Acta neurol. scandinav. 57, 438-442, 1978

The Neurochemical Institute, Copenhagen and *The Municipal Hospital, Neurological Department, Copenhagen, Denmark

Multiple sclerosis specific antigens in MS brains

S. C. RASTOGI, J. CLAUSEN AND T. FOG

Antigens in MS and non-MS brains were investigated by means of crossed immunoelectrophoresis. Two MS specific antigens were found: a measles antigen which was present in microsomes and cytosole, and an unidenti- fied antigen present only in cytosole.

The demyelination process in multiple sclerosis (MS) may be due to a chronic (viral) infection or to immunological abnormalities. A virus infection of the central nervous system (CNS) may cause formation of new (viral) antigens. However, even autoimmune processes may lead to lysosomal activation and conformational abnormalities of CNS antigens. Therefore, an attempt was made to trace “MS specific” CNS antigens. Results of preliminary investigations are reported in the present communication.

MATERIALS AND METHODS Chemicals

Freund’s complete adjuvant was from Difco Labs., U S A . and agarose A45 was from L‘Industrie Biologique Francaise, France. Myelin basic protein and measles antigen (prepared from infected vero cells, Oflner et al. 1973) were kind gifts from Dr. Halina Offner (The Neurochemical Institute, Copenhagen). All other chemicals were of highest obtainable purity from E. Merck, F.R.G.

Control and MS Brains

Brains from three MS patients (age 44-55 years, duration of disease 18-38 years) were collected 16 h after death and frozen (-70OC). Macroscopical inspection revealed plaques in the periventricular area and in white matter. Classical microscopical exami- nation revealed in these areas demyelination. When alive all the patients were clinically diagnosed as having “certain MS” (Schumacher 1965). Brains from four non-MS pa- tients (age 52-82 years) served as control material (no visible plaques were found in these brains).

Preparation of Brain Cytosole and Microsornes

A total of 3-4 g frontal and occipital lobe (both white and grey matter) of a brain were mixed (1:1, W/W) and a 25 % ( W N ) total hamogenate was prepared in 0.14 M sodium chloride using a Potter Elvehjem homogenizer. The homogenate was centrifuged (4OC) at 30,000 g-av (30 min) and the resulting supernatant was further centrifuged at 105,000g-av for 1 h in IEC ultracentrifuge. The supernatant (cytosole) was used for

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immunization, while for crossed immunoelectrophoresis (IE) it was concentrated to 20mg proteidml by freeze drying and dialyzed against 0.14 M sodium chloride. The 105,000 g pellet (microsornes) was suspended in 0.14 M sodium chloride at concentration 3 mg protein/ml (for immunization) or 20 mg proteinhl (for IE).

Preparation af Antisera

Albino rabbits (3000 g approx.) of both sexes were injected with 0.3 ml brain cytosole or microsomes (five animals in each group) subcutaneously on the back side. The first injection was followed by injection of 0.2 rnl Freund’s complete adjuvant (Clausen 1969). The immunization was repeated at least three times with a 1-week interval. A booster injection was given 4 weeks after the first injection and b b o d was collected 10 days after by puncturing the ear vein, the ear previously being irritated to blood accumu- lation with toluene. After coagulation and centrifugation, the antiserum obtained was stored frozen until use.

Figure 1 . Crossed I E of MS cytosole versus anti-MS cytosole absorbed with non-MS cytosole. For experimental conditions see text. Two immunoprecipitation arcs represent

the specific antigens in MS brain.

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Crossed Immunoelectrophoresis

Crossed IE of 25 1'1 cytosole (500 lig protein) was performed in 1 % agarose gel (5 ml gel on a 7x7 cmz glass plate) at pH 8.6, 0.075 M sodium barbital buffer containing 2 mM Calcium chloride (Laurel1 1966). IE was carried out (lO-lZ°C) at 250V for 3.5 h in first dimension and at 60 V for 18-20 h in the second dimension. The gel in the sec- ond dimension contained 11 % ( V N ) antiserum, 15 PI antiserum/cm2 gel. After IE, removal of unprecipitated proteins (pressing and washing the gel), staining (Commassie brilliant blue) and destaining (methanol-acetic acid-water) were performed as described by Axelsen er al. (1973). Experimental conditions for crossed IE of microsomes (in 0.1 % Triton X-1001B were changed, in that both buffer and electrophoretic gel con- tained 0.04 % Triton X-100, and IE was carried out for only 1.5 h in first direction.

Tandem technique was employed to determine the specificity of the antigens studied and the antiserum used was absorbed in order to eliminate normal CNS antigens: 1 ml anti-MS cytosole was incubated (37" C, 2 h) with non-MS cytosole (600, 1200, 2400 p g protein and the immunoprecipitate was removed by centrifugation at 2000 g for 10 min.

Figure 2. Tandem crossed immunoelectrophoresis of ( 1 ) non-MS brain cytosole, (2), MS brain cytosole, and (3) measles antigen versus anti-MS brain cytosole preabsorbed with non-MS cytosole. Two immuno-precipitation arcs were shown by MS brain cytosole,

one of which fused with that of measles antigen.

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RESULTS AND DISCUSSION

Crossed IE of MS and non-MS cytosole against their respective antiserum revealed the presence of at least seven immunoprecipitation arcs corresponding to seven normal CNS antigens. Two extra immunoprecipitation arcs were traced in MS cyto5ole. These arcs could not be traced when MS cytosole was electrophorized against anti-non-MS cytosole and vice versa. The two MS specific precipitation arcs persisted when the anti-MS cyto- sole used was preabsorbed with non-MS cytosole (2.4 mg proteidl ml antiserum) (Figure 1). Tandem crossed 1E of measles antigen and MS cytosole versus anti-MS cytosole preabsorbed with non-MS cytosole revealed that one of these two arcs fused with measles antigen (Figure 2).

Comparison of crossed IE of MS and non-MS microsomes versus their respective anti- serum revealed presence of only one extra immunoprecipitation arc by MS microsomes. This arc was found to correspond to a measles antigen when a series of experiments (similar to those made on cytosole) were performed. The antigen (other than measles antigen) similar to that traced in MS cytosole has not yet been observed in microsomes.

Besides MS, measles antigen may also be involved in some other diseases, for example, systemic lupus erythematosus (Beck & Clausen 1977) and subacute sclerosing panence- phalitis. It is not clear if the proliferation of measles virus is a primary phenomenon or

Drawing of Fig. 2.

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secondary to an immune depression (Valdimarsson et al. 1974). The second antigen traced in the present study has not yet been identified, it may be another viral antigen or a CNS protein with an abnormal conformational state. This specific antigen was not identical with myelin basic protein and attempts to demonstrate such an antigen in cerebrospinal fluid or serum of MS patients have failed.

REFERENCES

Axelsen, N. H., J. Krell & B. Weeke (1973): A manual of quantitative immunoelectro- phoresis. Methods and Applications. Scand. J. Immunol., Volume 2, Suppl. 1, Uni- versitetsforlaget, Oslo.

Beck, H. W. & J. Clausen (1977): An epidemiological study on paramyxovirus antibody titers in multiple sclerosis, systemic lupus erythematosus and rheumatoid arthritis. Zbl. Bakt. Hyg., I. Abt. Orig. A 238, 431-443.

Clausen, J. (1969): Immunochemical techniques for the identification and estimation of macromolecules, in: Laboratory techniques in biochemistry and molecular biology, (ed. T. S. Work & E. Work). North-Holland, Amsterdam.

Laurell, C.-B. (1965): Antigen-antibody crossed immunoelectrophoresis. Anal. Biochem.

Offner, H., T. Ammitzbell & J. Clausen (1973): The occurrence of measles antigen pre- cipitating antibodies in sera from patients with multiple sclerosis and their relatives. Acta Pathol. Microbiol. Scand. Sect. B 81, 157-164.

Schumacher, G. A. (1965): Problems of experimental trials of therapy in multiple scle- rosis Ann. N.Y. Acad. Sci. 122, 552-567

Valdimarsson, H., G. Agnarsdottir & P. J. Lachmann (1974): Cellular immunity in sub- acute sclerosing panencephalitis. Proc. Roy. SOC. Med. 67, 1125-1129

10, 358-364.

Received March 2, accepted March 9, 1978 S . C . Rastogi, Ph.D.

The Neurochemical Institute RHdmandsgade 58 2200 Copenhagen N Denmark