Evidence for conformationally different states of interleukin-10: binding of a neutralizing antibody enhances accessibility of a hidden epitope.

We present the mapping of two anti-human interleukin-10 (hIL-10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL-10-derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L-amino acids. The epitope-derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody-hIL-10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X-ray structure of IL-10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL-10 can adopt a conformational state other than that observed in the crystal structure.

Structure analysis of a fusogenic peptide sequence from the sea urchin fertilization protein bindin.

The structure of "B18", an 18-residue fusogenic peptide from the sea urchin fertilization protein bindin, was investigated in several membrane-mimicking environments with circular dichroism and nuclear magnetic resonance spectroscopy. The fully conserved peptide sequence represents the minimal functional part of the 24 kDa protein, which can bind to membranes and induce fusion of lipid vesicles. The B18 peptide undergoes a coil-helix transition in the presence of TFE, showing a transient tendency to self-associate. Its NMR structure in 30% TFE exhibits two helical regions at either side, connected by a flexible loop. In DPC and SDS detergent micelles, this loop becomes distinctly bent, presumably due to the high degree of curvature of the micelles. The loop contains a histidine-rich motif for binding zinc, which is required for the fusogenic function of the peptide. Therefore, we monitored the structural response of B18 and of recombinant bindin toward this ion. Like TFE, and in a mutually cooperative manner, zinc induces a partially helical structure in both the peptide and the protein. Complex formation via the histidine residues rigidifies the flexible loop and is accompanied by self-association of the molecules. The data suggest that the zinc-bound functional state is a continuous amphipathic alpha-helix, bearing some resemblance to a leucine zipper. Two hydrophobic patches on one face could favorably penetrate into a membrane, while two arginines on the other face could interact with lipid phosphate groups. The three-dimensional model of the B18 sequence thus contributes to a better understanding of peptide-induced vesicle fusion in general, and of the lipid-protein interactions of sperm bindin in particular.

Bioreaction network topology and metabolic flux ratio analysis by biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy.

Biosynthetically directed fractional 13C labeling of the proteinogenic amino acids is achieved by feeding a mixture of uniformly 13C-labeled and unlabeled carbon source compounds into a bioreaction network. Analysis of the resulting labeling pattern enables both a comprehensive characterization of the network topology and the determination of metabolic flux ratios. Attractive features with regard to routine applications are (i) an inherently small demand for 13C-labeled source compounds and (ii) the high sensitivity of two-dimensional [13C,1H]-correlation nuclear magnetic resonance spectroscopy for analysis of 13C-labeling patterns. A user-friendly program, FCAL, is available to allow rapid data analysis. This novel approach, which recently also has been employed in conjunction with metabolic flux balancing to obtain reliable estimates of in vivo fluxes, enables efficient support of metabolic engineering and biotechnology process design.

Neutralizing murine monoclonal anti-interleukin-10 antibodies enhance binding of antibodies against a different epitope.

Fifteen murine hybridoma lines that produce monoclonal antibodies against human interleukin-10 (IL-10), which is one of the most important regulatory cytokines of the immune system, were raised. Twelve of these antibodies, all in the class IgG1/kappa, recognize three groups of epitopes: A, B and C. All antibodies in these groups inhibit binding of antibodies of the same group to IL-10 and none of them inhibit binding of an antibody of another group. Two IgM/kappa antibodies and one IgG1/kappa antibody, with low affinity, have distinct binding properties and cannot be assigned to one of these groups. Antibodies from all three epitope groups inhibit the biological activity of IL-10. The three antibodies of group A neutralize IL-10 in an approximately equimolar ratio, at concentrations as low as 10 pM. In addition to their high neutralizing capacity, antibodies of group A enhance the binding of antibodies of group C to IL-10. The on-rate of binding of the two antibodies CB/RS/10 and 11 (group C) increased five- to seven-fold, when one of the antibodies CB/RS/1, 2 or 14 (group A) is bound to IL-10.

Chemical modification of recombinant HIV-1 capsid protein p24 leads to the release of a hidden epitope prior to changes of the overall folding of the protein.

It was found that the affinity of a monoclonal antibody directed against a recombinantly expressed HIV-1 capsid protein p24 (rp24) strongly increased after chemical modification of the Iysine residues of rp24 with different amounts of maleic anhydride. The extent and the sites of modification were analyzed by MALDI-TOF mass spectrometry. Unmodified rp24 and the differently modified rp24 samples were tested for binding the murine monoclonal antibody CB4-1 which recognizes the epitope GATPQDLNTML comprising residues 46-56 of rp24. An increase in the number of modified lysine residues led to enhanced binding affinity of CB4-1. Most pronounced effects were observed after substitution of the first amino groups: an average number of three modified residues per protein molecule increases the binding affinity by a factor of 23, but the substitution of the remaining nine residues increases the binding affinity only by a factor of 11. Fully modified rp24 variant proteins were bound by CB4-1 with Kd values comparable to that of the peptide epitope. Conformation and stability of the unmodified rp24, highly (rp24F, 9 residues; rp24G, 11 residues) modified, and fully modified protein (rp24I, 11 lysine residues and N-terminus) were analyzed by circular dichroism (CD) and fluorescence spectroscopy under different solvent conditions. Little difference in conformation and unfolding behavior was observed between the unmodified and highly modified rp24, which differ drastically in the antibody binding behavior. The fully modified sample, however, displayed a significant decrease in alpha-helical content. Thus, the epitope seems to be hidden (cryptotope) in the unmodified rp24 in a low-affinity binding conformation and becomes displayed at low levels of chemical modification which obviously induce subtle structural changes prior to changes of the overall folding observable by spectroscopic means.

Binding kinetics of an antibody against HIV p24 core protein measured with real-time biomolecular interaction analysis suggest a slow conformational change in antigen p24.

The interaction between HIV core protein p24 and the murine monoclonal antibody CB-4/1 or its Fab fragment showed unusual kinetics. Recombinant p24 was immobilised in a hydrophilic carboxymethyldextran matrix. At high concentration of CB-4/1 Fab the association of the antigen-antibody complex proceeds in two phases, while dissociation is mono-exponential. The antigen has a 'memory', i.e. shortly after dissociation of Fab-antigen complex the fast association phase is enhanced. Biphasic association was also found in solution. Experiments suggest a reversible change of binding properties in the epitope region with an overall time constant of about 100 s at room temperature. Intermediate steps with faster time constants must be involved. Slow conformational changes of p24 seem to be the most probable explanation. A simple model that provides a quantitative description of this process could not be found. Real-time analysis of antibody binding by surface plasmon resonance is a powerful method for studying such changes in the time domain of a few seconds to a few minutes.

Comparison of three human-murine heteromyeloma cell lines for formation of human hybridomas after electrofusion with human peripheral blood lymphocytes from meningococcal cases and carriers.

Peripheral blood lymphocytes from meningitis patients and healthy meningococcal carriers were fused by electrofusion with the three human-murine heteromyeloma cell lines CB-F7, K6H6B5 and H7NS. 934 hybridomas producing human immunoglobulins were obtained in 30 fusions. Heteromyeloma K6H6B5 yielded a significantly higher proportion of hybridomas producing IgG antibodies than did the two other cell lines. CB-F7 and K6H6B5 yielded comparable numbers of hybridomas whose supernatants reacted with homologous bacteria, whereas the cell line H7NS was less efficient.

A human monoclonal antibody with the capacity to neutralize Staphylococcus aureus alpha-toxin.

A human monoclonal antibody, CB STL-1, against staphylococcal alpha-toxin has been established by hybridoma technology. It is of IgG1 subclass with lambda light chain and possesses a dissociation constant of 8 x 10(-10) mol/l. 1 mg of purified antibody neutralizes the hemolytic activity of 800 micrograms/ml alpha-toxin in an in vitro hemolysis assay using rabbit erythrocytes. The antibody does not bind to overlapping (7 residues) decapeptides spanning the sequence of alpha toxin, thus it might bind to a conformational epitope. The epitope recognized by the antibody is not accessible in oligomeric toxin. The antibody binds both to the hydrophilic and amphipathic forms of the monomeric toxin Fab fragments of the antibody are stable and show no significant loss of activity. CB STL-1 was able to protect mice in vivo from i.p. challenge with alpha toxin. Thus, the antibody is a candidate for passive immunotherapy. The variable regions of the antibody secreted by CB STL-1 were sequenced and found to be encoded by a VH gene segment belonging to the VH1 family, and a Vlambda segment most likely belonging to the VlambdaIII subgroup. Further analysis concerning the third complementarity determining region (CDR3) of the heavy chain is presented.

Antigen-antibody binding and mass transport by convection and diffusion to a surface: a two-dimensional computer model of binding and dissociation kinetics.

The kinetics of binding and dissociation between a soluble analyte and an immobilized ligand on or near a surface are described numerically by an iterative computer model. The model is applied to a microflow chamber which is used for surface plasmon resonance measurements. It calculates diffusion perpendicular to the surface, flow parallel to the surface, and the interaction between any number of soluble and immobilized species. If the reaction between analyte and ligand is fast, binding and dissociation are influenced by the transport of the analyte to or away from the surface. In this case the measurement yields apparent association and dissociation rate constants which are not identical with the reaction rate of analyte and ligand. The transition between mass transport-controlled processes and reaction-controlled processes is described and attention is drawn to possible misinterpretations of experimental binding and dissociation curves. The measurement of rate constants higher than allowed by the conventional technique can be performed by elution of the analyte with a second analyte of low molecular weight.

Determination of antibody affinity by ELISA with a non-linear regression program. Evaluation of linearized approximations.

ELISA experiments based on competition between immobilized and soluble antibody for soluble antigen, and on the formation of a ternary complex of immobilized antibody, antigen and soluble antibody were used by Hoylaerts et al. (J. Immunol. Methods 126 (1990) 253-261) for the determination of dissociation constants. The dissociation constant was taken from linearized plots according to a theory that required several approximations. The effect of these approximations on the resulting dissociation constants has been investigated using two computer programs, CBEIA-C and CBEIA-S. Since most approximations that have to be made for linearization can be avoided by non-linear regression, the programs provide a more reliable basis for calculation of the dissociation constant. Experiments measuring formation of a ternary complex were found to be unsuitable for affinity determination.

CBEIA: programs for simulation of ELISA experiments and affinity determination.

Two computer programs for the analysis of results of enzyme immunoassays are presented. CBEIA-C simulates ELISA experiments based on competition between immobilized and soluble antibody for a soluble antigen (or vice versa). CBEIA-S simulates the formation of a ternary complex of immobilized antibody, antigen and soluble antibody. The programs provide a more reliable basis for affinity determination than linearized plots. Results obtained by the application of these programs are described in the accompanying paper on pp. 129-133).

Immortalization of magnetically separated human lymphocytes by electrofusion.

Human lymphocytes from peripheral blood (MNC) were separated on magnetic beads for the presence of different surface markers. Cells from positive and negative fractions were successfully immortalized by electrofusion with the heteromyeloma line CB-Fu2. B cells, which were separated on anti-CD 19 coated beads, could be immortalized at a rate between 10(-5) and 10(-4) even if the fusion was conducted with just a few hundred thousand cells. Comparison of the frequency of Ig-positive hybridomas in B cell, T cell, and unseparated MNC fusions indicated that also non-B cells may give rise to HAT resistant hybridoma clones, although the fusion frequency was low.

Development of specific human mab's by a small scale electrofusion technique: the influence of some physical and chemical factors on hybridoma yield of human peripheral blood lymphocytes XCB-F7 fusions.

A fusion chamber and an appropriate procedure are described which allow to fuse a sample of 15 to 25 microliters of cell suspension every two minutes. The cells can be observed throughout the process. They are not exposed to mechanical stress after the fusion pulse. Electrofusion between the heteromyeloma line CB-F7 and human mononuclear cells from peripheral blood of immunized donors is shown to provide stable hybridomas producing IgG against tetanustoxin. Pronase treatment, calmodulin, PEG, lanthanum and a number of variations in the fusion conditions were investigated as to whether they influence physical fusion of the cells, hybridoma yield, and immunoglobulin production.

Reversible electrical breakdown of lipid bilayers: formation and evolution of pores.

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.