Capillary electrophoresis-based assessment of nanobody affinity and purity

Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced “nanobody” EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50 mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0–12 nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (K_d) of 1.65, 1.67, and 1.75 nM for the three components, respectively; these values were comparable to the K_d of 2.1 nM obtained for the bulk EGa1 product using a cellular assay. CE with mass spectrometry (MS) detection using a BGE of 25 mM ammonium acetate (pH 8.0) revealed that the EGa1 sample comprised of significant amounts of deamidated, bisdeamidated and N-terminal pyroglutamic acid products. CE–MS using a BGE of 100 mM acetic acid (pH 2.8) in combination with a polybrene–dextran sulfate–polybrene capillary coating demonstrated the additional presence of minor products related to incomplete removal of the signal peptide from the produced nanobody. Combining the results obtained from affinity CE and CE–MS, it is concluded that the EGa1 nanobody product is heterogeneous, comprising highly-related proteins that exhibit very similar affinity towards EGFR.     

Capillary zone electrophoresis-based cytotoxicity analysis of Caco-2 cells

A capillary zone electrophoresis-based method to evaluate the cytotoxicity of substances to Caco-2 cells was established. The estimation of the injected cell number (500-5000) and the minor effect of injection condition on cytotoxicity determination were investigated. Caco-2 cells the best model of the intestinal absorptive epithelium, were treated with substances and then stained with Trypan Blue and fixed with paraformaldehyde. The treated Caco-2 cells were detected simultaneously at 590 nm and 214 nm, and the absorbance ratio of the two wavelengths (R(590/214)) can reflect simultaneously the loss of cell membrane integrity and the degradation/leak of intracellular components and indicate the cytotoxicity of substances. The cytotoxicity of the four substances sodium sulfite (Na(2)SO(3)), methyl mercury (MeHg), paclitaxel (PTX), and cadmium chloride (CdCl(2)) were determined and compared. There was no obvious cytotoxicity caused by 20 μM Na(2)SO(3) for 24 h treatment, and the toxicity of the other three toxicants was sequenced as: CdCl(2) > MeHg > PTX. The results are in good agreement with the references and the conventional Trypan Blue exclusion counting assay.     

Capillary electrophoresis-based separation techniques for the analysis of proteins

CE offers the advantages of high speed, great efficiency, as well as the requirement of minimum amounts of sample and buffer for the analysis of proteins. In this review, we summarize the CE-based techniques coupled with absorption, LIF, and MS detection systems for the analysis of proteins mostly within the past 5 years. The basic principle of each technique and its advantages and disadvantages for protein analysis are discussed in brief. Advanced CE techniques, including on-column concentration techniques and high-efficiency multidimensional separation techniques, for high-throughput protein profiling of complex biological samples and/or of single cells are emphasized. Although the developed techniques provide improved peak capacity, they have not become practical tools for proteomics, mainly because of poor reproducibility, low-sample lading capacity, and low throughput due to ineffective interfaces between two separation dimensions and that between separation and MS systems. In order to identify the complexities and dynamics of the proteomes expressed by cells, tissues, or organisms, techniques providing improved analytical sensitivity, throughput, and dynamic ranges are still demanded.     

Capillary electrophoresis-based methods for the determination of lipids--a review

Capillary electrophoresis (CE) is a high-resolution technique for the separation of complex biological and chemical mixtures. CE continues to emerge as a powerful tool in the determination of lipids. Here we review the analytical potential of CE for the determination of a wide range of lipids. The different classes of lipids are introduced, and the different modes of CE and optimization methods for the separation of lipids are described. The advantages and disadvantages of the different modes of CE compared to traditional methods like gas chromatography (GC) and liquid chromatography (LC) in the determination of lipids are discussed. Finally, the potential of CE in the determination of lipids in the future is illustrated.     

Capillary electrophoresis frontal analysis: principles and applications for the study of drug-plasma protein binding

Capillary electrophoresis is a well-established technique for the study of noncovalent interactions. Various approaches exist and capillary electrophoresis-frontal analysis provides an interesting alternative to the migration shift affinity capillary electrophoresis methods and conventional methods. The present work reviews the principles on which the frontal analysis method is founded. Advantages and limitations of capillary electrophoresis frontal analysis in comparison with both conventional and other capillary electrophoresis based methods for quantification of binding interactions are discussed. Investigations utilizing capillary electrophoresis-frontal analysis have focused on the interaction of drugs with plasma proteins. These studies, primarily addressing the binding of drugs to human serum albumin, alpha1-acid glycoprotein, and lipoproteins are reviewed together with some recent developments in capillary electrophoresis-frontal analysis methodology.     

Capillary electrophoresis immunoassays with conjugated quantum dots

Water-soluble CdTe quantum dots (QDs) and their conjugates with antibodies and antigenes were prepared by optimized procedures for applications in CE immunoassays. The QD size of 3.5 nm, excitation spectrum in the range of 300-500 nm, the maximum wavelength of the emission spectrum at 610 nm, quantum yield of 0.25 and luminescence lifetimes in the range of 3.6-43 ns were determined. The 0.1 M solution of TRIS/TAPS (pH 8.3) was found to be the optimum buffer for the separation of the antiovalbumin-ovalbumin immunocomplex from the free conjugates of QDs.     

Capillary membrane reactors: Performance and applications

Purpose of the work is the characterization of capillary membranes with entrapped cells, from a mechanical and a kinetic point of view.Polysulphone capillary membranes have been prepared by the phase inversion technique, extruding in a specially designed apparatus the polymer solution containing lyophilized cells of thermophilic Sulfolobus solfataricus.The mechanical properties of such capillary membranes were tested and compared to those of cell-free fibres in terms of bubble formation point and outburst under pressure, and of their behaviour under a stress—strain analysis.Fibres permeability to water and to the acetate buffer (pH 5) used in kinetic experiments was determined in the range of temperatures from 25°C to 70°C.Regarding the entrapped cells kinetic behaviour, an enzyme in particular was selected in bacterial enzymatic heritage, i.e. β-galactosidase, for the conversion of lactose to glucose and galactose. Kinetic assays were performed on bundles of fibres assembled in tube and shell reactors at different substrate concentrations and transmembrane pressures.     

Capillary electrophoresis-based analysis of phospholipid and glycosaminoglycan binding by human beta2-glycoprotein I

Human beta2-glycoprotein I (beta2gpI) is a phospholipid and heparin binding plasma glycoprotein involved in autoimmune diseases characterized by blood clotting disturbances (thrombosis) together with the occurrence of autoantibodies against beta2gpI. With the final goal of assessing autoantibody influence on binding interactions of beta2gpI we have studied the development of capillary electrophoresis (CE)-based assays for interactions of negatively charged ligands with beta2gpI. In the development of suitable conditions for analysis at neutral pH of this basic protein (pI about 8) we found the pH hysteresis behavior of fused silica surfaces useful since the protonated surface after an acid pre-wash counteracted protein adsorption efficiently in contrast to more laborious procedures including acrylamide/dimethylacrylamide coatings that did not permit analysis of this particular protein. This simple approach made estimates of heparin-beta2gpI interactions possible and the principle was shown also to work for detection of betagpI binding to anionic phospholipids. Utilizing the pH hysteresis effect may be a simple solution to the adsorption problems often encountered in analyses of proteins by CE.     

惯性微流体的应用与发展

根据流式细胞仪对样品“均一化”的需求,研制了一套基于微流控细胞分选技术的流式细胞仪样品预处理系统,装置可以实现对粒径小于100 μm细胞或微粒的驱动和分选. 系统主要包含微流控分选芯片和样品驱动模块两部分,通过聚焦不同位置,实现了对不同粒径细胞/颗粒的有效分离. 系统无需对细胞进行标记处理,经分选的细胞便于后续流式细胞仪检测. 经验证,系统能够有效去除牡蛎血淋巴细胞样品中的大粒子杂质,提高细胞样品的稳定性和均一性,增加流式细胞仪检测结果的准确性.

     

Direct optical sensors: principles and selected applications

In the field of bio and chemosensors a large number of detection principles has been published within the last decade. These detection principles are based either on the observation of fluorescence-labelled systems or on direct optical detection in the heterogeneous phase. Direct optical detection can be measured by remission (absorption of reflected radiation, opt(r)odes), by measuring micro-refractivity, or measuring interference. In the last case either Mach–Zehnder interferometers or measurement of changes in the physical thickness of the layer (measuring micro-reflectivity) caused, e.g., by swelling effects in polymers (due to interaction with analytes) or in bioassays (due to affinity reactions) also play an important role. Here, an overview of methods of microrefractometric and microreflectometric principles is given and benefits and drawbacks of the various approaches are demonstrated using samples from the chemo and biosensor field. The quality of sensors does not just depend on transduction principles but on the total sensor system defined by this transduction, the sensitive layer, data acquisition electronics, and evaluation software. The intention of this article is, therefore, to demonstrate the essentials of the interaction of these parts within the system, and the focus is on optical sensing using planar transducers, because fibre optical sensors have been reviewed in this journal only recently. Lack of selectivity of chemosensors can be compensated either by the use of sensor arrays or by evaluating time-resolved measurements of analyte/sensitive layer interaction. In both cases chemometrics enables the quantification of analyte mixtures. These data-processing methods have also been successfully applied to antibody/antigen interactions even using cross-reactive antibodies. Because miniaturisation and parallelisation are essential approaches in recent years, some aspects and current trends, especially for bio-applications, will be discussed. Miniaturisation is especially well covered in the literature.     

Directed antibody-engineering techniques and their applications in food immunoassays

The use of antibodies in immunodiagnostics has achieved new insights with recombinant technologies. This review summarizes the methods used to produce recombinant antibodies and those to tailor their properties. Finally, we address the advantages and the possibilities of recombinant antibodies in immunoanalytical applications through examples with the main focus on applications related to food quality and safety analysis.     

Low thermal mass gas chromatography: principles and applications

In gas chromatography (GC), temperature programming is often considered to be the second most important parameter to control, the first being column selectivity. A radically new GC technology to achieve ultrafast temperature programming with an unprecedented cool down time and low power consumption has recently become available. This technology is referred to as low thermal mass GC (LTMGC). Though the technology has its roots in resistive heating, which forms the basis of principle and design concept, the approach taken to achieve ultrafast heating and cool down time by LTMGC represents a significant break-through in GC. Despite some rectifiable shortcomings, LTMGC has proven to be an ideal methodology to deliver near/real time GC data, high precision, and high throughput applications. It is a new approach for modern high-speed GC. This paper documents the fundamental design principles behind LTMGC, performance data, and examples of applications investigated.     

Capillary electrophoresis-mass spectrometry: 15 years of developments and applications

Since its introduction in 1987, capillary electrophoresis-mass spectrometry (CE-MS) has developed to a well accepted multidimensional analytical approach complementary and/or competitive to classical MS-hyphenated separation techniques. The threefold combination of rapid developments of an exceptional separation technique, of selective mass detection possibilities, and of very mild ionization modes first allowed these progresses. This article shows the CE specificities that need to be well controlled/known, compared to classical and more routinely used liquid chromatography in the light of its coupling to MS. The major trends and developments over the last 15 years and most of the reviews and applications found in ISI Web of science and publisher databases are presented in a tabulated way. The reader can thus rapidly find existing CE-MS analysis techniques in his field of research and application (forensics, environment, bioanalytics, pharmaceutics, and metabolites).     

Developments in hollow fiber based liquid-phase microextraction: principles and applications

Hollow fiber liquid-phase microextraction (HF-LPME) offers an efficient alternative to classical techniques for sample preparation and preconcentration. Features include high selectivity, good enrichment factors, and improved possibilities for automation. HP-LPME relies on the extraction of target analytes from aqueous samples into a supported liquid membrane (SLM) sustained in the pores of the wall of a porous hollow fiber, and then into an acceptor phase (that can be aqueous or organic) in the lumen of the hollow fiber. After extraction, the acceptor solution is directly subjected to a chemical analysis. HP-LPME can be performed in either the 2- or 3-phases mode. In the 2-phase mode, the organic solvent is present both in the porous wall and inside the lumen of the hollow fiber. In the 3-phase mode, the acceptor phase can be aqueous and this results in a conventional 3-phase system compatible with HPLC or capillary electrophoresis. Alternatively, the acceptor solution is organic and this represents a 3-phase extraction system with two immiscible organic solvents that is compatible with all common analytical instruments. In HP-LPME methods based on the use of SLMs, the mass transfer occurs by passive diffusion, and high extraction yields as well as efficient extraction kinetics are obtained by applying a pH gradient. In addition, active transport can be performed by using carrier or applying an electrical potential across the SLM. Due to high analyte preconcentration, excellent sample clean-up, and low consumption of organic solvent, HF-LPME has a large application potential in areas such as drug analysis and environmental monitoring. This review focuses on the fundamentals of extraction principles, technical implementations, and future trends in HF-LPME.