Antibodies to White Matter Proteins and Viral Antigens in Multiple Sclerosis

Abstract

Protein antigens associated with multiple sclerosis (MS) have been described (Rastogi et al., 1978), and antibrain antibodies of several specificities have been found in cerebrospinal fluid (CSF) and sera of patients with multiple sclerosis (Ryberg 1978). These complement-fixing antibodies were of the IgGl or IgG2 subclass, reacting with protein A-containing Staphyloccus aureus Cowan I strain (Ryberg and Kronvall 1981). In the present study an analysis of antibrain antibodies in CSF of MS patients was therefore carried out by immunoprecipitation of the radiolabeled white matter antigen, followed by electrophoresis of the immunoprecipitates. Antibodies directed against labeled polypeptides of herpes simplex type 1, adeno type 5, mumps, measles, rubella, parainfluenza type 2, respiratory syncytial and cytomegalo viruses in CSF specimens of 8 MS patients and 8 controls were also analysed using immunoprecipitation and electrophoresis.Macroscopically normal-appearing WM was obtained by autopsy from four MS patients and four controls without known neurological diseases (death-autopsy intervals of 2-24 h and 4-72 h, respectively), Membranes were prepared without delay as described previously (Hukkanen et al. 1981). The membrane fractions labeled by Na125I using the chloramine T method (Hunter and Greenwood 1962) had specific activities ranging from 0.1 to 0.2 mCi/mg of protein. Lens culinaris lectin chromatography of the unlabeled membranes was performed as described previously (Hukkanen et al. 1981) and the glycoprotein fraction was radioiodinated as above. The viruses were grown and labeled by 35S-methionine as described by Vainionpas et al. (1978). For immunoprecipitation the radioiodinated membranes (lo6 dpm) were solubilized by 0.1% sodium dodecyl sulphate (SDS) in phosphate-buffered saline, pH 7.4 (PBS) for 30 min at +25OC. The virus-infected cell lysates and the lectin-isolated glycoprotein fractions were solubilized with 1% Triton X-100 (Tx-100) for imunoprecipitation. The solubilized antigen preparations were centrifuged at 8900 g for 15 min to remove insoluble material. 100 p1 of CSF or serum diluted to equal IgG concentrations was added and after an incubation of 4 hours at +37OC the complexes were precipitated with 100 1.11 of 10% staphylococcal suspension. The bacterial pellets were washed three times with PBS/O.OZ% Tx-100. In the precipitation of labeled glycoproteins the staphylococci were replaced by Protein A-Sepharose ( P h a mcia, Sweden), and the Sepharose gel was washed four times before extraction of the labeled antigen by 2% SDS and 5% beta-mercaptoethanol in 0.1 M Tris-HC1, pH 6.8. Electrophoresis of the immunoprecipitates was performed in 8.0-18.9% acrylamide gradient gels as described previously (Hukkanen 1982).Electrophoresis of the imunoprecipitates suggested that no specific binding of CSF IgG to the unfractionated membrane polypeptides occurs and qt? MS-associated autoantigen was revealed from MS white matter. Nonspecific precipitation of labeled membrane proteins by the CSF specimens was 0...