Cancer Cell Membrane Camouflaged Nanoparticles to Realize Starvation Therapy Together with Checkpoint Blockades for Enhancing Cancer Therapy.

Although anti-PD-1 immunotherapy is widely used to treat melanoma, its efficacy still has to be improved. In this work, we present a therapeutic method that combines immunotherapy and starvation therapy to achieve better antitumor efficacy. We designed the CMSN-GOx method, in which mesoporous silica nanoparticles (MSN) are loaded with glucose oxidase (GOx) and then encapsulate the surfaces of cancer cell membranes to realize starvation therapy. By functionalizing the MSN's biomimetic surfaces, we can synthesize nanoparticles that can escape the host immune system and homologous target. These attributes enable the nanoparticles to have improved cancer targeting ability and enrichment in tumor tissues. Our synthetic CMSN-GOx complex can ablate tumors and induce dendritic cell maturity to stimulate an antitumor immune response. We performed an in vivo analysis of these nanoparticles and determined that our combined therapy CMSN-GOx plus PD-1 exhibits a better antitumor therapeutic effect than therapies using CMSN-GOx or PD-1 alone. Additionally, we used the positron emission tomography imaging to measuring the level of glucose metabolism in tumor tissues, for which we investigate the effect with the cancer therapy in vivo.

Altered Expression Patterns of the Sumoylation Enzymes E1, E2 and E3 Are Associated with Glucose Oxidase- and UVA-Induced Cataractogenesis.

PURPOSE: Protein sumoylation is a well established regulatory mechanism that regulates chromatin structure and dynamics, cell proliferation and differentiation, stress response and cell apoptosis. In the vertebrate eye, we and others have shown that sumoylation plays an indispensable role in regulating eye development. During stress induction and aging process, the ocular tissues gradually loss their normality and develop major ocular diseases such as cataract and aging-related macular degeneration. We have recently demonstrated that sumoylation actively regulates differentiation of lens cells, whether this process is implicated in lens pathogenesis remains to be investigated. In this study, we have demonstrated that transparent mouse lenses treated with glucose oxidase and UVA irradiation undergo in vitro cataract formation, and associated with this process, the expression patterns of the 3 sumoylation enzymes have been found significantly altered. METHODS: Four-week-old C57BL/6J mice were used in our experiment. Lenses were carefully excised from eyes and cultured in M199 medium (Sigma 3769) for at least 12 hours. Transparent lenses (without surgical damage) were selected for experimentation. The lenses were exposed to UVA for 60 min or treated with 30 mU/mL glucose oxidase (GO, MP Biomedicals, 1673) to induce cataract formation. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: we have obtained the following results: 1) Both GO treatment and UVA irradiation can induce cataract formation in the in vitro cultured mouse lenses; 2) With GO treatment, the mRNAs and proteins for the 5 sumoylation enzymes were all significantly downregulated; 3) With UVA irradiation, the changes in the expression patterns of the mRNAs and proteins for the SAE1, UBA2 , UBC9 and PIAS1 were opposite, while the mRNAs were upregulated either significantly (for SAE1, UBA2 and UBC9) or slightly (PIAS1), the proteins for all 4 sumoylation enzymes were downregulated; For RanBP2, the UVA induced changes in both mRNA and protein are consist with the GO treatment. CONCLUSION: Under GO and UVA irradiation conditions, the expression levels of both mRNA and protein for the three major sumoylation enzymes were significantly changed. Our results suggest that altered expression patterns of the sumoylation enzymes are associated with oxidative stressinduced cataractogenesis.

A sensitive immunosensor via in situ enzymatically generating efficient quencher for electrochemiluminescence of iridium complexes doped SiO2 nanoparticles.

A sensitive electrochemiluminescent (ECL) sandwich immunosensor was proposed herein based on the tris (2-phenylpyridine) iridium [Ir(ppy)3] doped silica nanoparticles (SiO2@Ir) with improved ECL emission as signal probes and glucose oxidase (GOD)-based in situ enzymatic reaction to generate H2O2 for efficiently quenching the ECL emission of SiO2@Ir. Typically, the SiO2@Ir not only increased the loading amount of Ir(ppy)3 as ECL indicators with high ECL emission, but also improved their water-solubility, which efficiently enhanced the ECL emission. Furthermore, by the efficient quench effect of H2O2 from in situ glucose oxidase (GOD)-based enzymatic reaction on the ECL emission of SiO2@Ir, a signal-off ECL immunsensor could be established for sensitive assay. With N-terminal of the prohormone brain natriuretic peptide (BNPT) as a model, the proposed ECL assay performed high sensitivity and low detection limit. Importantly, the proposed sensitive ECL strategy was not only suitable for the detection of BNPT for acute myocardial infarction, but also revealed a new avenue for early diagnosis of various diseases via proteins, nucleotide sequence, microRNA and cells.

In situ deposition of Prussian blue on mesoporous carbon nanosphere for sensitive electrochemical immunoassay.

A Prussian blue (PB) functionalized mesoporous carbon nanosphere (MCN) composite was prepared for loading signal antibody and high-content glucose oxidase (GOD) to obtain a new nanoprobe for sensitive electrochemical immunoassay. The MCN nanocarrier with an average diameter of 180 nm was synthesized by using mesoporous silica nanosphere as a hard template in combination with a hydrothermal carbonization method. This hydrophilic carbon nanomaterial provided an ideal platform for in situ deposition of high-content PB to form the MCN-PB nanocomposite. Based on the step-wise assembly of polyelectrolyte and gold nanoparticles (Au NPs) on the negative-charged nanocomposite, signal antibody and high-content GOD were loaded on this nanocarrier to obtain the nanoprobe. After a sandwich immunoreaction at an Au NPs-modified screen-printed carbon electrode based immunosensor, the nanoprobes were quantitatively captured on the electrode surface to produce sensitive electrochemical response with a PB-mediated GOD catalytic reaction for immunoassay. The high loading of PB and GOD on the nanoprobe greatly amplified the electrochemical signal, leading to the development of a new immunoassay method with high sensitivity. Using human immunoglobulin G as a model analyte, excellent analytical performance including a wide linear range from 0.01 to 100 ng/mL and a low detection limit down to 7.8 pg/mL was obtained. Additionally, the immunosensor showed high specificity, satisfactory stability and repeatability as well as acceptable reliability. The PB-mediated GOD electrochemical system well excluded the conventional interference from the dissolved oxygen. Thus this immunoassay method provides great potentials for practical applications.

Enzymatically catalytic deposition of gold nanoparticles by glucose oxidase-functionalized gold nanoprobe for ultrasensitive electrochemical immunoassay.

A novel ultrasensitive immunoassay method was developed by combination of the enzymatically catalytic gold deposition with the prepared gold nanoprobe and the gold stripping analysis at an electrochemical chip based immunosensor. The immunosensor was constructed through covalently immobilizing capture antibody at a carbon nanotube (CNT) modified screen-printed carbon electrode. The gold nanoprobe was prepared by loading signal antibody and high-content glucose oxidase (GOD) on the nanocarrier of gold nanorod (Au NR). After sandwich immunoreaction, the GOD-Au NR nanoprobe could be quantitatively captured onto the immunosensor surface and then induce the deposition of gold nanoparticles (Au NPs) via the enzymatically catalytic reaction. Based on the electrochemical stripping analysis of the Au NR nanocarriers and the enzymatically produced Au NPs, sensitive electrochemical signal was obtained for the immunoassay. Both the GOD-induced deposition of Au NPs by the nanoprobe and the sensitive electrochemical stripping analysis on the CNTs based sensing surface greatly amplified the signal response, leading to the ultrahigh sensitivity of this method. Using carcinoembryonic antigen as a model analyte, excellent analytical performance including a wide linear range from 0.01 to 100 ng/mL and a detection limit down to 4.2 pg/mL was obtained. In addition, this immunosensor showed high specificity and satisfactory reproducibility, stability and reliability. The relatively positive detection potential excluded the conventional interference from dissolved oxygen. Thus this electrochemical chip based immunosensing method provided great potentials for practical applications.

Digital multimeter-based immunosensing strategy for sensitive monitoring of biomarker by coupling an external capacitor with an enzymatic catalysis.

A new digital multimeter (DMM)-based immunosensing system was designed for quantitative monitoring of biomarker (prostate-specific antigen, PSA used in this case) by coupling with an external capacitor and an enzymatic catalytic reaction. The system consisted of a salt bridge-linked reaction cell and a capacitor/DMM-joined electronic circuit. A sandwich-type immunoreaction with target PSA between the immobilized primary antibody and glucose oxidase (GOx)-labeled detection antibody was initially carried out in one of the two half-cells. Accompanying the sandwiched immunocomplex, the conjugated GOx could catalyze the oxidation of glucose, simultaneously resulting in the conversion of [Fe(CN)6](3-) to [Fe(CN)6](4-). The difference in the concentrations of [Fe(CN)6](3-)/[Fe(CN)6](4-) in two half-cells automatically produced a voltage that was utilized to charge an external capacitor. With the closing circuit switch, the capacitor discharged through the DMM, which could provide a high instantaneous current. Under the optimal conditions, the resulting currents was indirectly proportional to the concentration of target PSA in the dynamic range of 0.05-7 ng mL(-1) with a detection limit (LOD) of 6 pg mL(-1). The reproducibility, precision, and selectivity were acceptable. In addition, the methodology was validated by analyzing 12 clinical serum specimens, receiving a good accordance with the referenced values for the detection of PSA.

Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor marker.

A novel tracer, glucose oxidase (GOD)-functionalized hollow gold nanospheres encapsulating glucose oxidase (Au(shell)@GOD), was designed to label the ferrocenemonocarboxylic-grafted secondary antibodies (Fc@Ab(2)) for highly sensitive detection of tumor marker using carboxyl group functionalized multiwall carbon nanotubes as platform. Initially, Au(shell)@GOD was synthesized specially by reverse micelle approach, and then the labeling of antibody and the preparation of GOD-functionalized Au(shell)@GOD were performed by one-pot assembly of Fc@Ab(2) and GOD on the surface of Au(shell)@GOD. The ferrocene used to label antibodies acted as a mediator of electron transfer between GOD and electrode surface. The high-content glucose oxidase in the tracer (on the surface and in the cavity) could significantly amplify the amperometric signal for sandwich-type immunoassay. Using carcinoembryonic antigen (CEA) as model analyte, the designed tracer showed linear range from 0.02 to 5.0 ng mL(-1) with the detection limit down to 6.7 pg mL(-1). The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The new protocol showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and diagnosis of tumor disease.

Complexing of glucose oxidase with anti-glucose oxidase antibodies or the F(ab)'(2)/F(ab)' fragments derived therefrom protects both the enzyme and antibody/antibody fragments against glycation.

Incubation of Aspergillus niger glucose oxidase with glucose, fructose, or ribose results in remarkable inactivation of the enzyme. Glucose oxidase incubated with the sugars migrated as a diffuse band of low intensity and silver stained poorly after SDS-PAGE. Purified anti-glucose oxidase antibodies and F(ab)'(2) or F(ab)' derived therefrom were effective in restricting the inactivation of the enzyme induced by the sugars, providing up to 90% protection. The sugars also caused remarkable changes in the electrophoretic behavior of anti-glucose oxidase antibodies and the fragments, but complexing with glucose oxidase restricted the changes both in the enzyme and the antibody/antibody fragments.

Cloning and characterization of a single chain antibody to glucose oxidase from a murine hybridoma.

Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.

Immunoglobulin glycation with fructose: a comparative study.

BACKGROUND: Immunoglobulins undergo non-enzymatic glycation reaction with sugars both in vivo and in vitro. Effects of glycation on the ability of the antibodies to bind antigens are contradictory. Antibodies raised in various animals may also be exposed to high concentration of sugars that are added during freeze-drying/pasteurization for preservation. METHODS: IgG isolated from the sera of goat, human, rabbit, mouse, buffalo as well as IgY from hen egg yolk was subjected to in vitro glycation with fructose. The behavior of glycated IgG was investigated by SDS-PAGE, hyperchromicity at 280 nm, tryptophan fluorescence and new fluorescence. RESULTS: Marked variations were observed in the response of the immunoglobulins derived from various animals to incubation with fructose. Also, incubation of anti-glucoseoxidase (GOD) antibodies with fructose resulted in a rapid loss of their ability to bind the enzyme antigen as revealed by immunodiffusion and ELISA. DETAPAC and EDTA were quite protective but were unable to completely prevent the fructose-induced alterations. CONCLUSIONS: Immunoglobulins derived from goat, human, rabbit, mouse, buffalo and hen egg yolk undergo remarkable structural alterations on incubation with fructose. The susceptibility of the immunoglobulins to the modification however differed remarkably. The goat IgG was most recalcitrant while hen egg yolk IgY was most susceptible to the alterations. DETAPAC or EDTA restricted the fructose-induced alterations remarkably.

Preparation of a highly stable, very active and high-yield multilayered assembly of glucose oxidase using carbohydrate-specific polyclonal antibodies.

The purified oligosaccharide chains of Aspergillus niger glucose oxidase were coupled to BSA with the help of the cross-linking reagent glutaraldehyde. The neoglycoconjugate thus obtained was purified by concanavalin A-Sepharose chromatography and characterized by SDS/PAGE. Immunization of rabbits with the neoglycoprotein raised the glycosyl-specific anti-(glucose oxidase) polyclonal antibodies. Antibodies were purified by (NH(4))(2)SO(4) precipitation, followed by DEAE-cellulose chromatography. The IgG-Sepharose was prepared by covalently coupling the purified polyclonal antibodies to the CNBr-activated Sepharose 4B. The large assembly of glucose oxidase was made on the IgG-Sepharose by alternate incubation of glucose oxidase and glycosyl-specific anti-(glucose oxidase) polyclonal IgG. The immunoaffinity-layered assembled preparations were highly active and, after six alternate binding cycles with enzyme and glycosyl-specific IgG, the amount of enzyme immobilized could be raised 30-fold. The K (m) value of immunoaffinity-layered glucose oxidase preparations remained unaltered, while the V (max) slightly decreased as compared with the soluble enzyme. A layer-by-layer immobilization of glucose oxidase resulted in significant improvement in stability against high temperature, 4.0 M urea and very high concentrations of water-miscible organic solvents such as acetone, dimethylformamide, dioxan and tetrahydrofuran.

Isolation and characterization of the human monoclonal antibodies C10 in single-chain fragment variable (scFv) format to glucose oxidase from Aspergillus niger.

Despite biotechnological and clinical applications very few monoclonal antibodies (MAbs) directed to the enzyme glucose oxidase, have been produced so far because of the heavy side effects of the immunization schedule for conventional MAb preparation. In contrast, the phage display method allows for the selection of monoclonal human antibody fragments against any antigens, including toxic proteins. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human fragments recognizing glucose oxidase, we used the large synthetic ETH-2 library based on the principle of protein design. Phage displaying glucose oxidase reactive scFvs were obtained after three rounds of selection on glucose oxidase-coated immunotubes and subsequent amplification in TG1 E. coli cells. Eventually, one high reactive scFv clone was selected and further examined. The anti-glucose oxidase scFv C10 was found suitable for Western blot; Biacore analysis showed that the binding affinity of the glucose oxidase-reactive scFv is almost equal that of MAbs prepared with conventional hybridoma technology. Finally, the cDNA sequence of this human scFv may be exploited to generate bispecific antibodies to target in the tumor environment-specific toxic enzymatic reaction.

Vascular immunotargeting of glucose oxidase to the endothelial antigens induces distinct forms of oxidant acute lung injury: targeting to thrombomodulin, but not to PECAM-1, causes pulmonary thrombosis and neutrophil transmigration.

Oxidative endothelial stress, leukocyte transmigration, and pulmonary thrombosis are important pathological factors in acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Vascular immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) to the pulmonary endothelium causes an acute oxidative lung injury in mice.(1) In the present study we compared the pulmonary thrombosis and leukocyte transmigration caused by GOX targeting to the endothelial antigens platelet-endothelial cell adhesion molecule (PECAM) and thrombomodulin (TM). Both anti-PECAM and anti-TM delivered similar amounts of (125)I-GOX to the lungs and caused a dose-dependent, tissue-selective lung injury manifested within 2 to 4 hours by high lethality, vascular congestion, polymorphonuclear neutrophil (PMN) sequestration in the pulmonary vasculature, severe pulmonary edema, and tissue oxidation, yet at an equal dose, anti-TM/GOX inflicted more severe lung injury than anti-PECAM/GOX. Moreover, anti-TM/GOX-induced injury was accompanied by PMN transmigration in the alveolar space, whereas anti-PECAM/GOX-induced injury was accompanied by PMN degranulation within vascular lumen without PMN transmigration, likely because of PECAM blockage. Anti-TM/GOX caused markedly more severe pulmonary thrombosis than anti-PECAM/GOX, likely because of TM inhibition. These results indicate that blocking of specific endothelial antigens by GOX immunotargeting modulates important pathological features of the lung injury initiated by local generation of H(2)O(2) and that this approach provides specific and robust models of diverse variants of human ALI/ARDS in mice. In particular, anti-TM/GOX causes lung injury combining oxidative, prothrombotic, and inflammatory components characteristic of the complex pathological picture seen in human ALI/ARDS.

The antioxidant system beta-D(+) glucose-glucose oxidase-catalase: tests for pyrogenicity and antigenicity.

PURPOSE: beta-D(+) glucose-glucose oxidase (E.C.# 1.1.3.4)-catalase (E.C.# 1.11.1.6) (GO-CAT) is being investigated as a new antioxidant system for use in pharmaceutical solutions. This study reports the results of tests for pyrogenicity and antigenicity of GO-CAT derived from Aspergillus niger when used parenterally in autoclaved preparations. METHODS: The Limulus amebocyte lysate (LAL) method was used to test the pyrogenicity of native GO-CAT. Pyrogenicity/antigenicity was evaluated in vivo by injecting autoclaved GO-CAT into New Zealand white rabbits. Antigenicity was also evaluated by Ouchterlony and Western blotting. RESULTS: None of the native GO-CAT concentrations tested (up to 30.83 u/ml) produced a positive gel clot in the LAL test, thereby suggesting its non-pyrogenicity. The rabbits, which received seven injections of autoclaved GO-CAT over a period of eleven weeks, remained healthy during and after the GO-CAT injections. All Ouchterlony and Western blot assays using sera from rabbits injected with autoclaved GO-CAT were negative. Furthermore, autoclaved GO-CAT could not be detected in Ouchterlony assays using a mouse monoclonal antibody (GO40 mAb) to native A. niger glucose oxidase. Control samples containing native GO-CAT produced an antigen-antibody complex reaction in Ouchterlony assays against the GO40 mAb. Antigen-antibody complexes could be detected by non-denaturing PAGE in samples containing native GO-CAT/GO40 and boiled GO-CAT/GO40, but not in samples containing autoclaved GO-CAT/GO40. These results indicate autoclaved GO-CAT is neither pyrogenic nor antigenic. CONCLUSIONS: Based on these results, there is potential for the use of beta-D(+) glucose-glucose oxidase-catalase as an antioxidant system in pharmaceutical solutions, particularly in terminally autoclaved aqueous formulations for parenteral use.

A discussion of anti-Aspergillus niger glucose oxidase monoclonal antibody reactivity to red blood cells of several species.

We observed that mouse spleen cells from rosettes with autologous red blood cells (RBCs) and that rosette-formation was suppressed by anti-Aspergillus niger glucose oxidase monoclonal antibody (mAb). In the present study, we investigated whether RBCs of species besides mice have the structure recognized by anti-A. niger glucose oxidase mAb by using rosette-formation and complement-mediated hemolysis. Lysates of monkey and human RBCs did not suppress rosette-formation whereas autologous (mouse), rat and sheep RBC lysates partially suppressed rosette-formation. Those lysates exerted their suppressive activity after they had been treated at 56 degrees C for 30 min. A. niger glucose oxidase also suppressed rosette-formation with or without treatment at 56 degrees C for 30 min. Alternatively, anti-A. niger glucose oxidase mAb lysed mouse, rat and sheep RBCs but not human RBCs with complement. These findings suggest that the cell surfaces of mouse, rat and sheep RBCs have a structure which can be recognized by anti-A. niger glucose oxidase mAb while the cell surfaces of monkey and human RBCs do not.

Immunotargeting of glucose oxidase to endothelium in vivo causes oxidative vascular injury in the lungs.

Vascular immunotargeting is a novel approach for site-selective drug delivery to endothelium. To validate the strategy, we conjugated glucose oxidase (GOX) via streptavidin with antibodies to the endothelial cell surface antigen platelet endothelial cell adhesion molecule (PECAM). Previous work documented that 1) anti-PECAM-streptavidin carrier accumulates in the lungs after intravenous injection in animals and 2) anti-PECAM-GOX binds to, enters, and kills endothelium via intracellular H(2)O(2) generation in cell culture. In the present work, we studied the targeting and effect of anti-PECAM-GOX in animals. Anti-PECAM-GOX, but not IgG-GOX, accumulated in the isolated rat lungs, produced H(2)O(2,) and caused endothelial injury manifested by a fourfold elevation of angiotensin-converting enzyme activity in the perfusate. In intact mice, anti-PECAM-GOX accumulated in the lungs (27 +/- 9 vs. 2.4 +/- 0.3% injected dose/g for IgG-GOX) and caused severe lung injury and 95% lethality within hours after intravenous injection. Endothelial disruption and blebbing, elevated lung wet-to-dry ratio, and interstitial and alveolar edema indicated that anti-PECAM-GOX damaged pulmonary endothelium. The vascular injury in the lungs was associated with positive immunostaining for iPF(2alpha)-III isoprostane, a marker for oxidative stress. In contrast, IgG-GOX caused a minor lung injury and little (5%) lethality. Anti-PECAM conjugated with inert proteins induced no death or lung injury. None of the conjugates caused major injury to other internal organs. These results indicate that an immunotargeting strategy can deliver an active enzyme to selected target cells in intact animals. Anti-PECAM-GOX provides a novel model of oxidative injury to the pulmonary endothelium in vivo.

Selective binding of a monoclonal antibody to Aspergillus niger glucose oxidase by formaldehyde fixed human polymorphonuclear leukocytes.

BACKGROUND: Many of the procedures used in handling neutrophils may affect the expression of surface antigens, and hence their quantitation by flow cytometry. METHODS: Because the enzyme glucose oxidase of Aspergillus niger is absent in human tissues, an IgM against it (mAb GO) was used as negative control in a study involving the normal expression of neutrophil specific BH2-Ag in different age groups. RESULTS: When peripheral blood leukocytes (PBL) were freshly prepared, processed and stained with FITC-mAb GO without fixation or when the cells were stained with FITC-mAb GO prior to fixation with 2% formaldehyde, both median fluorescent intensity (MFI) and per cent of positively stained polymorphonuclear leukocytes (PMN) were similar to that obtained with a background sample without any antibody. However, when PBL were fixed after isolation with different concentrations of formaldehyde and for varying durations, MFI and per cent of positively stained PMN but not of monocytes or lymphocytes with FITC-mAb GO increased in a time and concentration dependent manner. Saturation was achieved at a finite concentration of the antibody. In a competition assay unlabelled mAb GO reduced binding of FITC-mAb GO to PMN by 79% and 95% at concentrations 100 and 200 times that of FITC labelled antibody, respectively. CONCLUSIONS: These observations strongly suggest that formaldehyde fixation causes the expression or accessibility of an epitope on PMN that is specifically recognized by the mAb GO.

Immunoaffinity layering of enzymes. Stabilization and use in flow injection analysis of glucose and hydrogen peroxide.

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.

Immunotargeting of glucose oxidase: intracellular production of H(2)O(2) and endothelial oxidative stress.

Extracellular and intracellular reactive oxygen species attack different targets and may, therefore, result in different forms of oxidative stress. To specifically study an oxidative stress induced by a regulated intracellular flux of a defined reactive oxygen species in endothelium, we used immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-(125)I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-(125)I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H(2)O(2) from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H(2)O(2) was generated intracellularly, whereas 30% of H(2)O(2) was detected in the cell medium. Catalase added to the cells eliminated H(2)O(2) in the medium but had little effect on the intracellular generation of H(2)O(2) by anti-PECAM-GOX. Both H(2)O(2) added exogenously to the cell medium (extracellular H(2)O(2)) and that generated by anti-PECAM-GOX caused oxidative stress manifested by time- and dose-dependent irreversible plasma membrane damage. Inactivation of cellular catalase by aminotriazole treatment augmented damage caused by either extracellular H(2)O(2) or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H(2)O(2), yet failed to protect cells against injury induced by anti-PECAM-GOX. Therefore, treatment of PECAM-positive cells with anti-PECAM-GOX leads to conjugate internalization, predominantly intracellular H(2)O(2) generation and intracellular oxidative stress. These results indicate that anti-PECAM-GOX 1) provides cell-specific intracellular delivery of an active enzyme and 2) causes intracellular oxidative stress in PECAM-positive cells.

Glucose 1- and 2-oxidases from fungal strains: isolation and production of monoclonal antibodies.

Monoclonal antibodies (Mabs) against purified glucose 2-oxidase (EC 1.1.3.10) from Coriolus versicolor were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. Hybrid growth was observed in 42% of culture wells and 30% of these (i.e. 30 culture wells) contained anti-glucose 2-oxidase activity. Three positive wells containing hybrid cell lines were selected and cloned twice by the limiting dilution method and two hybridoma clones (E1A5 and E1A6) secreting Mabs were selected at random for purification and characterisation purposes. Both cell lines secreted Mabs of IgM class which were purified by gel filtration chromatography on a Sephacryl S-200 column with a final recovery of 80% and a purification factor of 16. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 950 kDa. Mabs were highly specific for glucose 2-oxidase as determined by Western blotting. These Mabs also crossreacted with glucose 1- and 2-oxidases from other fungal sources (Phanerochaeta chrysosporium, Penicillium amagasakiense and Aspergillus niger) as determined by Western blotting and by ELISA. Both glucose 1- and 2-oxidases from C. versicolor, P. chrysosporium, P. amagasakiense and A. niger were purified by hydrophobic interaction chromatography on Sepharose 4B-triazine dye with a recovery of enzyme activity in the range 85-92%. Purified preparations of glucose oxidases from fungal strains were apparently homogeneous on native PAGE. Glucose 2-oxidases were more hydrophobic than glucose 1-oxidases as determined by their chomatographic behaviour on Sepharose 4B-Cibacron Red G-E which could be used to study their roles in lignin biodegradation.