Affinity capillary electrophoresis to evaluate the complex formation between poliovirus and nanobodies

It was demonstrated that nanobodies with an in vitro neutralizing activity against poliovirus type 1 interact with native virions. Here, the use of capillary electrophoresis was investigated as an alternative technique for the evaluation of the formation of nanobody–poliovirus complexes, and therefore predicting the in vitro neutralizing activity of the nanobodies. The macromolecules are preincubated offline in a specific nanobody‐to‐virus ratio and analyzed by capillary electrophoresis with UV detection. At low nanobody‐to‐virus ratios, a clear shift in migration time of the viral peak was observed. A broad peak was obtained, indicating the presence of a heterogeneous population of nanobody–virion complexes, caused by the binding of different numbers of nanobodies to the virus particle. At elevated nanobody‐to‐virus ratios, a cluster of peaks appeared, showing an additional increase in migration times. It was shown that, at these high molar excesses, aggregates were formed. The developed capillary electrophoresis method can be used as a rapid, qualitative screening for the affinity between poliovirus and nanobodies, based on a clearly visible and measurable shift in migration time. The advantages of this technique include that there is no need for antigen immobilization as in enzyme‐linked immunosorbent assays or surface plasmon resonance for the use of radiolabeled virus or for the performance of labor‐ and time‐intensive plaque‐forming neutralization assays.     

Reverse-phase high-performance liquid chromatography of virus proteins and other hydrophobic proteins

Summary So far most solvents generally used in reverse phase chromatography (RPC) for separation of peptides and water soluble polypeptides could not be utilized for hydrophobic proteins such as membrane proteins and structural polypeptides of viruses due to their insufficient solubility. But we have introduced a new RP-HPLC solvent system which was very useful in our studies on poliovirus polypeptides. Formic acid in high concentration is an extremely potent solvent for proteins, particularly those that are hydrophobic. Preliminary estimates are made of the concentration of formic acid which is required to completely dissolve hydrophobic proteins. For example, solubilization of structural polypeptides of poliovirus which are absolutely water insoluble requires 60% formic acid. Therefore, we used a proportion of 60% formic acid in all solvents for reversed phase chromatography and applied propanol-2 or acetonitrile as the organic modifiers for gradient elution. Using this mobile phase all four poliovirus polypeptides of three serological types were obtained in high purity by this rapid procedure. In each case, polypeptides were quantitatively eluted independent of the amount of protein (1–1000 μg) injected onto the columns. The solvents used were volatile and easily removed in a short evaporation step. Therefore this solvent system is suited for analytical and for micropreparative separation of proteins for chemical, biochemical and immunological studies. Rechromatography and electrophoresis in SDS-polyacrylamide gels of the separated polypeptides demonstrated that this solvent system with its high proportion of formic acid did not alter their primary structure. There may have been major changes in secondary and tertiary structure. In contrast, alterations of the elution characteristics were observed after reduction of disulfide bridges and several modifications of proteins. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Impact of the sample matrix composition on the signal enhancement in the capillary electrophoretic separation of poliovirus samples

The development of capillary electrophoretic applications aiming to provide reliable stability assessment of viral suspensions, to detect subviral particles from cell extracts or to study the interactions between virus particles and various biomolecules, cannot be done without a thorough understanding of the sample matrix contribution to the observed electrophoretic behaviour. The present study thoroughly investigates the effect of the sample matrix on the electrophoretic behaviour of poliovirus injected as sample plugs of 1%, 5% and 12% effective capillary length. The effect of the sample matrix for three different poliovirus batches was evaluated. Additionally, simulated samples, obtained from concentrated poliovirus suspensions of high purity and diluted with commonly used lab buffers in order to obtain samples with either high or low conductivities, were also investigated. The goal of the study was to obtain a better understanding of the effect of the sample matrix on the signal enhancement, in order to define a general approach allowing a repeatable capillary electrophoretic (CE) separation of poliovirus from complex samples. This study clearly demonstrates that the sample matrix has an important influence on the sensitivity of the CE poliovirus separations. Translation of these observations into routine practice involves several compromises and a set of rules in order to reduce day-to-day variation and to maximize sensitivity.