Advanced Analytical Methods for Pharmaceutical and Diagnostic Applications

Driving forces for the development of novel analytical technologies in the life-science industry are described. Technologies which either were developed in Bio-Analytical Research or brought to a reliability required for routine applications will be elucidated and, on the basis of practical examples, the impact of modern analytical technologies on the industrial research and development will be discussed: Optical biosensors based on evanescent excitation of luminescence allow for real-time monitoring of the binding of active compounds to specific biomolecular recognition sites. Molecular imaging technologies have the potential to gain rapid access to physical maps of genomic materials. Capillary electrophoresis or affinity gel electrophoresis are well suited for the fast determination of oligonucleotide mixtures in nl amounts of samples. Integrated capillary electrophoresis on chips will allow to multiplex capillary systems at low costs and results in high separation efficiencies. MALDI-TOF MS is an easy to operate non-scanning mass spectrometric instrumentation for the analysis of high molecular weight biopolymers such as immunoglobulins

Pemphigoid Antibody Mediated Attachment of Peripheral Blood Leukocytes at the Dermal-Epidermal Junction of Human Skin

It has been proposed that cutaneous inflammation and blister formation in bullous pemphigoid is caused by antibodies to the cutaneous basement membrane zone which activate complement, thereby, attracting leukocytes to the dermal-epidermal junction There is, however, no functional evidence which supports a role for pemphigoid antibodies in complement activation or leukocyte activity in skin. This study describes the in vitro attachment of human peripheral blood leukocytes to the dermal-epidermal junction of cryostat skin sections treated with 9/13 pemphigoid sera containing antibodies to the cutaneous basement membrane zone. A requirement for complement in the reaction was supported by the findings that only complement-fixing pemphigoid sera mediated the leukocyte response, a strong correlation existed between complement-fixation titers and leukocyte attachment titers and only leukocytes suspended in fresh serum but not buffer or heat in activated serum attached at the junction. A requirement for antibody was supported by the observation that IgG fractions of 4 pemphigoid sera were as effective as whole sera in mediating leukocyte attachment. The leukocyte response was shown to be specific for complement-fixing pemphigoid sera since it was not observed with non-complement-fixing sera or sera from 15 normal human and 22 non-pemphigoid disease controls. This study offers functional evidence for an interaction between pemphigoid anti-body, complement and leukocytes in the immunopathogenesis of bullous pemphigoid and demonstrates that complement fixing antibasement membrane zone antibodies may be important in initiating the cellular inflammatory events observed near the dermal-epidermal junction in viva

DNA and Protein Microarrays and their Contributions to Proteomics and Genomics

Knowledge in genomics and proteomics has exploded in the last two decades. This is in part due to key developments that have revolutionized the possibilities of bioanalytics such as the introduction of polymerase chain reaction (PCR) in the mid 80s that formed the base for the massively parallel sequencing of the genomes.A few years ago DNA and protein microarray analysis were added to the toolbox of life sciences analytics. These technologies already proved to be ideal tools for the identification of gene targets, the simultaneous measurement of the expression of a high number of genes or proteins, and the increase of the level of understanding of the biological functions of genes and proteins. A small number of experiments are now sufficient to obtain information on gene or protein expression which could not be obtained by using conventional bioanalytical technologies or which required an extremely high experimental effort. In the future applications, high sensitivity DNA and protein microarrays will allow low abundant genes and proteins to be monitored that so far have been inaccessible to current microarray technologies and thus will generate a new dimension of genomic and proteomic information

Chip Technology in Analytical Chemistry: Focal Point: Environmental Analytical Chemistry : ILMAC Congress: October 13, 1999

The promises of chip technology in analytical chemistry are attractive: nanoliter-size sample volumes, low reagent consumption, high degree of multiplexing, short analysis times, ultra-low detection limits, ease in portability of process and result. It is predictable that chip technology will result in a significant cut of costs per information unit obtained whilst at the same time will improve the quality and validity of data obtained.A new era of massive parallel information generation will pave the way to novel, much more efficient approaches in research and development. Chip-based technologies will expedite the identification and development of drug candidates and be the tools for more precise identification, monitoring, and treatment of both gene-based and infectious diseases. This is made possible, on one hand, by the massively parallel detection of a multitude of biological markers and genetic predisposition parameters so far inaccessible to analysis systems and, on the other hand, by making decisions based on a much broader basis of pharmacologically and clinically significant parameters.Research approaches for chip-based analytical systems started about ten years ago and are focused on two main streams: chips for fluidic handling and separations and chips for detection of analytes. On the detection side, products are already on the market, e.g. genechips enabling information to be obtained on the presence of genetic variations and genetic defects based on the binding of DNA to over 50 000 different oligonucleotide probes immobilized on one chip. On the fluidic and separation side several companies have developed systems that are close to commercialization.The workshop gave a broad overview covering the current technical status of chip-based analytical systems, key applications in clinical research, in drug metabolism, and in the field of infectious diseases and included a critical discussion about current limitations as well as the future potential and value of bioinformatics. The outline of chip-based combinatorial chemistry approaches and novel high density enzymatic assays showed that 'classical' chip technology is already expanding to new fields of applications. Abstracts by the authors are given below

Analysis of Drug/Plasma Protein Interactions by Means of Asymmetrical Flow Field-Flow Fractionation

Purpose. The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption. Methods. Field-Flow Fractionation is defined as a group of pseudo-chromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy. Results. Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), α2-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothio-cyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA. Conclusions. If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug / plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation tim

Study of Saccharomyces cerevisiae Yeast Cells by Field-Flow Fractionation and Image Analysis

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