AUTOMATED LIPOSOME-BASED FLOW INJECTION IMMUNOASSAY SYSTEM

A liposome-based flow injection immunoassay (FIIA) system for quantitation ofa clinical analyte, theophylline, and also, with very minor changes in the assay format, of an antibody to theophylline, has been developed. Automated sequential analyses were performed at room temperaturewith picomolesensitivity and a day-to-day coefficient of variation of less than 5 %. The system components include liposomesthat contain fluorophoresin their aqueouscentral cavities and an immobilized-antibody reactor column. The immunoreactor was regenerated hundreds of times over three monthsof continuoususe with no measurable loss of antibody activity. The special advantages ofusing flow injection analysis for immunoassays and ofusing liposomesin FIIA, compared to most current immunoassay techniques,are described

RAPID MICROBIAL TESTING IN ‘REAL’ SAMPLES

Three examples of electrochemical approachesto rapid assessmentof the microbiological Status of materials, especially foodstuffs, are described. One concerns the monitoring of overall reductive capacity for the determination of total microbial numbers in foodstuffs. A production prototype has now been developedfor the dairy industry. Another approachinvolvesthe useof a multiple, mediated amperometric glucose sensor to determine the glucose profile perpendicular to the surface of uncooked meat. This is affected by the surface microflora and hencethe principle can be used asthe basis of a meat freshness sensor. The final exampleis based on the exploitation of electrochemical immunoassay for identification of specific microbial markers

PROBING THE INTERACTION OF PHOSPHOLIPASE A2 AND PHOSPHOLIPIDS WITH RECOMBINANT DNA TECHNIQUES

The primary structure of eighty phospholipases A> from different sources is known. These sequences reveal a high degree of homology, and aboutthirty percent ofall residuesis fully conserved. In addition about twentyfive percentof the aminoacids is substituted by amino acids with similar properties like charge and/or polarity and hydrogen-bonding capacity. The use of recombinant DNAtechniques has proovento be a powerful techniqueto study the role of amino acid side chains in porcine pancreatic phospholipase Ap. The application of these techniques has madeit possible to create a deletion mutant with enhanced activity and improved crystallisation properties. It was shown that Leu-31 is important for the orientation of the substrate molecule and is probably also importantfor the shielding of the active site from excess water. Tyr- 69 was shownto be involved in the orientation of the substrate molecule through an interaction with the phosphate group ofthe substrate. A numberof absolute conserved residues like Tyr-52 and Tyr-73 are important for folding and/or stability of the protein. Theresults of the studies described here support the mechanism of catalysis of phospholipase A2 that was proposed already ten years ago

MEDIA ENGINEERING IN THE CATALYSIS BY LIPASES

Two reactions catalyzed by lipases have been studied, namely i) Transesterification between ethyl butyrate and glycerol in a two-phase system. The optimal water content in the mixture was 5%. Only monobutyrin was obtained. ii) Hydrolysis of p-nitrophenyl esters in microemulsions of sodium bis-2-ethylhexyl in heptane; its specificity constant was an order of magnitude lower than in aqueous mediun

COMPARATIVE ANALYSIS OF LIPASES IN VIEW OF PROTEIN DESIGN

The consensus sequences containing the active serine residue of 21 lipases were examined for structural properties by secondary structure prediction and hydrophobicity plots. Mostof the G-X-S-X-G peptides were found to form a turn structure andto be buried,i.e. inaccessible to water. The structural characters of a second serine containing consensus peptide describedin literature were compared to those of the G-X-S-X-G sequences. In addition, we investigated,if a correlation of the structural features of these peptides to the substrate specificity (regio specificity and fatty acid specificity) can be found

Biosensors based on capacitance measurements and 2D-microelectrophoresis on lipid membranes

A great numberofbiological receptor moleculesare located within lipid bilayers. How can one use them for biosensor applications? Biological receptors should be reconstituted into their natural environment- e.g. the lipid bilayer - to achieve proper function. It is therefore necessary to develop (1) methodsfor the preparation of high resistance lipid/protein membranesonsolid supports and (2) appropriate transducers for the conversion of the chemicalto an electrical signal. In our case we apply capacitance measurement. Amplification of the sensor signal is a further important requirement for biosensors to achieve sufficient signal/noise ratios of the sensor. We suggestthe lateral 2D-microelectrophoresis. This could be a useful tool for the separation and local accumulation of different receptors

MEMBRANE MATERIALS FOR AMPEROMETRIC AND POTENTIOMETRIC THICKFILM BIOSENSORS

Thickfilm transducers are reliable devices for the construction of biosensors. Polymer mixtures are described which can be used in screen printing processes and allow to immobilize biopolymers,e.g., enzymes, effectively. Potentiometric membranes with improved adhesionto the thickfilm electrodes are presented. Examples from the field of potentiometric (urea, penicillin) and amperometric (glucose, sucrose, ascorbic acid) enzyme sensors are reported and compared to conventional electrodes. The application of a 4-channel multisensor for the determination of glucose, sucrose and ascorbic acid is described

THE RELEVANCE OF GLYCOSYLATION TO THE PRODUCTION OF RECOMBINANT GLYCOPROTEINS

It is being increasingly recognised that many polypeptides of therapeutic interest, which in their native form are glycoproteins, need to be glycosylated in order to be of benefit in vivo..A consideration of polypeptide glycosylation therefore becomes relevant throughoutthe development and production of recombinant glycoproteins, principally for the following reasons. First, cell lines differ in their glycosylation characteristics, and the same polypeptide expressed in two different cell lines will generally be glycosylated differently. As a consequence, a recombinant glycoprotein is usually glycosylated differently to the native form, and such 'non-physiological' glycosylation can have profoundeffects on functional activity, physicochemical properties, and pharmacokinetic behaviour in vivo . A limited set of oligosaccharide determinants has been identified, the members of which influence the pharmacokinetic and immunogenic properties of a glycoprotein. It can therefore prove valuable to screen anycell line chosen for the production of a recombinant polypeptide, for expression of such determinants. Second, to ensurethat any changes in culture method (for example, during scale-up) are not associated with alterations in glycosylation, and that batch-to-batch uniformity is maintained during production,it is necessary to follow the glycosylation pattern of the secreted protein. Third, individual glycoformsof a polypeptide can differ with respect to functional properties. Identification of an improved product may, in some cases, involve nothing morethan isolation of a particular glycosylation variant. These and other aspects of the glycosylation of recombinant glycoproteins are discussed ir this paper

MICROSCOPIC MULTICHANNEL SPECTROMETER FOR LIGHT ABSORPTION MEASUREMENTS OF PIGMENTS INSIDE OF MAMMALIAN CELLS

During the evolution process most cells have learned to use oxygen for establishing higher functional regulations. For this purpose different proteins have been developed which are able to react with oxygen; as for instance hemoglobin and myoglobin for oxygen transport and storage or cytochromes, which act in the respiratory chain as electron carriers to reduce oxygen to water for generating energy. Also other proteins are known, like oxidases, which are involved in the reduction of oxygen. The question was therefore addressed, whether monitoring of the activity of these proteins in dependence on the oxygen partial pressure can be used to construct a biosensor for oxygen

An Algorithm for the Protein Docking Problem

We have implemented a parallel distributed geometric docking algorithm that uses a new measureforthe size of the contact area of two molecules. The measure is a potential function that counts the “van der Waals contacts” between the atoms of the two molecules ( the algorithm does not compute the Lennard-Jones potential). An integer constant c, is added to the potential for each pair of atoms whose distance is in a certain interval. For each pair whose distance is smaller than the lower boundofthe interval an integer constant c, is subtracted from the potential (cg < c,). The number of allowed overlapping atom pairs is handled by a third parameter N. Conformations where more than N atom pairs overlap are ignored. In our “real world” experiments we have used a small parameter N that allows small local penetration. The algorithm almost always found good (rms) approximations of the real conformations that were among the best five geometric dockings. In 42 of 52 test examples the best conformation with respect to the potential function was an approximation of the real conformation. The running time of our sequential algorithm is in the order of the running time of the algorithm of Norel et al. [NLW+]. Theparallel version of the algorithm has a reasonable speedup and modest communication requirements