Complexes between monoclonal antibodies and receptor fragments with a common cold virus: determination of stoichiometry by capillary electrophoresis.

Complex formation between monoclonal antibodies or soluble receptor fragments and a human rhinovirus is quantified by relating the concentration of the antibody or receptor under equilibrium conditions to the initial concentration of the virus. Within a given concentration range of the reactants, the shape of the resulting curve depends only on the value of the dissociation constant of the particular system studied. Using antibodies and receptor fragments, cases for high, low, and intermediate affinity were investigated. For high-affinity systems, the curve approximates a decaying straight line and the binding stoichiometry can be accurately determined from the intercept with the x-axis. For the case of intermediate affinity, the curve can be linearized at low virus concentrations with the receptors present in large excess. Extrapolation of this line allows derivation of the binding stoichiometry from the intercept with the x-axis, although with less accuracy. For intermediate affinities, an estimate of the dissociation constant can be obtained from fitting the curve to the data points measured. Finally, in the case of low affinity none of the binding parameters can be quantified, although a rough estimate of the lower limit of the dissociation constant is possible. The method was applied for two different monoclonal antibodies, a Fab fragment and a receptor fragment, binding to human rhinovirus serotype 2. Thirty copies of the monoclonal antibody 8F5 were found to bind to the virion, which is in agreement with data from electron cryomicroscopy. The complex between monovalent human very-low-density lipoprotein receptor encompassing repeats 2 and 3 and human rhinovirus serotype 2 showed 60 receptor molecules bound per virion.

VLDL receptor fragments of different lengths bind to human rhinovirus HRV2 with different stoichiometry. An analysis of virus-receptor complexes by capillary electrophoresis.

The formation of complexes between the minor receptor group human rhinovirus HRV2 and two recombinant soluble receptor fragments derived from the human very low density lipoprotein receptor (VLDLR) and containing ligand-binding repeats 1-3 (MBP.VLDLR(1-3)) or 1-8 (MBP.VLDLR(1-8)) fused to the carboxyl terminus of the maltose-binding protein was analyzed by affinity capillary electrophoresis. At low molar ratios of receptor/virus, the peaks corresponding to substoichiometric complexes were broad indicating heterogeneity. When the receptors were present in molar excess with respect to the virus, the peaks were sharp, suggesting saturation of all binding sites. For the determination of the stoichiometry, constant amounts of receptor were incubated with increasing amounts of virus, and the peak areas corresponding to free receptor were measured and plotted versus total virus concentration. Extrapolation of the linear part of the resulting curve to zero concentration of free receptor enabled quantitation of the molar ratios of the components present in the complex. Using this method, we determined that about 60 molecules of MBP.VLDLR(1-3) but only about 30 molecules of MBP.VLDLR(1-8) were bound per virion.

Affinity capillary electrophoresis for the assessment of complex formation between viruses and monoclonal antibodies.

The formation of complexes of human rhinovirus (serotype HRV2 and HRV14) with nonaggregating neutralizing monoclonal antibodies was investigated by affinity capillary electrophoresis. The method is based on preincubation of virus with antibody, followed by CE analysis. At low antibody-to-virus ratios, peaks corresponding to the complexes were broad, pointing to the presence of a heterogeneous population of virions with various numbers of antibodies bound; at a high molar ratio between virus and antibody, the peak became narrow again, indicating saturation of the 60 equivalent viral epitopes with the antibodies being attached bivalently. As SDS was used as an additive in the background electrolyte to allow for separation, its influence on complex formation was investigated. Once formed, HRV2-antibody complexes were found to be stable in the presence of the detergent but complex formation in buffer containing SDS was severely impaired. HRV14-antibody complexes were rapidly dissociated by SDS. The method proved to be useful for a rapid assessment of complex formation and might allow for an estimation of the binding stoichiometry.

Capillary electrophoresis with postcolumn infectivity assay for the analysis of different serotypes of human rhinovirus (common cold virus).

Differentiation of virus serotypes with capillary zone electrophoresis was demonstrated. For four serotypes of human rhinovirus (HRV2, HRV14, HRV16, HRV49), different electrophoretic mobility was achieved at pH 8.3 (borate/boric acid buffer, 100 mmol/L). Addition of detergent (Triton X-100-R, deoxycholate, and/or SDS) to the background electrolyte was required for reduction of wall adsorption and improvement of peak shape. A major nonviral contaminant, present in all virus samples, was best separated from the viral peaks with 10 mmol/L SDS as additive. The method allowed detecting serotypes HRV16 and HRV49 in crude, partially purified virus preparations. An infectivity assay carried out off-line with fractions collected at the capillary outlet enabled the sensitive and biospecific identification of the peaks of HRV2 and HRV14.

Separation and biospecific identification of subviral particles of human rhinovirus serotype 2 by capillary zone electrophoresis.

During infection, human rhinoviruses undergo structural rearrangements of their capsid proteins from D-antigenic native virus (sedimenting at 150S upon sucrose density gradient centrifugation) to C-antigenic A-particles (sedimenting at 135S) and B-particles (sedimenting at 80S); the latter remain after release of the viral genomic RNA into the cytosol. Subviral particles with very similar properties can also be produced in vitro upon exposure to elevated temperatures or to low-pH buffers. This paper reports on the successful separation of native virus and 80S B-particles by capillary zone electrophoresis. Separation was carried out in an untreated fused-silica capillary (50 microns i.d., total length 50.0 cm, effective length 41.5 cm) at 20 degrees C and monitored with UV detection. The separation buffer was 100 mmol/L boric acid/borate (pH 8.3) and contained 0.5% sodium deoxycholate, 0.05% SDS, and 0.5% Triton X100R; the detergents were required to prevent viral aggregation and adsorption to the capillary wall. The analytes were identified from their characteristic spectra as determined by fast spectral scanning. Final confirmation was obtained by comparison of electropherograms from samples prior and after immunodeplition with antibodies specifically precipitating D- or C-antigen. The present method enables one to easily monitor and quantify these structural changes and thus to determine the most favorable conditions for complete conversion of native virus to 80S B-particles.

Analysis of common cold virus (human rhinovirus serotype 2) by capillary zone electrophoresis: the problem of peak identification.

Different preparations of human rhinovirus serotype 2 (HRV2), a common cold virus, were analyzed by capillary zone electrophoresis (CZE) in untreated fused-silica capillaries using borate buffer (100 mmol/L, pH 8.3) and sodium dodecyl sulfate (10 mmol/L) as additive to prevent wall adsorption. The electropherograms showed one major peak at 205- and 254-nm detection wavelengths. The identity of the peak as originating from native virus was confirmed by several indirect methods. Heating to 56 degrees C is known to lead to release of the genomic RNA from the viral capsid; this treatment resulted in the disappearance of the major peak and the emergence of a new predominant peak that was identified as RNA by enzymatic digestion. As expected, RNase treatment of the unheated sample remained without effect as the viral genome is inaccessible in the native viral shell. A monoclonal, virus-aggregating antibody was used for immunodepletion of native virus; again, the major peak disappeared upon removal of viral aggregates by centrifugation prior to CZE analysis. In combination, these results allowed for the unambiguous identification of the main peak as native HRV2 and of the minor peaks as contaminants present in various amounts in the different viral preparations. It is demonstrated that CZE allows for an extremely easy and rapid assessment of conformational state and purity of virions in a given viral preparation.

Affinity mode of capillary isoelectric focusing for the characterization of the biotin-binding protein actinavidin.

Different modes of capillary isoelectric focusing (CIEF) with salt mobilization and zwitterionic additive to cathodic mobilizer were applied to characterize the biotin-binding protein actinavidin and its affinity properties. The analysis is performed in a neutral coated capillary with completely eliminated electroosmotic flow. Synthetic pI standards with absorption in the visible region were used in all runs and pI determinations were based on a fitted nonlinear calibration graph. CIEF of highly purified actinavidin in native conditions was revealed in about 12 peaks in pI range 6.2-7.5, with three major forms having pI 6.80, 6.86, 6.90. CIEF of a mixture of actinavidin and increasing concentrations of two affinity ligands (biotin and biotinylated oligonucleotide) demonstrated drastic changes in the number of protein isoforms. In the latter case it resulted in only one peak (pI 5.05) when the ligand was in excess. This method, which can be named affinity CIEF, was found to be well-suited for studying structural changes, occurring in receptor proteins upon binding with a ligand. CIEF of the protein, performed under denaturing conditions (6 M urea in a solution of carrier ampholytes) is also reported. It was revealed in three isoforms with a pI more acidic than that of native actinavidin. It is demonstrated that careful selection of denaturing conditions was necessary for the reproducible results.

Analysis of the biotin-binding protein actinavidin using affinity capillary electrophoresis.

Affinity capillary electrophoresis (ACE) was applied to study the bioaffinity of ligand-receptor interaction between the microbial biotin-binding protein actinavidin and biotin. The ACE method is based on short time incubation of a mixture of actinavidin and increasing concentrations of biotinylated oligonucleotide (bio-ON), which was found to be an effective affinity ligand. Separation of intermediate loading forms of actinavidin from unbound ligand in the presence of micellar phase and by capillary zone electrophoresis enabled the quantitation of free bio-ON, permitting the evaluation of the biotin-binding capacity of actinavidin in absence and presence of sodium dodecyl sulfate (SDS). Although in the latter case actinavidin lost a part of its binding capacity (not more than 12%), it was still possible to develop an indirect, noncompetitive assay for the determination of actinavidin in culture liquid, utilizing the combination of micellar electrokinetic capillary chromatography (MEKC) and ACE. Due to the affinity interaction, actinavidin in the sample decreases the amount of bio-ON added, enabling quantitation of the protein. SDS, which is required in this assay to prevent protein adsorption to the capillary wall, greatly enhances the reproducibility and peak shape. Actinavidin levels determined are in agreement with those obtained by commonly used solid-phase analysis. The limit of detection was about 500 ng/mL. Thus the proposed method was found to be well suited for the evaluation of actinavidin affinity and monitoring of its levels in cultivation process.

Determination of the anticancer drug prospidin in human tissue by high-performance capillary electrophoresis using derivatization.

A high-performance capillary electrophoresis (HPCE) method is described for the quantitative determination of the anticancer drug prospidin in human tissue after its derivatization with diethyldithiocarbamic acid sodium salt (DDTC). It was found that absorption of prospidin and its derivatives on the capillary wall due to the strong positive charge in the drug molecules could be eliminated by increasing the methanol content in the run buffer up to 50% and increasing the pH value up to 11.2. While studying the conditions of the interaction between prospidin and DDTC, a molar excess of the latter of 1:9 and 1.5 h of reaction time were found to be enough for complete derivatization. Sample preparation included homogenization of freshly cut papilloma species and deproteinization by methanol addition. Detection was by ultraviolet (UV) absorption at 254 nm. Due to its speed and high performance in separation, HPCE was found to be well suited for the fast checking of drug therapy in clinical practice.