Isolation of viruses using specific immunoadsorbents

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6. SH~;PHIZRD, MhNKO,

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R. J., WAKEMAN, R. J., and RoR. It., virology 36, 150-152 (1968).

T. KAMEI M. RUBIO-HCIGRTOS

C.

MATSUI

Plant Pathology Laboratory Faculty of Agriculture Nagoya University Magoya, Japan; Institute “Jaime Ferran” de Microbiologia Madrid, Spain Accepted January 9, 1969

Isolation

of Viruses using Specific lmmunoadsorbents

Since the first report by Campbell and his co-workers (1) water insoluble antigen derivatives are currently used for the isolation of the corresponding homologous antibodies (2). Although the possibilities of water-insoluble antibodies were pointed from earlier studies, little work in this area has been undertaken (2). Recently, the preparation of protein polymers, using either ethyl-chloroformate (3) or glutaraldehyde (4) as polymerizing agents, has been described. The water insoluble protein derivatives obtained by these methods were found to be efficient and specific immunoadsorbents and were used to isolate specific antibodies from a number of proteins and to isolate protein antigens from mixtures (3, 4). It was then of interest to investigate if insolubilized immune-serum could also be used for the isolation of virus particles, thus providing a new method for virus purification. In this paper, we are reporting the isolation of rat K virus (Parvo-virus Ratti) and poliovirus from infected cultures using the homologous insolubilized antisera. Antisera were freed, before insolubilization, by successive absorptions, of all contaminating antibodies, except those directed against the virus. No virus particles could be isolated or absorbed when insoluble heterologous antisera were used. The preparation of isolated virus were found to be biologically active and to be, when tested by double

diffusion in agar or acrylamide electrophoresis, free of contaminating protein antigens of the culture medium. The strain of the rat K virus used in the present study was that already reported (5). The crude or semi-purified stock virus preparations were titrated by the plaque assay method on rat embryonic cells according to a technic described elsewhere (5). The semipurified K virus preparation, containing low quantities of extraneous protein, was obtained after sucrose density gradient ultracentrifugation of a crude virus preparation which has been treated twice with fluorocarbon. The poliovirus (strain Type 1 of Sabin) was grown on monkey cells (BSC-1) cultures and the infectivity of the virus was titrated by the plaque assay. The complete details of culture medium, preparation and propagation of rat (~7)or monkey cells have been already described (6). Rabbit antisera against whole normal bovine serum (NBS) or the constituents of the complete culture medium’ (CM) were obtained by using immunization procedures described elsewhere (3, 4). The anti-K virus antiserum was obtained by immunizing five young rabbits with a semipurified K virus preparation titrating l.lO* PFU/ml. On the first day, each animal received in the hind foot pad 0.1 ml of virus preparation emulsified in 0.1 ml of complete Freund’s adjuvant and 0.5 ml of virus preparation by intravenous route. After 4,5 days, each animal received intravenously 0.5 ml of virus preparation; the injections were repeated weekly for a period of 1 month. Rabbits were bled S days after the last injection. After the antisera were pooled, the titer was found to be of 13.000 neutralizing units/ml. Rabbit anti-poliovirus y-globulin fraction titrating 2.500 units/ml was obtained from the Institut Pasteur, Paris. The anti-K virus and anti-poliovirus antisera were found, by double diffusion in agar, to contain antibodies directed against a number of proteins present in the noninfected complete culture medium. Antisera 1 The complete culture medium consists in a mixture of rat embryo cells suspended in modified Eagle’s medium containing 10c/c of tryptose phosphate (Broth) and 1070 calf serum.

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AND ELUTION OF RAT HETEROLOGOUS

K

1

VIRUS .IND INSOLUBILIZED

POLIOVIRUS ANTISERA

ON

HOMOLOGOUS

Rat K virus Insolubilized

Anti

Virus

antisera

K virus Found Found

Anti-poliovirus Found Found Snti-whole bovine and anti-complete medium

serum culture

Found

Added in the supernates in the eluates Added in the supernates in the eluates Added in the supernates

monospecific for the viruses were obtained when these multispecific antisera were adsorbed on insoluble polymers of NBS and CX. Insolubilization of the NBS (10 ml) containing the CM (70 mg of dialyzed and lyophilized CM) and adsorption of the anti-K virus antiserum with this immunoadsorbent were carried out following procedures described in details elsewhere (3, 4). The monospecific anti-K virus rabbit serum was then polymerized with either ethylchloroformate (3) or glutaraldehyde (4) and employed for the isolation of the viruses. In order to achieve this, normal or concentrated crude virus preparations were mixed with the immunoadsorbent. The mixture was stirred gently for 1 hour at room temperature and then the immunoadsorbent was stashed by successive centrifugations (3, 4). Elution of the adsorbed virus was carried out by successive centrifugations, using either cold 2 M sodium iodide in Tris buffer 0.05 11, pH 9 (7), or cold 2.5 M magnesium chloride in Tris buffer 0.05 M, pH 7.5 (4). In Table 1 are summarized the results obtained by using the polymer-immunoadsorbents for the isolation of viruses. The data included in this table represent mean values of five experiments. It can be seen that the method is highly specific. Thus, when a preparation of K virus is allowed to react with an anti-K virus polymer, a considerable absorption of the virus by t,he polymer is noted. In contrast, when the

Viral titer

Percentage

5.1 x 105 103 4.8 x 105 5.4 x 106 5.7 x 106 -

100 <1 94 100 NlOO 100 9G

4.6 X lo4 4.4 x 104

AND

Poliovirus Viral titer 1.3 x 1.5 x 2.3 X 105 7.7 x -

PeFient-

10’ 107

100 NlOO

10’

100 <0.5 33 -

10”

anti-poliovirus polymer is used, virtually no adsorption of the K virus by the immunoadsorbent is observed. The same observation holds true when poliovirus if employed. Adsorption capacity for the viruses by the immunoadsorbents seems to be quite satisfactory for K virus and poliovirus (Table 1). However, as different ways of titration are used for K virus and poliovirus, no correlation can be established between their antiserum titer and the adsorption capacity of their corresponding immunoadsorbents. By using polymerized antibody and protein antigen, it was established that higher the titer of the initial antiserum was, the more antigen could be adsorbed by the immunoadsorbent. One can then expect a similar relationship for virus-antivirus systems. Effective elution of adsorbed K virus could be achieved with 2 Al sodium iodide in Tris buffer 0.05 M, pH 9 or with 2.5 M magnesium chloride in Tris buffer 0.05 M, pH 7.5. Recovery of adsorbed K virus was estimated close to 100 % (Table 1). Elution of poliovirus with 2 M sodium iodide seems to be less effective as only 30% of the virus could be recovered. Different eluting fluids such as urea, guanidinium salts, acid or alkaline pH, have to be tried if it is found with other viruses that solutions of sodium iodide or magnesium chloride affect the infectivity of the viruses. The polymerized insoluble antivirus antibodies after elution of adsorbed virus can

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be washed with saline and used again without apparent loss in adsorptive capacity for virus. If the virus is not completely eluted, the immunoadsorbent is washed two to three times with cold glycine-hydrochloric acid buffer 0.2 M, pH 2.2 (3), neutralized and then washed with saline. In general, this procedure has proven effective in removing residual virus. Virus immunoadsorbents so treated have been used as often as three times without losing their capacity. Insoluble antiprotein antibodies have been used ten times and no appreciable loss in their adsorption capacity was noted. It is then probable that a polymerized antivirus serum can be used many times for the isolation of a virus. This might mean then that purification of a virus using an immunoadsorbent is a relatively easy task once a high quantity of the immunoadsorbent was prepared. No differences in shape of virions were noted between the starting preparations of K virus and those isolated with immunoadsorbents when the preparations were examined under electron microscope. Preparations of 1~ virus or poliovirus isolated on immunoadsorbent were tested by double diffusion and ring test with the anti-r\;BSCM antiserum. Both tests were negative and suggested that no appreciable amounts of contaminating proteins mere present in these preparations. Electrophoresis in acrylamide gels of the purified virus preparations gave similar results. No precipitating reaction was noted between the immunoadsorbent-isolated virus and its homologous antiserum. However, at the moment one cannot say whether a nonprecipitating antibody or a weak concentration of virions are responsible for the lack of precipitation. When the polymerized antiserum was not exhaustively adsorbed with NBS-CM polymer or the virus was eluted before washings gave an optical density of 0 at 280 rnl.r, traces of proteins of the CM could be de-

tected. In this case, the contaminating substances could be removed by mixing the purified virus preparation with a polymerized anti-NBS-CM antiserum. Virtually none of the viruses were nonspecifically adsorbed by this treatment (Table 1). This procedure offers an alternative method for the isolation of viruses using immunoadsorbents and was successfully employed in the present work for the purification of rat K virus. In general, antisera containing antibodies directed against the constituents of the complete culture medium can be prepared and polymerized. When the virus does not wear host antigens, then these polymers can be used to adsorb all the unwanted constituents thus leaving the virions in the supernate. This technic could be of particular interest when fragile viruses are to be isolated. REFERENCES D. H., LCJESCHER, E. and LERMAN, L. S. (1951). Proc. N&Z. ilcad. Sci. U.S. 37, 575-578. SILMAN, I. H. and KATCH~LSKI, E. A. (1966). Ann. Rev. Uiochem. 35,873-908. AVRAME~~S, S. and TERNYNCK, T. (1967). J. Biol. Chem. 242, 1651-1659. AVR.IMKQS, S. and TERNYNCK. (1969). Immunochemistry. 6, no. 1. BRMLOVSKY, C. (1966). Ann. Inst. Pasteur 110,49-59. TOURNIER, P., C.~SSINGENB, It., WICKER, R., COPPEY, J. and Sua~sz, H. (1967). Znt. J. Cancer 2, 117-132. AVRAMEAS, S. and TERNYNCK, T. (1967). Biothem. J. 102, 37C-39C.

1. CAMPBELL,

2. 3.

4. 5. 6.

7.

STR.4TIS AVRAMEA~ CARLOS BRAILOVSKY THERI~SE TERNYNCK

Znstitut de Recherches Scientifiques sur le Cancer BP. No. 8 Villejuij-Val de Marno France Accepted .Tnnu,nru 98. 1969