Rudolf Dernick

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.

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.

N-AglB 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.

Induction of neutralizing antibodies by all three structural poliovirus polypeptides

Structural polypeptides VP1, VP2, and VP3 of poliovirus, type 1, strain Mahoney, induced low, but clearly demonstrable levels of neutralizing antibodies in rabbits. Half-neutralization titers of antisera for 1000 PFU/ml of poliovirus were in the range of 0.5 to 1.5 log10 units. Polypeptides were isolated by three independent procedures including different dissociation and separation methods to circumvent their denaturation and possible contamination by neighboring fractions. The best and fastest method was dissociation of poliovirus by formic acid and separation of polypeptides by reverse-phase high-performance liquid chromatography.

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.

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.

Evidence for several unrelated neutralization epitopes of poliovirus, type 1, strain Mahoney, provided by neutralization tests and quantitative enzyme-linked immunosorbent assay (ELISA)

With the aid of 11 neutralizing monoclonal antibodies which were investigated in four different neutralization tests, evidence was provided that several unrelated epitopes for neutralizing antibodies exist on the surface of poliovirus type 1. All monoclonal antibodies were able to neutralize poliovirus infectivity prior to and after virus adsorption to host cells. The quantitative enzyme-linked immunosorbent assay is introduced as a second independent system for the determination of antibody specificity. Antibodies could be divided into four groups according to their reaction patterns.

Monospecific Antisera Against Capsid Polypeptides of Poliovirus Type 1 Distinguish Antigenic Structures of Poliovirus Proteins

Pure poliovirus polypeptides, namely VP1, VP2, VP3 and VP4, have been isolated by isoelectric focusing after dissociation of poliovirus by urea. When injected into rabbits, all four polypeptides produced monospecific antisera which were used for the characterization of poliovirus particles and poliovirus-infected cells. The specificity of these antisera was determined by immunoprecipitation of polypeptides obtained by dissociation of poliovirus with SDS, followed by characterization by polyacrylamide gel electrophoresis. The antisera revealed differences in the antigenic sites of native poliovirus particles, heated poliovirus particles and naturally occurring empty capsids. Only VP3 antiserum reacted with native poliovirus and showed some neutralization, whereas all antisera precipitated heated virus and empty capsids. These antisera reacted also with the appropriate precursors of the capsid polypeptides demonstrating their usefulness for an analysis of the cleavage pathway by monospecific antibodies and revealed a second protomer (90 kilodalton) polypeptide for the capsid proteins of poliovirus particles.

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.

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.