A sulfatide-reactive human monoclonal antibody obtained from a multiple sclerosis patient selectively binds to the surface of oligodendrocytes.

In a previous paper, we described the production of a sulfatide-reactive IgM antibody-secreting B cell line that was obtained by Epstein-Barr virus transformation of peripheral B cells from a patient with multiple sclerosis (MS) (Uhlig and Dernick, 1989). In the present study, we demonstrate that this human monoclonal antibody (humAb) DS1F8 selectively binds to the surface of living oligodendrocytes in mixed brain cell cultures of newborn rats. Since a mouse mAb reactive with sulfatide was shown to inhibit oligodendrocyte progenitor differentiation, autoantibodies with binding specificities similar to DS1F8 could play a role in the demyelinating process in the CNS.

Monoclonal autoantibodies derived from multiple sclerosis patients and control persons and their reactivities with antigens of the central nervous system.

Peripheral blood B lymphocytes of multiple sclerosis (MS) patients and control persons were transformed with Epstein-Barr virus. Antibody production of transformed cells against isolated human myelin was investigated by enzyme-linked immunosorbent assay (ELISA). Cells producing reactive antibodies were cloned and propagated to produce monoclonal antibodies (mAbs). These mAbs did also react with acetone fixed frozen sections of normal human white matter, as determined by indirect immunofluorescence staining. Some of the mAbs derived from MS patients and a control person with a central nervous system cyst agglutinated liposomes made from lipids of a chloroform/methanol extract of human myelin, whereas mAbs derived from four glioma patients were negative in these tests. The reactive antibodies were investigated further using agglutination tests with liposomes made from pure auxiliary lipids (cholesterol and lecithin) or containing in addition either galactocerebroside, sulfatide or a mixture of bovine brain gangliosides. The great majority of myelin liposome agglutinating antibodies reacted with all types of liposomes, including those made from pure auxiliary lipids. Investigations by ELISA suggest that phospholipids are the reactive components, at least for some of these mAbs. Some antibodies reacted with liposomes containing galactocerebroside or sulfatide, others only with sulfatide containing liposomes. Antibodies showing these specificities were only obtained from MS patients.

Rapid analytical and preparative separation of structural polypeptides of poliovirus by reverse-phase high-performance liquid chromatography.

A reverse-phase high-performance liquid-chromatography (RP-HPLC) technique was developed for rapid (20 min) and effective separation of the structural polypeptides of poliovirus of strains of all three serological types. The method is useful for quantitative analysis as well as for isolation of polypeptides on a micropreparative scale for chemical, biochemical and immunological studies. All four virus polypeptides (VP1 to VP4) were obtained quantitatively in high purity by this one-step procedure. The solvents used were volatile and easily removed by evaporation, giving dry, amorphous polypeptides free of any additives. The separation mechanism of this RP-HPLC is based on the hydrophobicity of the protein surface. It is distinct from other separation methods such as molecular sieving and isoelectric focusing. Therefore, differences in primary and secondary structure of a polypeptide can be detected by RP-HPLC.

Antigenic structure of poliovirus.

The molecular basis for future developments of poliovirus vaccines is the detailed knowledge of the structure of the virion. Different types and forms of poliovirus particles are detected in virus preparations. Physical and chemical properties characterize the virion better than its electron microscopic image. The compact poliovirus particle is dissociated only by strong denaturizing agents into four polypeptides and RNA. The best analytical method to separate and characterize these polypeptides is the two-dimensional gel electrophoresis. Isoelectric focusing in urea sucrose gradients gives excellent separations also on a preparative scale. The antigenicity of poliovirus particles is the consequence of a complex cleavage pathway leading from the originally translated polyprotein to virus particles. Bearing in mind the need for a special conformation of the virus protein for N-antigenicity, ways are discussed of how to produce a non-infectious poliovirus vaccine in the future.

Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera.

Viral polypeptides were prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by copper staining and electroelution from gel slices. Poliovirus capsid polypeptide VP 1 isolated by this procedure induced monospecific antibodies in rabbits, i.e., antisera reacting only with the homologous polypeptide. Our results demonstrate the applicability of the described copper staining method as a rapid visualization step for preparing viral proteins after SDS-PAGE.

Characterization of a solvent system for separation of water-insoluble poliovirus proteins by reversed-phase high-performance liquid chromatography.

Formic acid in high concentration is an extremely potent solvent for proteins, particularly for hydrophobic ones. 60% Formic acid, necessary for solubilization of structural polypeptides of poliovirus and other proteins, modified at the cysteines, was used together with 2-propanol or acetonitrile as organic modifier for gradient elution in reversed-phase high-performance liquid chromatography. Several reversed-phase columns were tested. In each case, polypeptides were eluted quantitatively. It was demonstrated that this solvent system, with its high proportion of formic acid, did not affect the size, hydrophobicity and charge of the separated polypeptides. By injection into rabbits of poliovirus polypeptides, obtained in high purity by chromatography in the new solvent system, monospecific antibodies were induced, the specificity of which was determined by immunoprecipitation.

Intertypic cross-neutralization of polioviruses by human monoclonal antibodies.

Human B cell lines producing monoclonal antibodies (mAbs) reactive with poliovirus type 1 were generated by transformation with Epstein-Barr virus (EBV) B 95-8 of tonsillar lymphocytes from several immune donors. EBV-transformed cells were cloned in semisolid agarose. Some neutralizing (Nt) human mAbs recognized and neutralized only poliovirus type 1, whereas other Nt mAbs neutralized either poliovirus type 1 and 2 or all three serotypes. mAbs reactive with poliovirus type 1 and 3 but not with type 2 were not detected. Immunoprecipitation of radiolabeled poliovirus type 1 with cross-reactive human Nt mAbs was inhibited competitively by preincubation of mAbs with cold poliovirus type 3 and/or 2.

Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels.

A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05-0.1 ng protein per band. Total staining time with the new procedure is 75 min.

Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem Development Unit.

Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate-polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.

From host components of virus particles to anti-myelin human monoclonal antibodies obtained by Epstein-Barr virus transformation of lymphocytes from multiple sclerosis patients.

Human monoclonal antibodies against viruses and host components were obtained after transformation of human B cells by Epstein-Barr virus, their cloning and propagation. B cells were mainly obtained from peripheral blood of healthy individuals or patients with neurological diseases, especially with multiple sclerosis (MS). The obtained autoantibodies reacted with cell components, i.e. with the nucleus, cytoplasm or cytoskeleton. Using myelin in the screening procedure, about 30 monoclonal antibodies were obtained reacting with human brain cells or myelin components. Anti-myelin antibodies were further examined to determine their antigenic targets in a liposome agglutination assay and an anti-lipid ELISA. Most of these antibodies reacted with phospholipids, some reacted with glycolipids. Some of the tested autoantibodies were obtained only from MS patients. Differences in their reaction with galactocerebroside between MS- and non-MS-persons were observed. The regression of transformed B cells was more pronounced and faster in MS-patients than in healthy individuals. The idea that host components in enveloped viruses could be responsible for autoimmune diseases, especially for MS, is put into a historical perspective and discussed in the paper.

Effective blotting of ultrathin polyacrylamide gels anchored to a solid matrix.

Ultrathin polyacrylamide gels bound on glass plates or plastic sheets cannot be removed from their support without destruction. Therefore electrophoretic transfer methods are not applicable. We have developed a fast diffusion blotting procedure which is very simple and does not need any equipment like blotting chamber or power supply. Furthermore, no special buffer solutions are required. The method is universally applicable to ultrathin sodium dodecyl sulfate, native as well as isoelectric focusing polyacrylamide gels.

Native horizontal ultrathin polyacrylamide gel electrophoresis of proteins under basic and acidic conditions.

The preparation of homogeneous ultrathin native polyacrylamide gels, using a basic as well as an acidic buffer system is described. The basic buffer system consists of Tris-HC1/Tris-glycine, the same buffer as in sodium dodecyl sulfate (SDS)-gel electrophoresis but without SDS. The acidic system uses potassium acetate, pH 4.3, as gel buffer and beta-alanine, pH 4.6, acetic acid as electrolytes. The gels are covalently bound on glass plates. Binding of acidic gels requires a special pretreatment of glass plates. The whole procedure is simple and extraordinarily fast: 100-120 min from the start of gel preparation to the end of electrophoresis. Coomassie staining is done in 40 min and silver staining in 90 min. The native gels are excellently suited for diffusion blotting. Further attractive properties of these gels are easy handling, simple drying and dimensional stability.

Molecular basis of antigenic structures of poliovirus: implications for their evolution during morphogenesis.

Neutralizing monoclonal antibodies against poliovirus type 1 were obtained after conventional immunization or combined in vivo-in vitro immunization. Antibody binding sites were determined by sequence analysis of neutralization-resistant mutants. Site 3 variants had several amino acid substitutions in previously unidentified positions for neutralization resistance. Evidence for a linkage of subsites 3a and 3b is presented. Some site 3b antibodies as defined previously precipitated 14S subunits, although with reduced titers.

Reversed-phase high-performance liquid chromatography of virus proteins and other large hydrophobic proteins in formic acid containing solvents.

The excellent dissolving capacity of formic acid together with a propanol-2 gradient is utilized in a new system for reversed-phase high-performance liquid chromatographic separation of poliovirus polypeptides and a variety of large proteins. Differences in elution characteristics were detected between reduced and non-reduced proteins containing disulphide bridges as well as proteins modified at cysteinyl residues. The retention coefficients of single amino acids were used to calculate those of proteins. The correlation of calculated coefficients with actual retention times indicates that some proteins are bound via their full, unfolded length to the reversed-phase support, whereas others partly preserved their secondary structure. Treatment of proteins with sodium dodecyl sulphate prior to injection dissociates these structural elements and leads to an increase in retention times. The high resolution of the system described should be applicable to the isolation and characterization of components of mixtures of proteins, particularly those of water-insoluble proteins of membranes or viruses, on the analytical and semi-preparative scales.

Evidence for a complex structure of neutralization antigenic site I of poliovirus type 1 Mahoney.

We have selected neutralization escape mutants by using a monoclonal antibody (nt-MAb) against a sequential epitope between amino acids 93 through 104 (neutralization antigenic site I) of poliovirus type 1 Mahoney. The majority of mutants were also resistant against five strain-specific nt-MAbs which recognized conformation-dependent epitopes, suggesting that the neutralization antigenic site I must be involved in the formation of such epitopes. An analysis of all mutants by the binding of nt-MAbs and by isoelectric focusing of VP1 allowed discrimination of five classes of mutants. Sequence analysis of mutant RNAs revealed point mutations and deletions in the antibody-binding site.

N-AgIB of poliovirus type 1: a discontinuous epitope formed by two loops of VP1 comprising residues 96-104 and 141-152.

Analysis of resistant mutants to neutralizing monoclonal antibodies revealed a discontinuous neutralization epitope on VP1 of poliovirus type 1, Mahoney. The epitope has the unique property of being also part of a sequential epitope within neutralization antigenic site I (N-AgI). It is formed by residues in the loop 96-104 connecting the B and C strand and in the loop 141-152 connecting the D and E strand of VP1. Because of strong analogy to neutralization immunogen IB (NImIB) of human rhinovirus 14 (HRV-14) we have called this site N-AgIB of poliovirus type 1.

Evidence for conformational changes of poliovirus precursor particles during virus morphogenesis.

Antisera raised against isolated structural polypeptides VP1, VP2 and VP3 of poliovirus type 1, strain Mahoney, reveal a differential reaction against mature virus and its precursor particles. During virus morphogenesis antigenic sites recognized by VP1 and VP2 antisera are lost stepwise from the surface of precursor particles. These sites are cross-reacting between serotypes and are also lost from precursor particles of type 2 (MEF-1) and type 3 (Saukett). They are absent on the surface of mature virus of all three serotypes. In contrast, the VP3 antiserum recognizes sites expressed maximally on the surface of infectious virus of type 1 (Mahoney). This antiserum did not show significant intertypic cross-reactions with virus particles, empty capsids or 14S particles of poliovirus types 2 and 3. However, it does recognize intertypic cross-reacting sites, like the VP1 and VP2 antisera, on denatured polypeptides and 5S particles of each serotype.

Binding of neutralizing monoclonal antibodies to empty capsids of poliovirus can be blocked by monospecific antisera to structural polypeptides VP1 and VP2.

Binding of two neutralizing monoclonal antibodies (Nt-mAbs) to natural empty capsids (NEC) of poliovirus, type 1, was blocked to the extent of 83 per cent to 98 per cent by monospecific rabbit antisera directed against the structural polypeptides VP1 and VP2. Monospecific antisera against VP3 or VP4, however, did not show this blocking effect. It is therefore assumed that VP1 and VP2 are located close together at the antigenic sites for the two mAbs.

A new antigenic site of poliovirus recognized by an intertypic cross-neutralizing monoclonal antibody.

A monoclonal antibody (mAb 7J6) neutralizing poliovirus type 2 (PV2) and poliovirus type 1 (PV1) was obtained after immunization of BALB/c mice with infectious PV2, strain MEF-1. Preincubation of mAb 7J6 with PV1 inhibited its binding to PV2 and vice versa. Neutralization-resistant variants of PV2 and PV1 were selected. Nucleotide sequencing of the RNAs of some variants revealed mutations in the loop of amino acid residues 239 to 245 in VP2 and in the loop of amino acid residues 195 to 207 in VP3. This is the first evidence that these two loops contribute to a neutralization antigenic site (N-Ag) for poliovirus. Moreover, this new site on PV2 induced intertypic cross-neutralizing antibodies.

Binding site of neutralizing monoclonal antibodies obtained after in vivo priming with purified VP1 of poliovirus type 1 is located between amino acid residues 93 and 104 of VP1.

Three hybridomas obtained after in vitro stimulation of spleen cells of mice primed in vivo with purified VP1 of poliovirus type 1 (Mahoney) with the homologous virus produced antibodies which reacted with VP1 and immunoprecipitated and neutralized only the homologous virus. Evidence for the location of their binding sites was obtained by inhibition of virus neutralization and virus binding by a synthetic peptide comprising the amino acid sequence 93-104 of VP1 of poliovirus type 1 (Mahoney).