Abstract: Commercial cholesterol esterase from bovine pancreas and cholesterol oxidase from Brevibacterium recombinant type have been immobilized individually and co-immobilized on to arylamine glass beads (pore diameter, 55 nm) through diazotization. A method for discrete analysis of total cholesterol in serum was developed employing individually immobilized cholesterol esterase (0.36 mg/50 mg of glass beads) and cholesterol oxidase (0.41 mg/50 mg of glass beads) or co-immobilized cholesterol esterase and cholesterol oxidase (0.56 mg/100 mg of glass beads). Peroxidase from horseradish immobilized on to arylamine glass (0.9 mg/50 mg of glass beads) was common in both the cases. 4-Aminophenazone (0.25 mg/1.5 mL of reaction mixture) and phenol (0.5 mg/1.5 mL of reaction mixture) were used to form dye. In the method, cholesterol ester is hydrolyzed by cholesterol esterase to free fatty acid and cholesterol, which is oxidized by cholesterol oxidase to cholestenone and H2O2. H2O2 is determined enzymatically with horseradish peroxidase by additive coupling of 4-aminophenazone with phenol, and the resulting quinioneimine dye is measured at 520 nm, (epsilon = 4.0 x 10^-4). The lower detection limit of the method was 42.8 mg/l for individually immobilized enzymes and 21.4 mg/l for co-immobilized enzymes. Within-day and between-day coefficients of variation were < 1.5% and < 4.0% respectively for individually immobilized enzymes and < 1.0% and < 2.5% respectively for co-immobilized enzymes. A good correlation (r = 0.99) was found between total serum cholesterol obtained by the present method and a commercial enzo-kit method employing free enzymes. The individually immobilized/co-immobilized enzymes did not show much loss of activity after their 300 uses, when stored at 4°C in distilled water. The co-immobilized enzymes showed better efficiency in terms of sensitivity, linearity and precision compared with individually immobilized enzymes.

Abstract: The application of a chemiluminometric method for the online monitoring of a hybridoma cell culture is described. Enzyme sensors for glucose, lactate, glutamine, glutamate and ammonia, based on oxidase- catalyzed reactions, were developed and connected to a flow injection analysis (f.i.a.) biosensor. H2O2 produced by the oxidase-catalyzed enzyme reaction was detected by luminol chemiluminescence with a fiber- optic H2O2 biosensor. The system has been used to monitor animal cell cultures. A continuous hybridoma cell cultivation for the production of monoclonal antibodies is presented as an example. It was possible to monitor the bioprocess over a period of 15 days. A complete analysis of all five components could be performed within 42 min. The enzyme sensors were stable during the whole cultivation time without significant loss of activity. The computer-controlled biosensor f.i.a. works with good reliability. The precision for all five components ranged between 2.2 and 4.5%. It was possible to determine glutamine in one step using an anti-interference enzyme reactor. Endogenous glutamate was completely removed up to a level of 0.5 mM.

Abstract: Glutamate dehydrogenase(I) and L-glutamate oxidase(II) were immobilized (separately) on glass beads by the method of Weetall ('Immobilized Enzymes, Antibodies and Peptides', Dekker, New York, NY, 1975). A 12-mm layer of immobilized I and an 8-mm layer of immobilized II were packed in series in a column (2 cm x 2 mm) for use in a flow injection system. The flow injection system was used in the chemiluminescent determination of NH3 as described by Tabata et al. (J. Appl. Biochem., 1984, 6, 251). The calibration graph was rectilinear up to 0.3 mM NH3. Glutathione interferes; ascorbic acid interferes less than in other methods. Results agreed well with those obtained by a manual kit (r = 0.992).