ABSTRACT
Biological active compounds such as insulin, heparin, progesterone and labeled-LH were entrapped in glutaraldehyde cross-linked bovine serum albumin (BSA) and human serum albumin (HAS) microspheres. Studies were carried out for their binding capacity and biodegradability using new proteolytic enzymes. Effects of proteolytic enzymes such as trypsin, chymotrypsin, papain and pronase-E on microspheres were studied in order to understand the biodegradability of the cross-linked proteins. It has been observed that labeled-LG was entrapped 60% in BSA and HAS microspheres. Labelled-LH-BSA, Labelled-LH-HAS and insulin microspheres were injected into mice and rabbits. It was observed that these cross-linked microspheres were biodegradable and the process appeared to be slow one, useful for sustained release of hormones. It was also observed that these albumin microspheres exhibit fluorescence at 495 nm.
Subject(s)
Drug Delivery Systems , Fluorescein-5-isothiocyanate/administration & dosage , Hormones/administration & dosage , Insulin/administration & dosage , Serum Albumin, Bovine/administration & dosage , Serum Albumin/chemistry , Animals , Biodegradation, Environmental , Cattle , Coconut Oil , Cross-Linking Reagents/pharmacology , Delayed-Action Preparations , Endopeptidases/pharmacology , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/pharmacokinetics , Glutaral/pharmacology , Hormones/chemistry , Hormones/pharmacokinetics , Humans , Insulin/analogs & derivatives , Insulin/chemistry , Insulin/pharmacokinetics , Luteinizing Hormone/administration & dosage , Luteinizing Hormone/chemistry , Luteinizing Hormone/drug effects , Luteinizing Hormone/pharmacokinetics , Mice , Microspheres , Mustard Plant , Olive Oil , Plant Extracts , Plant Oils , Rabbits , Serum Albumin/drug effects , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/pharmacokineticsABSTRACT
In vitro testing is useful for detecting pollen and fungal-allergen sensitivities in nasobronchial-allergy patients. However, allergen-coated discs or solid-phases supplied in kits do not contain all the relevant extracts. The present study was aimed to develop a safe and credible procedure of allergen coating on paper discs for allergy diagnosis by ELISA. Paper discs were coated with indigenous allergens using poly(vinyl alcohol) and glutaraldehyde. ELISAs were carried out to standardize the allergen-coated discs using skin-test-positive-patients' sera. The cut-off value (A) for ELISA was worked out by analysing normal human sera (NHS) and is based on the mean+/-2 S.D. for the NHS value. The allergen-coated discs, i.e. Centre for Biochemical Technology (CBT) discs, demonstrated excellent comparison with plate-binding method and Pharmacia discs [Prosopis juliflora (mesquite), critical value 0.879 at 5% probability+/-0.599, n=11, is significant]. Inter- and Intra-assay coefficients of variations ranged from 2.5-14.05% to 4. 8-11.5% with different allergens. Results of skin tests and ELISA with CBT discs showed 60-70% correlation.
Subject(s)
Allergens , Enzyme-Linked Immunosorbent Assay/methods , Hypersensitivity/diagnosis , Reagent Kits, Diagnostic , Evaluation Studies as Topic , Humans , Paper , Pollen/immunology , Reagent Kits, Diagnostic/standards , Skin TestsABSTRACT
We have developed a biostrip for determination of urea in serum. The test strip is based on enzymatic assay where urease has been immobilized on the chromatographic paper along with chromogen, phenol red. The chromogen is easily soluble in water and does not require other components for the color change. Serum urea reacts with urease and water to liberate ammonia and carbon dioxide. The liberated ammonia changes the pH of the reaction medium, which is monitored by the chromogen phenol red. A single step working reagent strip has been developed and the reaction is completed within 50 seconds at room temperature. With this test strip urea concentration is measured in serum as low as 0.15 g/L. The speed and convenience of determining urea in serum by this strip instantly makes it well suited for individuals, physicians and emergency centres.
ABSTRACT
The present study describes a new immobilization support for the preparation of enzyme membrane in the presence of organic solvents. The support was composed of Formvar solubilization in organic solvents with enzyme. More than 90% of the enzyme was immobilized on the membrane. The membrane was prepared by mixing 4% Formvar in organic solvents and 1% cholesterol oxidase was immobilized by entrapment technique. Practically no leaching of the entrapped enzyme was observed. The resultant immobilized enzyme membrane was stored at 4 degrees C for 10 days without losing its activity. The pH and temperature stabilities were greater than those of the native enzyme. The immobilized enzyme membrane has a long life due to its hydrophobic nature, as compared with other membranes.
Subject(s)
Cholesterol Oxidase/chemistry , Enzymes, Immobilized/chemistry , Polyvinyls/chemistry , Cholesterol Oxidase/metabolism , Colorimetry , Enzyme Stability , Enzymes, Immobilized/metabolism , Hydrogen-Ion Concentration , Organic Chemicals/chemistry , Solvents/chemistry , TemperatureABSTRACT
A method based on pseudoaffinity chromatography has been developed for the separation of lactate dehydrogenase (LDH), pyruvate kinase (PK) and aldolase from rabbit muscle extract using cross-linked guar (CLG) and cross-linked pectin (CLP) as the matrices, and dyes as the ligands. Screening of several dyes revealed that dyes No. 1014 and No. 1015, immobilized on CLG and CLP displayed a higher affinity for LDH and PK. Aldolase was not retained on any of the dye columns. It was observed that 1014-CLP and 1014-CLG columns retained 90% and 55% LDH activities, respectively, whereas 1015-CLP and 1015-CLG retained 83% LDH and 72% PK. A coupled-column system comprising 1014-CLP and 1015-CLP or 1014-CLG and 1015-CLG could separate LDH, PK, and aldolase from a mixture of these enzymes, as well as from rabbit muscle extract. Enzymes were found to be homogeneous on polyacrylamide gel electrophoresis. The method has been found to be simple and economical.
