1967] MUNRO: MOLECULAR GEL COLUMN 2 4 9

U S E OF M O L E C U L A R G E L C O L U M N TO P R E P A R E P O T A T O VIRUS X I N O C U L A FOR A N T I S E R A 1

JAMES MUNRO

I NTRODUCTIO N

A simple technique has been devised at Fredericton for those who have need of potato virus X antiserum to make their own virus prepara- tions for inoculations into rabbits. The method makes use of a molecular gel column to separate or filter the virus from crude plant sap, and dialysis with polyethylene glycol to reduce the amount of liquid in the virus suspension obtained from the gel column. Gel filtration is a method of selective molecular separations by size made possible by the use of modified dextrans. This techniques is used to separate plant viruses from molecules and small macromolecules of other plant substances normally found in virus-infected plant sap.

MATERIALS AND METHODS

Preparation o] the Gel

Experience has shown that Sephadex G2002 prepared according to the manufacturer's (Pharrnacia (Canada) Montreal, P.Q.) directions provides a gel of suitable pore size for expeditious and convenient separa- tions of potato virus X. A glass tube, 90 cm long and 1.5 cm in diameter (Sephadex Laboratory Column K 15/90) 2 is used to hold the gel in a column. The most suitable height of gel column for this size of glass tube is about 75 cm. This amount of swollen gel is prepared with 4 g of Sephadex G200 in 270 ml of distilled water.

Preparation of the Glass Tube

The glass tube is aligned and fixed into a vertical position on a support stand with a double buret holder (Fig. 1). Above the junction of the capillary outlet at the bottom of the glass tube a plug of glass wool is inserted after a little distilled water has been poured in to prevent air bubbles. A layer of glass beads (Superbrite, type 130-5005, Minnesota Mining and Manufacturing Co.) 2 is placed on top of the glass wool to level it off.

Packing the Column in the Tube

The gel is stirred with enough added distilled water to remove air bubbles from the preparation and poured into the vertical glass tube. The pouring is careful and gradual so that excess water can be drained through the open capillary outlet and removed by suction from above the settling gel. Pouring is continued intermittently as the gel packs and the surplus fluid drains until a packed gel column of 75 cm is obtained. The length of tube above the packed gel is then filled with distilled water

1Contribution No. 229, Research Station, Canada Department of Agriculture, Fred- ericton, New Brunswick, Canada. Accepted January 9, 1967.

"Mention of commercial products and specific equipment throughout this paper is not an endorsement by the Canada Department of Agriculture over similar products or companies not named.

250 AMERICAN POTATO JOURNAL [Vol . 44

FIG. 1.--Left : Tube used to concentrate virus fluid by dialysis. Right : Gel columns prepared for routine operation.

1967] M U N R O : MOLECULAR GEL C O L U M N 2 5 1

and attached by plastic tube to a reservoir. Continuous flow of distilled water through the capillary outlet is maintained for at least 24 hours to wash out particles of matter that do not form part of the gel. The gel column is now ready for use.

The reservoir remains connected as long as the gel column is main- tained except for the brief periods when the virus sap is poured into the tube and is passing down into the gel. The plastic tubing connecting the reservoir with the top of the tube may be clamped to stop flow when the gel column is not in use.

Preparation of Virus .~ap Greater amounts of the potato X virus can be obtained from Nicotiana

glutinosa L., tobacco and tomato plants than from other common hosts (1, 3). However, experience has shown that young Samsun tobacco plants provide extracts that contain much virus and relatively little of other unwanted substances. These substances include proteins and nucleoproteins so close in size to vir~s particles that they are not separated by filtration through the gel column. The virus fluid collected from the column therefore contains other antibody producing substances. But the value of that part of antiserum produced against potato virus X is not lessened by the presence of other antibodies in the total bulk of antiserum.

Leaves from rapidly growing young Samsun tobacco plants, heavily inoculated with potato virus X, are picked when symptoms are fully developed. The petioles and main veins are discarded because most virus is concentrated in the leaf blades. The sap is extracted by grinding the leaves in a domestic meat grinder, and by squeezing the mascerated leaves through four layers of muslin. The expressed sap is frozen overnight, thawed, and centrifuged at 3000 rpm for 10 minutes. As changes in the exposed sap that may affect the stability of the virus particles are rapidly taking place, gel filtration should be started immediately. About 10 ml of prepared sap is required for each gel column.

Passage of Virus Through Gel Column Before the prepared sap is poured into the column tube, disconnect

the reservoir of distilled water and withdraw by suction all water in the tube above the gel. Add a few drops of a blue dextran (Blue Dextran 2000, Pharmacia (Canada), Montreal, p .Q.)2 to the top of the gel, carefully fill the tube with physiological saline solution (.85% NaC1) and attach to a reservoir of the solution for continuous flow. The free flow of saline solution through the gel column is indicated by the movement of the blue dextran. When the first drops of blue liquid pass out of the capillary at the bottom of the tube, the free liquid in the gel column is saline solution. The gel column is now ready to receive the prepared plant sap.

Immediately prior to pouring the prepared plant sap into the colunm tube, disconnect the saline solution reservoir and float a disc of filter paper down the tube on to the surface of the gel column across its diameter. After the disc is in place, withdraw all saline solution above it by suction, then pour the sap in gently. The disc should be about one cork borer size less than the inner diameter of the column tube. This disc causes a

252 AMERICAN POTATO JOURNAL [Vol. 44

momentary holdback of the virus sap before it penetrates the column, and then permits a uniform descent into the gel.

During preliminary attempts to use this technique, several drops of blue dextran should be added to the prepared virus sap before it is poured into the column tube. The blue dextran marks the movement of the high molecular weight fractions through the gel column. Although inoculum colored with this dye is not harmful to the rabbbit, it should not be used in inoculum preparations. Use the blue dextran in preliminary tests but not when producing virus inoeulum.

The saline solution reservoir should remain unattached until all the virus sap passes through the paper disc. The disc is then gently rinsed with a few drops of saline solution from a pipette. When this has passed through the paper disc into the gel, the tube is filled gently with saline solution and then attached to the reservoir for continuous flow until the virus fluid is collected.

Collection of Virus Fhdd When a gel column is used in a glass tube of this size, one drop of

fluid will pass out of the gel column every 12-15 seconds, depending on how tightly the gel is packed. There are 21 drops in a ml from the capillary outlet of this kind of tube.

About 35 ml will pass through the gel column before the simultaneous appearance of the blue dextran in the drops and the detection of the virus. The virus can be detected by using suitably diluted virus X antiserum in slide tests (4) with single drops of fluid collected immediately before and after the first appearance of the faint blue color in the drops. The total amount of virus fluid obtained will be about 20 ml - - twice the original volume of virus sap poured into the column tube.

Concentration of Virus Fluid The virus fluid as obtained from the gel column is too low in virus

content to be used as intraveinal inoculum for antiserum production. As with all other known methods to purify virus from plant saps, there is some loss, and so the concentration of virus per ml in the fluid collected from the gel column, is less than half of that in the original plant sap. It is therefore necessary to concentrate the virus solution by reducing the fluid bulk. When this is reduced from 20 ml to 1 ml, the concentration is sufficient for the solution to be used as inoculum for intravenous in- jections. About 2 ml of this inoculum is used for each inoculation and this amount is obtained if two gel columns are used simultaneously.

The virus solution is concentrated by a method developed by Kohn (2) based upon dialyses against substances of high molecular weight. The virus solution is put into a glass tube which has a small downward protuberance below its floor (Fig. 1). The protuberance holds 2 ml and the glass tube itself has an area of dialysing surface that will take the fluid well up the sides of the tube when the dialysing tubing is put into i t . .

An amount of polyethylene glycol, equal to about 20% of the virus fluid volume, is put into the dialysing tubing. The length of tubing extends over the rim of the glass tube to allow for the rising column of dialysed fluid. The bottom of the dialysing tubing reaches down to

1967] MUNRO: MOLECULAR GEL COLUMN 253

the original floor of the glass tube just above the protuberance. As there is free passage of electrolytes across the membrane, the concentrated virus fluid bulk is in inoculable physiological saline solution when the process is completed. The approximately 40 mls of virus solution obtained from two gel columns is reduced to a bulk of 2 ml, being the amount of fluid still remaining in the protuberance. If four gel columns are prepared at the same time in similar glass column tubes, 2 ml concentrations of virus inoculum can be prepared for use on alternate days.

Dlscussloi~

When the virus sap passes into the gel column, the large macro- molecules and virus particles move rapidly through the gel, leaving the brown colored plant substances behind. The continuous flow of distilled water from the reservoir maintained after the virus fluid has been collected moves this material out through the column in about 24 hours. The presence of large quantities of these plant substances has no apparent deleterious effect upon the gel. In this work, gel columns have been maintained in frequent use with plant sap for more than 3 months. However, if continuous flow is broken for periods of several hours while the brown plant substances are still in the gel column, the gel disintegrates and large cavities appear.

In this relatively crude use of the gel column, the flow of fluid through the column tube from the moment the virus sap penetrates the gel until the virus is detected in a blue drop, may be called the void volume. When this becomes constant, the dyed dextran is omitted and need not be used again until the gel column is remade or replaced.

Other refinements to the techniques are optional and seed potato certification agencies can rely upon antiserum made against virus inoculum prepared in this way. Antisera have been obtained by this method with titres up to 4,000 and in practice, chloroplast agglutination tests of potato leaves can be made with these antisera diluted to 1/100.

Although the most important and perhaps most difficult part of antisera production procedures has been the preparation of the (antigens) virus inocula, it should be noted that some individual animals produce more antibody than others under standard immunizing conditions.

SUMMARY

A simple technique has been developed to obtain purified potato virus X in physiological saline solution by passage through a gel column. The virus solution is then concentrated by dialysis with polyethylene glycol for use in intravenous injections for the production of potato virus X- immune rabbit antiserum.

I:~ESUMEN

Se ha desarrollado una t6cnica sencilla para obtener el virus X de patata purificado en una solucidn salina fisiSldgica por pasarse por una columna de gelatina. Luego, se concentra la soluci6n del virus por di/tlisis con el glicol polietileno para usarse en intervenciones intravenosas para la produccidn del antisuero de conejo inmune del virus X de patata.

254 AMERICAN POTATO JOURNAL [Vol. 44

LITERATURE CITED

1. Bawden, F. C. and A. Kleczkowski. 1948. Variations in the properties of potato virus X and their effects on its interaction with ribo-nuclease and proteolytic enzymes. J. Gen. Microbiol. 2: 173-185.

2. Kohn, J. 1959. A simple method for the concentration of fluids containing proteins. Nature 183: 1055.

3. Loring, Hubert S. and W. M. Stanley. 1937. Isolation of crystalline tobacco mosaic virus protein from tomato plants. J. Biol. Chem. 117: 733.

4. Munro, James. 1954. Maintenance of virus X-free potatoes. Amer. Potato J. 31 : 73-82.