Highly sensitive determination of DAO activity by oxidation of a luminescence reagent.

A highly sensitive method for measuring the activity of the enzyme diamine oxidase (DAO) independent of the type of substrate is described. The principle of the assay is to determine the amount hydrogen peroxide generated as a reaction product during oxidation of diamines by DAO. PSatto, a highly sensitive luminescence reagent, was used to generate a signal depending on the hydrogen peroxide concentration based on the action of horseradish peroxidase. DAO is specifically captured from a sample by an antibody immobilized to microwell plates, and the substrate is added to the bound enzyme. Various diamines were used as substrates; the peroxide produced is directly proportional to the amount of DAO bound to the specific antibodies. With this very sensitive method, it is possible to detect pmol amounts of generated hydrogen peroxide in plasma matrix corresponding to the biological activity of DAO.

Overexpression and purification of a recombinant chimeric HIV type 2/HIV type 1 envelope peptide and application in an accelerated immunobased HIV type 1/2 antibody detection system (AIBS): a new rapid serological screening assay.

A chimeric HIV-2/HIV-1 envelope sequence containing an immunodominant region of HIV-2 gp36 and the corresponding region of HIV-1 gp41 was constructed and overexpressed in Escherichia coli. The recombinant product (rp21/18) was purified and applied in a novel antibody-screening assay. Characteristics in the design of this new principle are as follows: (1) the overall assay time is about 30 min; (2) the assay procedure includes three manipulation steps; and (3) the test shows a reliable performance with respect to sensitivity and specificity. The diluted serum sample and the protein G-horseradish peroxidase conjugate are added simultaneously into a coated (hybrid antigen HIV-1/2) and blocked microtiter plate well. The in-batch incubation of serum sample with protein G-horseradish peroxidase saves two manipulation steps that are normally necessary in the five-step procedure of a classical ELISA. AIBS was evaluated with commercially available seroconversion panels and with random negative serum samples from a blood bank. Seroconversion results demonstrated that AIBS has equivalent sensitivity to ELISAs and the third generation assays. The specificity was determined on a total blood donor population of 5012 (Red Cross Vienna, Austria). The repeat reactive rates for donor population were 0.02%. AIBS represents a general immunometric system (not only HIV antibodies). The entire assay procedure of AIBS evaluated for HIV-1/2 screening, including result reporting, can be performed automatically by several commercially available systems. Depending on these systems AIBS is potentially useful in laboratories or blood banks that have both high- and low-volume testing.

Determination of salicylate in beverages and cosmetics by use of an amperometric biosensor.

A fast and selective enzymatic method for the determination of salicylate in beverages and cosmetics has been developed. The enzyme salicylate hydroxylase was immobilised covalently onto a glassy carbon working electrode of a wall-jet cell coupled with a flow-injection analysis system. The salicylate is enzymatically converted to catechol, which can be detected amperometrically on the glassy carbon electrode at +0.45 V. The response of the biosensor is linearly proportional to the concentration of salicylate between 725 nmol/l and 700 micromol/l. A high sample throughput (60 h(-1)) is possible, and the biosensor is stable for more than three months. Sample pretreatment for beverages and hair lotions is easy and fast. For creams, an extraction of salicylate is necessary. Relative standard deviations are less than 5.5% and the recoveries are between 95 and 105%.

Drug-protein conjugates: haptenation of 1-methyl-10 alpha-methoxydihydrolysergol and 5-bromonicotinic acid to albumin for the production of epitope-specific monoclonal antibodies against nicergoline.

Two types of monoclonal antibodies were used for the determination of nicergoline in biological matrices. The antibodies were prepared with the hydrolysis products 5-bromonicotinic acid and 1-methyl-10 alpha-methoxydihydrolysergol after hemisuccinoylation to haptens. The current amide bond-generating methods (mixed anhydride-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, and dicyclohexylcarbodiimide/N-hydroxysuccinimide methods) were used in bovine serum albumin (BSA)-coupling techniques and yielded conjugates that were haptenated to varying extents. The conjugates exhibiting 23 mol of 1-methyl-10 alpha-methoxydihydrolysergol (MMD) or 41 mol of 5-bromonicotinic acid (BNA) per mole of BSA were used for both immunization of mice and for coating the wells of the microtiter plates to select hybridomas and investigate specificity of the obtained antibodies. The results of hapten-inhibition ELISA using antigen-coated wells indicate that the supernatant of MMD-specific hybridoma exhibited 50% inhibition of antibody binding at 17 +/- 2 micrograms of MMD and at 24.5 +/- 2 micrograms of nicergoline, and the BNA-specific hybridoma exhibited similar inhibition at 147 +/- 6 micrograms of BNA and 500 +/- 30 micrograms of nicergoline. A main requirement for analytical purposes is that two different types of monoclonal antibodies recognize two different epitopes on nicergoline and its main metabolite, as shown by hapten-inhibition ELISA.

A rapid fluorimetric test for lysozyme with purified fluorescamine-labelled peptidoglycan.

A sensitive and rapid fluorometric lysozyme assay is described. It is based on the hydrolysis of fluorescamine-labelled peptidoglycan from Micrococcus luteus cell walls. Lysozyme levels as low as 0.1 microgram can be detected.

Quantitative determination of pectic substances as an example of a rhamnopolysaccharide assay.

A quantitative enzymatic assay for rhamnopolysaccharides is described. The procedure is shown with pectic substances as an example. The test is based on the enzymatic degradation of the macromolecules to liberate L-rhamnose. This sugar can be quantitatively determined with the help of L-rhamnose dehydrogenase under concomitant reduction of NAD, thus allowing the quantitative evaluation of the original pectin.

Determination of alpha-human atrial natriuretic peptide (alpha-hANP) in urine using combination HPLC with RIA strongly indicates non-immunoreactive metabolites.

Employing HPLC coupled with RIA, it was shown that alpha-human atrial natriuretic peptide is excreted in urine. Freshly collected urine had to be acidified to obtain reproducible results. When prepurified urine was subjected to HPLC (ion exchange and reversed phase) the subsequent quantification of alpha-hANP immunoreactive material in the eluate showed 10- to 30-fold greater amounts of alpha-hANP after treatment with HPLC; substances with the same elution parameters as synthetic alpha-hANP were detected, but they gave no response in the RIA.

Novel transducers for nano-optical biosensor chips based on biological and synthetic polymers with analyte-dependent swelling/shrinking behavior.

Analyte-dependent swelling/shrinking properties of ultrathin polymer layers are an appropriate means for the detection of various analytes. Optical metal nanoclusters can be used to determine the change of the layer's thickness, which is shown by a change in the color of the chip. By using different cross-linking agents and different polymers (biological or artificial as well) it was possible to design various sensitive layers showing different swelling/shrinking behaviors. Sensitivity on various analytes could be observed, since the different types of polymers employed differed in structure, functional groups, or biorecognitive properties.

Stabilisation and determination of the biological activity of L-asparaginase in poly(D,L-lactide-co-glycolide) nanospheres.

The preservation of biological activity of protein drugs in formulations is still a major challenge for successful drug delivery. The enzyme L-asparaginase, which exhibits a short in vivo half-life and is only active against leukaemia in its tetrameric form, was encapsulated in poly(D,L-lactide-co-glycolide) nanospheres by the (w/o)/w-emulsion solvent evaporation technique in presence of various potential stabilisers. Elucidation of the preparation steps revealed that the enzyme is denaturated at the aqueous/organic interface and by sonication. The preparation of L-asparaginase nanospheres with trehalose, PEG 400, and glycerol as components of the inner aqueous phase yielded colloidal formulations with increased biological activity as determined by an improved protocol for quantification of the active enzyme encapsulated. After lyophilisation the enzyme activity and particle size distribution were retained only by use of Pluronic F68 as a lyoprotectant. Despite the unaltered particle size and improved biological activity, the release profile of the enzyme was strongly altered by coencapsulation of the stabilisers resulting in increased first bursts. In consequence, the biological activity of L-asparaginase during preparation and storage can be improved by combining appropriate additives but concurrently the release profile is influenced.

High-throughput assays on the chip based on metal nano-cluster resonance transducers.

High throughput transducers using metal cluster resonance technology are based on surface-enhancement of metal cluster light absorption. These devices can be used for detection of biorecognitive binding, as well as structural changes of nucleic acids, proteins or any other polymer. The optical property for the analytical application of metal cluster films is the so-called anomalous absorption. An absorbing film of clusters positioned 10--400 nm to a mirror surface reacts in a similar way to a reflection filter. At a certain distance of the absorbing layer to the mirror the reflected electromagnetic field has the same phase at the position of the absorbing cluster as the incident fields. This feedback mechanism strongly enhances the effective cluster absorption coefficient. The system is characterised by a narrow reflection minimum whose spectral position shifts sensitively with the interlayer thickness, because a given cluster-mirror distance and wavelength defines the optimum phase. Based on this principle a set of novel tools including biochips and micro arrays is presented, which enabled us to transduce binding, as well as changes of protein-, DNA- and polymer-conformation, quantitatively into an optical signal which can be observed directly as a colour change of a sensor-chip surface.

Temperature-dependent behavior of immobilized alkaline phosphatase. I. Role of working conditions and pH during coupling.

Alkaline phosphatase from hog intestine was immobilized to controlled-pore glass under various conditions. The specific activity of the enzyme was not diminished by immobilization. The influence of temperature and pH on the behavior and the stability of the immobilized enzyme preparations is discussed and compared to that of the native enzyme.

Temperature-dependent behavior of immobilized alkaline phosphatase II. temperature pulse : A tool to enhance enzymatic activity for prolonged periods.

The specific activity of immobilized alkaline phosphatase can be considerably increased by exposure to a brief temperature pulse. The thus-pulsed preparations show a hysteresis of higher activity at room temperature varying from 10 min to several days, depending upon their pretreatment and the conditions of the reaction mixture during the pulse. In almost all cases the pulse can be repeated many times to increase the activity.

Immobilization ofmyo-inositol-1-phosphate synthase containing active, self-regenerating coenzyme (NAD(+)) on the same matrix.

myo-Inositol-1-phosphate synthase (EC 5.5.1.4) from rat testes, an NAD(+)-containing enzyme, which convertsD-glucose 6-phosphate to 1L-myo-inositol 1-phosphate, could be immobilized together with its cofactor and bovine serum albumin by crosslinking with glutaraldehyde at pH 4.5. The enzyme bound to the gel showed a specific activity of 5.6% of that of the native enzyme, but the activity could be increased to 21% by pretreatment with urea.

Influence of urea and organic solvents on the activity of immobilizedmyo-inositol-1-phosphate synthase containing active, self-regenerating coenzyme (NAD(+)) on the same matrix.

myo-Inositol-1-phosphate synthase (EC 5.5.1.4.) from rat testes, an NAD(+)-containing enzyme that convertsD-glucose 6-phosphate to 1L-myo-inositol-1-phosphate was immobilized together with its cofactor and bovine serum albumin by crosslinking with glutaraldehyde at pH 4.5. The cofactor is reduced and reoxidized during the reaction cycle, thus forming a self-regenerating system with respect to the cofactor. The behavior of this immobilized enzyme/cofactor system in presence of organic solvents and urea and the activating effect of these compounds on the enzymatic activity were studied and discussed in the paper.

Degradation of bacterial cell walls by immobilized lysozyme.

Lysozyme was immobilized by two different methods in two different ways in order to obtain a preparation with an activity as high as possible toward a macromolecular substrate. The enzyme was bound as a Schiff base to a silicate carrier by using oxidized dextrans of different lengths as spacer and also was bound to controlled pore aminoglass via pyridino-4-aldehyde and BrCN. The latter preparations had activities up to 2.5% of the free lysozyme.

Studies on the immobilization of glucuronidase (Part 2). Cleavage of hardly soluble substrates in organic solvents.

Naturally occurring glucuronides and glucosides dissolved in organic solvents can be split with the help of beta-glucuronidase (EC 3.2.1.31) immobilized on controlled pore glass. To protect the enzyme against denaturation by the organic solvents and to promote hydrolytic cleavage of substrates, two methods were used: (a) Immobilization via crosslinking with aged glutaraldehyde in presence of bovine serum albumin; and (b) Adsorption of wet enzyme to the carrier in the presence of organic solvents.

Studies on the immobilization of glucuronidase (Part 1). Covalent immobilization on various carriers (a comparison).

beta-Glucuronidase (EC 3.2.1.31) was immobilized on various organic and inorganic carriers by different methods. Optimum coupling conditions have been worked out. The immobilization were characterized and compared to each other. Parameters resulting in most stable preparations with high activities are discussed.

Immobilization of active facilitated glucose transporters (GLUT-1) in supported biological membranes.

Membrane fragments or membrane proteins within a lipid mixture were immobilized over metal electrodes. This procedure has been developed to study biological membranes without interference from cell machinery. To obtain a smooth hydrophilic biomembrane support and a mode of binding of the membrane, either a crosslinked gel or an aromatic polyamine-polymer doped with avidin was deposited at the metal electrode by electropolymerization. This layer (less than 10 nm thick) also served as a submembrane compartment. The facilitated glucose transporter (GLUT-1) purified from human erythrocytes was integrated into a lipid membrane containing artificial biotinylated lipids and reacted with the activated surface of the glucose sensitive electrode. It was demonstrated that the lipid layer was attached to the polymer-containing avidin and could only be removed by detergent extraction. The presence of an active membrane transporter was demonstrated by electrochemical detection of glucose in the submembrane compartment, and by inhibition of glucose transport with the specific inhibitor Cytochalasin-B.