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A method for enhancing the stability of thiol-containing enzymes immobilised on nylon.

The limited stability of immobilised enzymes is a major factor restricting their use in clinical chemistry. The immobilisation procedure described here affords a simple method of enhancing the stability of certain enzymes immobilised on nylon. Studies with alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase have indicated that a significant increase in stability can be obtained using this immobilisation procedure as compared with conventional procedures. As anticipated the immobilisation procedure was unsatisfactory for oxidases such as glucose oxidase.

Alcohol Oxidoreductases↗

Characteristic biochemical differences in human T and B lymphocytes separated on nylon wool.

T and B lymphocytes from human tonsils were separated on a nylon wool column. T. cells are enriched in the nonadherent, B cells in the adherent fraction. Several enzymes and other markers were tested in separated and non separated lymphocyte populations. Certain enzymes and other properties can be used as T or B lymphocyte markers because of their preferential occurrence and because of the advantages of their estimation (simple, quick methods, objective evaluation). The following characteristics were considered as markers on the basis of our results: (I) acid phosphatase, Na+-K+-activated ATPase, BAEE-peptidase and chromium labeling in T lymphocytes; (II) 5'-nucleotidase, FITC-IgG binding, N-acetyl-D-glucosaminidase, thymidine and valine incorporation in B lymphocytes.

B-Lymphocytes↗

The use of nylon fibre as a method of separating human lymphocyte subpopulations.

When the mononuclear cell population separated from normal human blood is passed through a nylon fibre column, monocytes and surface Ig bearing cells are retained; the eluted cells are a mixed properties. It appears that a proportion of the T cells eluted form EA-rosettes, and the eluted cell subpopulation includes non-T cells which share with B cells the ability to bind both EA and aggregated human IgG.

Cell Separation↗

Non-selective lymphocyte isolation from human blood by nylon wool filtration and density centrifugation.

A new method was employed to isolate lymphocytes from human peripheral blood. Continuous flow filtration through a nylon wool filter, at a flow rate of 1.4 ml/min, produced a lymphocyte yield of 90.5% and a purity of 96% without any shift in the B-T cell ratio. Ficoll-Isopaque with a specific gravity of 1.085 g/ml instead of 1.077 g/ml could be used to remove the erythrocytes. An overall recovery, including defibrination, filtration, Ficoll-Isopaque centrifugation and washing step, of 74.5% was achieved.

B-Lymphocytes↗

Lymphocyte membrane alterations caused by nylon wool column separation.

Use of the nylon wool column method to separate lymphocytes results in preparations which, while fully viable, have grossly depleted cellular levels of phospholipid, cholesterol and sialic acid. These membrane effects, which relate mainly to T cells, indicate that the method is unsuitable for use in biochemical analysis of lymphocytes.

B-Lymphocytes↗

Characteristics of nylon fiber adherence-separated chicken splenocytes.

Separation of chicken splenocytes by nylon fiber adherence yielded two distinct subpopulations. The non-adherent cells responded well to T cell mitogens, while the recoverable adherent cells did not. The non-adherent subpopulation showed a marked decrease in the number of surface immunoglobulin-bearing cells as compared to the adherent population. Cell diameter distribution was skewed toward larger sizes in the adherent subpopulation, and smaller sizes in the non-adherent subpopulation. There was no significant difference between the groups, however, in the number of cells which bore Fc receptors, stained for non-specific esterase, or actively phagocytosed. Therefore, the non-adherent subpopulation appeared to be enriched for T cells, the adherent subpopulation for B cells, and neither group for macrophages.

Animals↗

Human monocyte adherence measured by the nylon fibre microcolumn technique.

A simple rapid method for the measurement of human monocyte adherence using nylon fibre microcolumns is described. The kinetics indicate the optimal contact time to be 30 min for monocytes, compared with 10 min for neutrophils. The optimal temperature is 37 degrees C; significantly low values were obtained for 4 degrees C and 45 degrees C, while intermediate values were obtained for 25 degrees C. Monocyte adherence was more sensitive to inhibition by fluoride than cyanide, suggesting that energy for adherence is mainly derived from the glycolytic pathway. The addition of phorbol myristate acetate enhances monocyte adherence. Significant decay in monocyte adherence occurred after isolation from whole blood for 24 h or longer.

Cell Adhesion↗

The use of nylon wool for the isolation of T lymphocyte subpopulations.

A simple method for the affinity isolation of lymphocyte subpopulations using nylon wool as a solid phase matrix is described. This matrix was coated with an acid-treated IgG fraction of polyclonal antibodies against mouse immunoglobulins. Using this approach, a simple isolation procedure for lymphocyte subpopulations, yielding purities higher than 95% was developed. The method can be used on a large scale for positive and negative cell selection, with very little non-specific binding. The isolated populations are fully functional.

Animals↗

The adherence of Giardia lamblia trophozoites to nylon fibre microcolumns: effect of temperature, fluoride, cyanide and metronidazole.

A simple rapid method for the measurement of adherence of Giardia lamblia trophozoites is described. Microcolumns are prepared from 10 mg amounts of nylon fibre packed into 100 microliter size disposable pipette tips. The kinetics show that 30 min is the optimum contact time for the experiments. Maximum adherence occurred at 37 degrees C, minimal at 4 degrees C, and intermediate at 25 degrees C, suggesting that adherence is an energy-dependent process. Adherence was fluoride-sensitive but cyanide-insensitive, suggesting that this parasite derives its energy from the glycolytic pathway, and not the oxidative pathway. Metronidazole, one of the mainstays for chemotherapy of giardiasis, produced significant inhibition of adherence at 10 micrograms/ml and 100 micrograms/ml concentrations after one hour pre-incubation.

Adhesiveness↗

Routine analysis with immobilized enzyme nylon tube reactors.

The basic strategy involved in the design, development, and application of immobilized enzyme nylon tube reactors for routine analysis is described in this chapter, touching on some of the attractive features of these methods. Extensive data (Tables I and II) and the references provide details which may be needed based on specific methods.

Alcohol Dehydrogenase↗

Synthesis of esters using a nylon-immobilized lipase in batch and continuous reactors.

Direct esterifications using a nylon-immobilized lipase from Candida cylindracea were carried out in batch and continuous-flow reactors. The immobilized enzyme was effective in catalyzing the synthesis of ethylpropionate, isoamylpropionate, and isoamylbutyrate. With ethanol dissolved in hexane as a substrate, the maximum initial esterification rate was 0.02 mole/(h x g of immobilized protein), but the enzyme was stable only when the substrate concentrations were lower than 0.2 M. With isoamyl alcohol in hexane as a substrate, esterification rates as high as 0.085 mole/(h x g of immobilized protein) were observed and the immobilized enzyme was stable over a much broader concentration range. However, in this case, the use of a solvent, such as hexane, was not necessary for esterification, and the enzyme could be employed in equimolar acid/alcohol mixtures. A packed-bed reactor was operated successfully for the continuous synthesis of esters. The reactor was stable for long periods of time, and the steady-state performance could be accurately predicted on the basis of batch reaction experiments.

Candida↗

DNase activity of micrococcal endonuclease covalently immobilized on nylon and polystyrene.

Water-insoluble nucleases were prepared by immobilizing the endonuclease from S. aureus onto the surface of nylon-66 and polystyrene spheres. The activation phase of the synthetic supports was optimized to define optimal conditions of pH, temperature, and Ca2+ concentration for using immobilized enzymes. The activity, evaluated by hydrolysis of high-molecular-weight and supercoiled DNA, indicates that both derivatives are highly stable for storage and further use. Immobilization of the enzyme is much more effective when the covalent binding is performed on polystyrene. By using different activation methods with these matrices, a set of immobilized nucleases with various levels of enzymatic activity can be prepared. The possibility of working in a wide range of enzymatic activity and at low temperature and Ca2+ concentrations in different buffers makes these immobilized nucleases very useful for investigating accessible DNA regions in chromatin structure.

Biotechnology↗

Visualization and analysis of mural thrombogenesis on collagen, polyurethane and nylon.

Visualization and analysis of mural thrombogenesis on collagen-coated glass, polyurethane and nylon was discussed. Epi-fluorescent video microscopy was used to visualize thrombotic events at a protein or polymer surface in contact with flowing whole blood. Digital image processing was used to analyse the real-time microscopic images obtained, resulting in measurements of morphological features and the number of platelets that compose each thrombus. The three-dimensional structures of the thrombi were also estimated. A photodiode was used to measure integrated end-point platelet accumulation at different axial positions along the surface. Additionally, the convection-diffusion equations were solved to estimate the concentrations of adenosine diphosphate, thromboxane A2, and thrombin generated by activated platelets at the blood-contacting surface.

Adenosine Diphosphate↗

In vitro and in vivo studies on antithrombogenicity of poly(propylene oxide) segmented nylon 610 in relation to its crystalline-amorphous microstructure.

In vitro and in vivo antithrombogenicity of poly(propylene oxide) segmented nylon 610 was investigated. For the in vitro simulation of thrombosis on copolymer surfaces, a modified microsphere column method evaluated the thrombosis time of the copolymer column. For the in vivo evaluation of the antithrombogenicity of copolymer surfaces, small diameter tubing, precoated with copolymer, was implanted in rabbits as an arteriovenous shunt to determine the time of patency for the copolymer tubing. In both the in vitro and in vivo tests, the copolymer having a particular size and distribution of crystalline and amorphous microphases on its surface exhibited excellent antithrombogenicity; the copolymer with a long period of approximately 12 nm and a crystallite thickness of 6.5 nm suppressed the activation process of adhering platelets to show thromboresistant property. This paper emphasizes the importance of controlling the crystalline-amorphous microstructure for the antithrombogenicity of polymer surfaces with a semicrystalline microstructure.

Animals↗

The enzyme coupling process in urease immobilization on O-alkylated nylon tubes.

Coupling of Jack bean urease (EC 3.5.1.5) to the inside surface of type 6 nylon tubes, activated by high-temperature O-alkylation with dimethyl sulphate and modified subsequently with lysine and glutaraldehyde, was investigated to establish optimal experimental conditions for the coupling process. For the system described, the most active immobilized urease derivatives were prepared with 2 mg/ml of the solubilized urease solution and use of higher enzyme concentrations proved wasteful. Although urease coupling without thermal denaturation of the solubilized enzyme was achieved at 20 degrees C, derivatives prepared at 37 degrees C yielded maximal activity over the 3 h coupling period. Also, longer incubations of the enzyme solution in the tube were unnecessary under these conditions. Optimal pH for the coupling process was 6.5, one at which the solubilized enzyme was most stable.

Alkylation↗

An evaluation of serum sources used in the separation of T- and B-lymphocytes by nylon-wool columns.

Gamma-globulin-free (GG-) newborn-calf, mouse and horse, as well as newborn-calf and calf serums were evaluated as substitutes for fetal bovine serum in the classical nylon-wool procedure used for the separation of T- and B-lymphocytes. The efficacy of the cell separation was evaluated by non-specific esterase staining and cytotoxicity tests for cell surface Thy 1.2 antigen and surface immunoglobulins. The results of these studies indicate that GG- newborn-calf, mouse and horse but not newborn-calf or calf serum could be substituted for fetal bovine serum. The results of these studies also indicate that the ratio of adherent to non-adherent cells is a useful indicator of the efficacy of the cell separation. A ratio of non-adherent to adherent less than 1.0 is indicative of a successful separation.

Animals↗

Detection of anti-RNA antibodies in systemic lupus erythematosus by ELISA using nylon as solid support.

A highly sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) for the detection of anti-RNA antibodies is described. The assay procedure involves adsorption of total cellular RNA on nylon beads which could be conveniently stored for a considerable period of time without loss in antigenicity. Sixty-four percent of systemic lupus erythematosus (SLE) sera were positive for anti-RNA antibodies with fluorogenic substrate against 48% with colorigenic substrate.

Antibodies, Antinuclear↗

A nylon membrane enzyme immunoassay for rapid diagnosis of influenza A infection.

A new membrane-enzyme immunofiltration assay (MIFA) was developed for rapid diagnosis of influenza A infection. The pretreated specimens were dispensed into a 1.2 micron Biodyne B nylon membrane-bottomed microplate and vacuum filtration was applied. Blocking solution, peroxidase-conjugated anti-influenza A nucleoprotein monoclonal antibody, washing buffer and substrate were added in that order. The assay was completed within 30 min. Out of 103 nasopharyngeal swabs collected in transport medium, 31 isolates of influenza A virus were obtained and 22 specimens were detected directly by the MIFA technique. The 9 isolation-positive MIFA-negative specimens required 6 days or more for viral detection in cell culture, and probably contained a very low quantity of virus. The 72 cell culture negative specimens were also negative by MIFA. Comparison with a classical immunocapture assay (ICA) gave a better sensitivity for MIFA, as only 15/103 specimens were positive by ICA. MIFA is a rapid test with 71% sensitivity and 100% specificity. It was also very useful to test the cell culture supernatants, as a sensitivity of 100% was obtained with MIFA when the immunofluorescence technique was positive. The same technique could be readily carried out on the same plate for other respiratory viruses since capture antibody is not used.

Adolescent↗