PREPARATION AND CHARACTERIZATION OF COVALENTLY LINKED ENZYME-ANTIBODY CONJUGATES AND THEIR USE IN MEASURING MINUTE QUANTITIES OF PROTEIN ANTIGENS

The discovery and characterization of oncofetal proteins have led to significant advances in early cancer diagnosis and therapeutic monitoring of patients undergoing cancer chemotherapy. These tumor-associated antigens are presently measured by sensitive, specific immunoassay techniques based on the detection of minute amounts of labeled antigen or antibody incorporated into immune complexes, which must be isolated from free antigen and antibody.^ Since there are several disadvantages with using radioisotopes, the most common immunolabel, one major objective was to prepare covalently coupled enzyme-antibody conjugates and evaluate their use as a practical alternative to radiolabeled immune reagents. An improved technique for the production of enzyme-antibody conjugates was developed that involves oxidizing the carbohydrate moieties on a glycoprotein enzyme, then introducing antibody in the presence of polyethylene glycol (PEG). Covalent enzyme-antibody conjugates involving alkaline phosphatase and amyloglucosidase were produced and characterized.^ In order to increase the sensitivity of detecting the amyloglucosidase-antibody conjugate, an enzyme cycling assay was developed that measures glucose, the product of maltose cleavage by amyloglucosidase, in the picomole range. The increased sensitivity obtained by combined usage of the amyloglucosidase-antibody conjugate and enzyme cycling assay was then compared to that of conventional enzyme immunoassay (EIA).^ For immune complex isolation, polystyrene tubes and protein A-bearing Staphylococcus aureus were evaluated as solid phase matrices, upon which antibodies can be immobilized. A sandwich-type EIA, using antibody-coated S. aureus, was developed that measures human albumin (HSA) in the nanogram range. The assay, using an alkaline phosphatase-anti-HSA conjugate, was applied to the determination of HSA in human urine and evaluated extensively for its clinical applicability.^ Finally, in view of the clinical significance of alpha-fetoprotein (AFP) as an oncofetal antigen and the difficulty with its purification for use as an immunogen and assay standard, a chemical purification protocol was developed that resulted in a high yield of immunochemically pure AFP. ^

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167 ± 0.14 mmol/g of liberated amine groups vs. 598 ± 60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)-propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2-heparin, 1D2-LMWH or 1D2-rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (+/-heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2-heparin or 1D2-LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2-rapamycin were not affected by injury. Arteries exposed to 1D2-heparin or 1D2-rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation. (C) 2004 Elsevier B.V. All rights reserved.

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.

The cellular response to transglutaminase-cross-linked collagen

Collagen, type I, is a highly abundant natural protein material which has been cross-linked by a variety of methods including chemical agents, physical heating and UV irradiation with the aim of enhancing its physical characteristics such as mechanical strength, thermal stability, resistance to proteolytic breakdown, thus increasing its overall biocompatibility. However, in view of the toxicity of residual cross-linking agents, or impracticability at large scales, it would be more useful if the collagen could be cross-linked by a milder, efficient and more practical means by using enzymes as biological catalysts. We demonstrate that on treating native collagen type I (from bovine skin) with both tissue transglutaminase (TG2; tTG) and microbial transglutaminase (mTG; Streptoverticillium mobaraense) leads to an enhancement in cell attachment, spreading and proliferation of human osteoblasts (HOB) and human foreskin dermal fibroblasts (HFDF) when compared to culture on native collagen. The transglutaminase-treated collagen substrates also showed a greater resistance to cell-mediated endogenous protease degradation than the native collagen. In addition, the HOB cells were shown to differentiate at a faster rate than on native collagen when assessed by measurement of alkaline phosphatase activity and osteopontin expression. © 2005 Elsevier Ltd. All rights reserved.

Stabilisation of isocyanate cross linked polybutadiene binder

Isocyanate cross-linked hydroxy terminated polybutadiene is used as a binder for solid rocket propellant. Rocket motors containing this propellant require a storage life of at least 20 years. During storage it has been found that the important rubbery properties of the binder can be lost due to oxidative cross-linking of the polybutadiene chains. This could cause catastrophic failure when the rocket motor is required. At present the bis-hindered phenol Calco 2246 is used as a thermal oxidative stabiliser, but it's performance is only adequate. This has led to the search for a more efficient stabiliser system. To hasten the evaluation of new antioxidant systems the use of dynamic thermal analysis was investigated. Results showed that a tentative relationship existed between predictions by thermal analysis and the long term oven ageing for simple single antioxidant systems. But for more complex systems containing either autosynergistic or mixed antioxidants no relationship was observed suggesting that results for such an "accelerated" technique cannot be used for the purpose of extrapolation for long term performance. This was attributed to the short time and more aggressive condition used (hjgher temperature and oxygen rich atmosphere in thermal analysis) altering the mechanism of action of the antioxidants and not allowing time for co-operative effect of the combined antioxidant system to form. One potential problem for the binder system is the use of an diisocyanate as a cross-linking agent. This reacts with the hydroxyl hydrogen on the polymer as well as other active hydrogens such as those contained in a number of antioxidants, affecting both cross-linking and antioxidant effectiveness. Studies in this work showed that only antioxidants containing amine moieties have a significant affect on binder preparation, with the phenolic antioxidants not reacting. This is due to the greater nucleophilicity of the amines. Investigation of a range of antioxidant systems, including potentially homo, hetero and autosynergistic systems, has highlighted a number of systems which show considerably greater effectiveness than the currently used antioxidant Calco 2246. The only single antioxidant which showed improvement was the partially unhindered phenol y-Tocopherol. Of the mixed systems combinations of the sulphur containing antioxidants e.g. DLTP with higher levels of chain-breaking antioxidants, especially Calco 2246, were the most promising. Also the homosynergistic mix of an aromatic amine and a phenol was seen to be very effective but the results were inconsistent. This inconsistency could be explained by the method of sample preparation used. It was shown that the efficiency of a number of antioxidant.s could be dramatically improved by the use of ultrasound during the mixing stage of preparation. The reason for this increase in performance is unclear but in the case of the homosynergistic amine/phenol mix both more efficient mixing and/or the production of a novel mechanism of action are suggested

Self-assembly of cross-linked β-cyclodextrin nanocapsules

Stirring of perthiolated β-cyclodextrin in water yields cross-linked hollow capsules ca. 50 nm in diameter, which can be used for encapsulation and controlled release of large molecules as shown using Reichardt's dye. © 2009 The Royal Society of Chemistry.

Covalent/crystallite cross-linked co-network hydrogels : an efficient and simple strategy for mechanically strong and tough hydrogels

Covalent/crystallite cross-linked co-network hydrogels have been prepared using epoxy and PVA through a cyclic freezing-thawing process. The PVA/epoxy hydrogels show enhanced mechanical strength and toughness. PVA/epoxy hydrogels with 4 wt% epoxy loading display maximum tensile strength and toughness of 1.1 MPa and 2838 kJ/m3 respectively. The fracture toughness of PVA/epoxy hydrogels ranges from 160 to 450 J/m2. Radius of gyration and fractal information of the hydrogels were obtained by fitting the SAXS data to the Guinier and power law models. The enhanced mechanical properties are attributed to the increase in covalent bonding and decrease in crystallite distribution with an increase in epoxy content. However a larger hysteresis is shown for PVA/epoxy hydrogels due to irreversible destruction of covalent bonds between epoxy and PVA.

Avaliação da promiscuidade catalítica de soroalbuminas em sínteses orgânicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass

Background: Perennial Ryegrass is a major cause of rhinitis in spring and early summer. Bahia grass, Paspalum notatum, flowers late into summer and could account for allergic rhinitis at this time. We determined the frequency of serum immunoglobulin (Ig)E reactivity with Bahia grass in Ryegrass pollen allergic patients and investigated IgE cross-reactivity between Bahia and Ryegrass. Methods: Serum from 33 Ryegrass pollen allergic patients and 12 nonatopic donors were tested for IgE reactivity with Bahia and Ryegrass pollen extracts (PE) by enzyme-linked immunosorbent assay (ELISA), western blotting and inhibition ELISA. Allergen-specific antibodies from a pool of sera from allergic donors were affinity purified and tested for IgE cross-reactivity. Results: Seventy-eight per cent of the sera had IgE reactivity with Bahia grass, but more weakly than with Ryegrass. Antibodies eluted from the major Ryegrass pollen allergens, Lol p 1 and Lol p 5, showed IgE reactivity with allergens of Ryegrass and Canary but not Bahia or Bermuda grasses. Timothy, Canary and Ryegrass inhibited IgE reactivity with Ryegrass and Bahia grass, whereas Bahia, Johnson and Bermuda grass did not inhibit IgE reactivity with Ryegrass. Conclusions: The majority of Ryegrass allergic patients also showed serum IgE reactivity with Bahia grass PE. However, Bahia grass and Ryegrass had only limited IgE cross-reactivity indicating that Bahia grass should be considered in diagnosis and treatment of patients with hay fever late in' the grass pollen season.

Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

Optimization process of polyester polymer mortars modified with recycled GFRP waste aggregates – application of factorial experiment design-

Glass fibre-reinforced plastics (GFRP), nowadays commonly used in the construction, transportation and automobile sectors, have been considered inherently difficult to recycle due to both: cross-linked nature of thermoset resins, which cannot be remolded, and complex composition of the composite itself, which includes glass fibres, matrix and different types of inorganic fillers. Presently, most of the GFRP waste is landfilled leading to negative environmental impacts and supplementary added costs. With an increasing awareness of environmental matters and the subsequent desire to save resources, recycling would convert an expensive waste disposal into a profitable reusable material. There are several methods to recycle GFR thermostable materials: (a) incineration, with partial energy recovery due to the heat generated during organic part combustion; (b) thermal and/or chemical recycling, such as solvolysis, pyrolisis and similar thermal decomposition processes, with glass fibre recovering; and (c) mechanical recycling or size reduction, in which the material is subjected to a milling process in order to obtain a specific grain size that makes the material suitable as reinforcement in new formulations. This last method has important advantages over the previous ones: there is no atmospheric pollution by gas emission, a much simpler equipment is required as compared with ovens necessary for thermal recycling processes, and does not require the use of chemical solvents with subsequent environmental impacts. In this study the effect of incorporation of recycled GFRP waste materials, obtained by means of milling processes, on mechanical behavior of polyester polymer mortars was assessed. For this purpose, different contents of recycled GFRP waste materials, with distinct size gradings, were incorporated into polyester polymer mortars as sand aggregates and filler replacements. The effect of GFRP waste treatment with silane coupling agent was also assessed. Design of experiments and data treatment were accomplish by means of factorial design and analysis of variance ANOVA. The use of factorial experiment design, instead of the one factor at-a-time method is efficient at allowing the evaluation of the effects and possible interactions of the different material factors involved. Experimental results were promising toward the recyclability of GFRP waste materials as polymer mortar aggregates, without significant loss of mechanical properties with regard to non-modified polymer mortars.

A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed TrP(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Immobilization of papain on chitin and chitosan and recycling of soluble enzyme for deflocculation of saccharomyces cerevisiae from bioethanol distilleries

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.