals to be immunized for antiserum production isdetermined 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 theresearcher’s needs (see Table 1).

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Table 1.

Animal Maximum volume ofantiserum

(ml obtainable)

Possibility of production ofmonoclonal antibodies

Remarks

Rabbit 500 No The best choice for the production of polyclonalantibodies, 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 monoclonalantibodies

Hamster 20 No A good choice for the production of polyclonalantibodies when the antigen is a limit factor.

Guinea pig 30 No Difficult to bleed

CIP employs rabbits in its polyclonal antisera production process, usingthe following steps:

a. First immunization: Multiple intradermal (4 weeks beforeproduction).

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

c. Second immunization: Intramuscular (to supply the necessaryboost in antigen numbers).

Procedure

Step a): First immunization

1. Immunogen preparation

*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 uniformand dense suspension is obtained. It is advisable to keep this on ice toprevent degradation.

2. Inoculation

Shave an area on the rabbit's back. This has to be large enough to allow40 or 50 inoculation sites for approximately 20 �l of immunogen in each.

Use a 1 ml syringe and a 26 G �/�" needle. Inoculate intradermally onboth sides of the back using the backbone as a reference. Takeappropriate precautions to avoid infection in the inoculated area.

10 mM Phosphate buffer pH 7.4 0.5 mlpurified virus 100–200 µgFreund’s adjuvant* 0.5 ml

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Step b): Bleeding

The fourth week after inoculation, the first bleeding can proceed in orderto test the titer (specificity) of the antibody produced. The titer isdetermined 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 alamp to warm the ear and swell the vein).

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

Stop the bleeding by pressing the cut with a sterile piece of cotton. Pressfor 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 boosterimmunizations in order to collect the maximum amount of antiserum.Booster immunizations should be given every 6 weeks and the bloodcollected 10 days after each booster.

Table 2. Main methods for rabbit immunization

Route Abbreviation Maximum volume Adjuvant Immunogenrequirement

Remarks

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 toinoculate slowrelease

Intravenous iv 1 ml no Soluble, ionic and non-ionic*

Ineffective forprimaryimmunizations

Intraperitoneal Ip Not advisable inrabbits

Detergents

*Less than 0.2% salts or 0.3 M urea.

Microprecipitation Test

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

a) Materials:

� Petri dishes

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� Lidded plastic boxes

� Wax 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 thesupernatant into a test tube.

Prepare battery of 12 test tubes which contain 1ml of saline solution eachand 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 mlfrom 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 thistube 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 agrid design. Write in the schematic diagram as shown below.

With a syringe, dispense one drop of each dilution of both the antiserumand antigen in the center of each square so that each antiserum dilutionwill be tested against each dilution of the clean and infected sample. Besure 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 thedishes in a wet chamber or in a large container with a lid lined with wetpaper.

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

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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

Ag½¼1/81/161/321/641/1281/2561/5121/10241/2048

Recommended LiteratureMathews, R.E.F. 1957. Plant Virus Serology, Cambridge University

Press, Church Army Press. Cowley, Oxford.

Nowotny, 1969. Basic Exercises in immunochemistry. A LaboratoryManual. Springer-Verlag. Berlin, Germany.

Harlow, E. and D. Lane. 1988. Antibodies: a laboratory manual. ColdSpring Harbor Laboratory. New York, USA.