Glutaraldehyde Coupling Research Articles

Antibody immobilization onto glow discharge treated polymers.

Previous studies have shown that certain glow discharge treated polymers strongly retain adsorbed albumin and fibrinogen. On the basis of this phenomenon, we have investigated the possibility of immobilizing antibodies on glow discharge treated surfaces for diagnostic immunoassay applications. As a model for antibody immobilization, bovine IgG was immobilized on the following polymers: polyethylene (PE), tetrafluoroethylene glow discharge treated PE (TFE/PE), poly(ethylene terephthalate) (PET), TFE/PET, poly(tetrafluoroethylene) (PTFE), ethylene glow discharge treated PET (E/PET) and hexamethyldisiloxane glow discharge treated PET (HMDS/PET). IgG was radiolabeled with 125I and immobilized by either of the following two methods: (a) physical adsorption of IgG on untreated and glow discharge treated polymers or (b) physical adsorption of albumin followed by chemical coupling of IgG to albumin by glutaraldehyde. IgG concentration as well as adsorption times were varied in order both to optimize the immobilization conditions and to investigate the adsorption and retention mechanisms. To evaluate the efficiency of the immobilization techniques, blood plasma, Tween-20, and sodium dodecyl sulfate (SDS) were used to elute the adsorbed IgG layer. We found that IgG was successfully immobilized on the fluorocarbon glow discharge treated surfaces by using either the physical adsorption or the glutaraldehyde coupling method, although the former is more efficient than the latter method.

Toxin detection using capacitance measurements on immunospecies grafted onto a semiconductor substrate

The feasibility of direct immunosensors based on capacitance measurements on silicon-silica heterostructures is shown for an immunosystem based on G type immunoglobulin. The first results obtained for the detection of enterotoxin B from Staphylococcus Aureus are presented. Capacitance measurements on immunosystems succeed only if the successive biomolecular layers grafted onto the heterostructures are electrically insulating, so they are strongly dependent on the coupling procedure used for antibody fixation on the silanized silica layer. Two coupling procedures were investigated. One is based on a cyanosilane [γ-cyanodecyldimethyl(dimethylamino)silane]; in this instance antibody fixation proceeds through a non-covalent strong interaction between the cyano groups and residual hydroxyl groups of the glycoproteins. However, in spite of the high fixation rate of antibodies, checked by ELISA tests, no capacitive response was obtained. The second procedure involved covalent grafting of the antibody onto an amino-silanized heterostructure (δ-aminobutyldimethylmethoxysilane) through a special glutaraldehyde coupling technique, allowing good electrical characteristics of the biomolecular layers. A typical capacitance decrease of 400 pF cm −2 was obtained for a toxin concentration of 10μg ml −1.

An improved conjugation method for controlled covalent coupling of synthetic peptides to proteins using glutaraldehyde in a dialysis method

Controlled and efficient conjugation of synthetic peptides to proteins, for use in immunization or in assay procedures, is a prerequisite for the immunological applications of synthetic peptides. This study describes a new method of conjugating synthetic peptides to proteins in such a way that no homopolymers of synthetic peptides or proteins occur. To achieve this, the protein is first activated with glutaraldehyde and subsequently excess glutaraldehyde is removed. Then coupling of the synthetic peptide to the activated protein occurs while subsequetly the surplus reactive glutaraldehyde groups on the protein are blocked with lysine. Excess free peptide and lysine is then removed by dialysis. This improvement not only results in better defined conjugates when compared to classical glutaraldehyde coupling, but also in the consumption of smaller amounts of synthetic peptide during conjugate formation. When used for immunization we obtained similar and sometimes even better responses with the glutaraldehyde based conjugates than with succinimidyl (MBS) conjugates of the same peptides. The performance of the modified conjugates in ELISA procedures, immunization and immunocytochemistry suggests that they are superior to conjugates formed by classical glutaraldehyde coupling.

Adsorption of charged substrates and products on an enzyme reactor prepared by glutaraldehyde coupling on alkylamine derivatives of Ti(IV)-coated porous silica beads

Ti( IV) coating of porous silica beads, followed by derivatization with 1,6-diaminohexane and activation with glutaraldehyde was tested for the immobilization of glutamate decarboxylase ( l-glutamate 1-carboxylyase, EC 4.1.1.15 ). The enzyme column prepared with the immobilized glutamate decarboxylase was designed for the preparation of 1 μmol γ-[ 13 N] aminobutyric acid, a new tracer for positron emission tomography. Preliminary results, indicating high immobilization yields of active enzyme with good long term stabilities, led to a more detailed investigation of the Ti( IV) coating. When a column, containing about 1 g of enzyme-loaded beads was used for the synthesis of γ-[ 13 N] aminobutyric acid ( GABA) from l -[ 13 N] glutamate, most of the 13 N activity remained adsorbed onto the column. The elution patterns of l-glutamate and GABA from columns of glutamate decarboxylase, immobilized on Ti ( IV) coated silica beads, were investigated by using an h.p.l.c. u.v. detector. Different treatments of the Ti( IV) coated supports were tested to improve the desorption kinetics of GABA and l-glutamate. None of these methods gave a satisfactory improvement of the elution patterns of GABA and l-glutamate. The results indicate that the Ti ( IV) coated silica beads have a large adsorption capacity, even though the enzyme is covalently linked. The described immobilization method is not recommended for enzymes having charged substrates or products and in which a small amount of substrate has to be applied onto a reactor containing a large amount of Ti( IV) coated support. The method can be applied when the enzyme reactor is operated in steady state conditions with continuous supply of substrate.

Retrograde axonal transport of asialoglycoproteins in mouse trigeminal neurons in vivo and in rat dorsal root ganglia neurons in vitro

Different terminal sugars of the glycoprotein orosomucoid were exposed by sequential glycosidase digestions. The orosomucoid and its different derivatives were conjugated to horseradish peroxidase by a two-step glutaraldehyde coupling procedure, injected into the snout of 12-day-old mice or exposed to dorsal root ganglia neurons from embryonic rats, cultivated in a two chamber system. A marked increase in transport of the conjugates in the trigeminal and dorsal root ganglia neurons was observed histochemically after removal of sialic acid, exposing galactose as the terminal sugar. Quantitative hydrolysis of galactose residues resulted in reduced uptake. The data suggest the presence of a galactose-recognition molecule in the axon-terminal membrane, involved in retrograde axonal transport.

The antithrombotic effect of prostaglandin E1 immobilized on albuminated polymer matrix.

For intravascular implantation, a biofunctional surface seems to retard surface thrombosis upon synthetic materials. Prostaglandins, like PGI2, PGE1, and PGD2, etc., are believed to stimulate membrane-bound adenyl cyclase and thereby raise intracellular levels of c-AMP within platelets, which inhibit platelet adhesion and aggregation. A new procedure is suggested for the immobilization of prostaglandin E1 on an albuminated polymer matrix, through glutaraldehyde coupling. Materials thus prepared show dramatic antiplatelet effects, with regard to platelet adhesion, when compared with albumin-immobilized surfaces. The affinity of various modified surfaces toward platelet adhesion is studied, using washed platelets suspended in Tyrode's solution. Octane contact angle studies are used to develop an understanding of the varied nature of bound substrates at equilibrium on polymer surfaces. These are studied at the solid/liquid interface, which is closest to in vivo conditions. The plasma recalcification time demonstrates the anticoagulant properties of various surfaces. A possible role of PGE1 in reducing platelet activity in the presence and absence of vitamin C is discussed. This technique may be used in the development of non-thrombogenic surfaces on existing biomedical polymers. Simultaneous pharmaceutical modification of the blood with vitamin C may enhance the blood compatibility of the surface.

High-performance displacement chromatography of nucleic acid fragments in a tandem enzyme reactor—liquid chromatograph system

Separation of the reaction mixture in the ribonuclease T1-catalyzed synthesis of GpU from cyclic GMP in the presence of a large excess of uridine was carried out by displacement chromatography on octadecyl-silica columns with 0.25 M n-butanol in the carrier buffer (50 mM phosphate, pH 3.0) as the displacer. The tandem reaction and separation was performed with an instrument assembled from parts customary in analytical high-performance liquid chromatography (HPLC). The enzyme was immobilized on microparticulate amino-silica by glutaraldehyde coupling and used in a short packed-bed reactor. The conditions of the reaction that reaches equilibrium after approximately half of the cyclic GMP is converted and the results of displacement chromatography were evaluated by HPLC analysis. The synthesis took place in a 2.5 mm long × 4.6 mm I.D. packed-bed reactor used in the recirculating mode and the product was introduced directly into the chromatographic unit consisting of two 4.6 mm I.D. columns, 150 and 250 mm in lengths, in series. The bulk of the uridine separated by frontal chromatography in the first column was withdrawn and displacement development in the second column was used in order to isolate the product from other components. Nearly 100 mg of GpU with a purity of 99.7% could be achieved in a chromatographic experiment of 2.4 h. By returning unreacted starting materials to the reactor the process can be carried out in a continual fashion and the system readily leads itself to automation. The combination of similar enzyme reactors with the liquid chromatograph operated in the displacement mode offers an efficient means for laboratory-scale separation of complex biochemical substances.

Enzymatic glucose electrodes.

Enzymatic electrodes are described for the specific and sensitive determination of glucose in blood. The probes are based on the conversion of B-D-glucose by glucose oxidase chemically attached to the surface of a Pt electrode. The peroxide liberated is sensed by measurement of the current generated at +0.7 volts vs. S.C.E. The enzyme, chemically attached to pig intestine via glutaraldehyde coupling is quite stable for months, even at room temperature. Other glucose electrodes developed at the University of New Orleans, using Pt (measurement of the O2 consumed at - 0.7 volts vs. S.C.E.) and I- probes are also described.

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K m value of the immobilized α-amylase was decreased by immobilization while V max was unaltered. E a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.

Fixed-time kinetic enthalpimetry: improved sensitivity for enthalpimetric enzyme activity determinations in homogeneous and heterogeneous systems

A fixed-time (integral) method is described for the enthalpimetric determination of enzyme activity. The method involves the determination of residual unreacted substrate after a fixed incubation time with the sample. Results are presented for the determination of cholinesterase in aqueous solution and in 0.1cm 3 samples of reference sera using 30-min incubation periods. Results are correlated with a spectrophotometric procedure. A precision of 1.8% relative standard deviation is reported for serum assays. Preliminary data are also presented for the enthalpimetric determination of cholinesterase activity after immobilization onto non-porous glass beads by carbodiimide and glutaraldehyde coupling procedures.

Bacterial dextranase immobilised on zirconia coated alkylamine glass using glutaraldehyde

Bacterial dextranase has been immobilised on zirconia coated alkylamine glass through the process of glutaraldehyde coupling. The immobilised enzyme preparation exhibited 62% of the initial enzyme activity with a conjugation yield of 18 mg/g support. K m of the immobilised enzyme exhibited a decline in its value as compared to the soluble enzyme while V max remained unaltered. E a of the enzyme was decreased upon conjugation. The soluble enzyme had its optimal pH at 5.4 while the alkylamine conjugated dextranase was optimally active in the pH range 5.2–6.2. The immobilised enzyme has also been characterised through its pI by a new method. The industrial importance of this work is discussed.

A comparison of different enzyme-antibody conjugates for enzyme-linked immunosorbent assay

Calf intestinal alkaline phosphatase (CIAP)- and horseradish peroxidase (HRP)-linked goat antibody (Ab) conjugates have been prepared by two procedures. One procedure is by glutaraldehyde coupling of the proteins; the other is by intermolecular disulfide bond formation of the appropriately modified proteins. The conjugates, specific for rabbit IgG, were tested for their effectiveness as reagents in enzyme-linked immunosorbent assays for ragweed antigen E specific rabbit antibody. The CIAP-Ab conjugate prepared by glutaraldehyde coupling and the HRP-Ab conjugate made by disulfide bond formation were equally effective reagents for immunoassay, but the HRP-Ab conjugate obtained by glutaraldehyde coupling was definitely less useful than the other two conjugates. The assay procedure utilized an antigen E coupled paper disc as the immunosorbent and it was sensitive in the range of 0.5–100 ng per test. Inhibition of the enzyme-linked immunosorbent assay was used as a sensitive technique for measuring antigenic activity of antigen E or its derivatives.

Immobilization of proteinases on Chitosan

Trypsin, pronase and subtilisin were immobilized on chitosan by glutaraldehyde coupling. Significant retention of activity was observed when synthetic substrates as well as casein were used. The specific activities of the bound proteinases ranged from 38% to 79% of their initial specific activities. The pH-activity profile of trypsin was slightly shifted toward alkaline values, and its thermal stability was increased. Immobilized trypsin was found to be less sensitive to its natural inhibitors than the soluble enzyme.

The immobilization of adenine nucleotides on polysaccharides by using glutaraldehyde coupling and borohydride reduction.

Adenine nucleotides were immobilized on modified Sepharose 4B or Dextran T40 with glutaraldehyde and reduced with KBH4. Binding was dependent on pH and the nature of the amino group on the modified polysaccharide. ATP bound to soluble dextran retained coenzyme activity with glycerol kinase. Binding is proposed to occur via a Schiff base.

Insulin and epidermal growth factor-urogastrone: Affinity crosslinking to specific binding sites in rat liver membranes

Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with (125)I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with (125)I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization.

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. II. Urease in a packed-column differential reactor system.

Urease from Jack bean was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column differential reactor. To facilitate comparison, the urease was immobilized by both diazo and glutaraldehyde coupling. The kinetic properties of immobilized urease were similar to those of the soluble enzyme and different immobilization methods did not appreciably alter the kinetic properties. The affects of three different amino acid activators appear to follow predictions obtained from a relatively simple competitive model, except at very low substrate levels.

LACTOSE HYDROLYSIS IN MILK AND MILK PRODUCTS BY BOUND FUNGAL BETA-GALACTOSIDASE

The β-galactosidase of Aspergillus niger was immobilized by glutaraldehyde coupling to porous glass beads and the bound enzyme evaluated for its applicability to hydrolysis of lactose in milk and milk products. Lactose in sweet whey and skim milk was hydrolyzed at approximately one-third the rate in acid whey. Non-lactose solids inhibited β-galactosidase activity. Greater efficiency of lactose hydrolysis was obtained with the bound enzyme in column operations than in stirred batch reactors.

Réactions synthétiques par la ribonucléase T 1

Separation of the reaction mixture in the ribonuclease T1-catalyzed synthesis of GpU from cyclic GMP in the presence of a large excess of uridine was carried out by displacement chromatography on octadecyl-silica columns with 0.25 M n-butanol in the carrier buffer (50 mM phosphate, pH 3.0) as the displacer. The tandem reaction and separation was performed with an instrument assembled from parts customary in analytical high-performance liquid chromatography (HPLC). The enzyme was immobilized on microparticulate amino-silica by glutaraldehyde coupling and used in a short packed-bed reactor. The conditions of the reaction that reaches equilibrium after approximately half of the cyclic GMP is converted and the results of displacement chromatography were evaluated by HPLC analysis. The synthesis took place in a 2.5 mm long × 4.6 mm I.D. packed-bed reactor used in the recirculating mode and the product was introduced directly into the chromatographic unit consisting of two 4.6 mm I.D. columns, 150 and 250 mm in lengths, in series. The bulk of the uridine separated by frontal chromatography in the first column was withdrawn and displacement development in the second column was used in order to isolate the product from other components. Nearly 100 mg of GpU with a purity of 99.7% could be achieved in a chromatographic experiment of 2.4 h. By returning unreacted starting materials to the reactor the process can be carried out in a continual fashion and the system readily leads itself to automation. The combination of similar enzyme reactors with the liquid chromatograph operated in the displacement mode offers an efficient means for laboratory-scale separation of complex biochemical substances.