Recombinant ß-Glucocerebrosidase specific immunoaffinity ligands selected from phage-displayed combinatorial scFv libraries.

Antibodies specific to ß-Glucocerebrosidase were selected from phage displayed naïve scFv libraries. Biopannings were performed against recombinant human protein ß-Glucocerebrosidase immobilized on polystyrene surface, specific phages were eluted with 50% ethylene glycol in citrate buffer (pH 6.0). Several specific binders were discovered and converted to full-size hIgG1 antibodies leading to highly stable binders with dissociation constants (Kd) in the range 10-40 nM. The antibodies were used further as ligands for affinity chromatography, where efficient and selective recovery of biologically active ß-Glucocerebrosidase from cultured media of Chinese hamster ovary cells was demonstrated. ß-Glucocerebrosidase was purified to nearly homogeneous state and had specific activity comparable to the commercially available preparations (40-44 U/mg protein). The obtained immunoaffinity sorbents have high capacity and can be easily regenerated.

Immobilization of lipase Eversa Transform 2.0 on poly(urea-urethane) nanoparticles obtained using a biopolyol from enzymatic glycerolysis.

In this work, the free lipase Eversa® Transform 2.0 was used as a catalyst for enzymatic glycerolysis reaction in a solvent-free system. The product was evaluated by nuclear magnetic resonance (1H NMR) and showed high conversion related to hydroxyl groups. In sequence, the product of the glycerolysis was used as stabilizer and biopolyol for the synthesis of poly(urea-urethane) nanoparticles (PUU NPs) aqueous dispersion by the miniemulsion polymerization technique, without the use of a further surfactant in the system. Reactions resulted in stable dispersions of PUU NPs with an average diameter of 190 nm. After, the formation of the PUU NPs in the presence of concentrated lipase Eversa® Transform 2.0 was studied, aiming the lipase immobilization on the NP surface, and a stable enzymatic derivative with diameters around 231 nm was obtained. The hydrolytic enzymatic activity was determined using ρ-nitrophenyl palmitate (ρ-NPP) and the immobilization was confirmed by morphological analysis using transmission electron microscopy and fluorescence microscopy.

Enzymes as Immunotherapeutics.

Enzymes are attractive as immunotherapeutics because they can catalyze shifts in the local availability of immunostimulatory and immunosuppressive signals. Clinical success of enzyme immunotherapeutics frequently hinges upon achieving sustained biocatalysis over relevant time scales. The time scale and location of biocatalysis are often dictated by the location of the substrate. For example, therapeutic enzymes that convert substrates distributed systemically are typically designed to have a long half-life in circulation, whereas enzymes that convert substrates localized to a specific tissue or cell population can be more effective when designed to accumulate at the target site. This Topical Review surveys approaches to improve enzyme immunotherapeutic efficacy via chemical modification, encapsulation, and immobilization that increases enzyme accumulation at target sites or extends enzyme half-life in circulation. Examples provided illustrate "replacement therapies" to restore deficient enzyme function, as well as "enhancement therapies" that augment native enzyme function via supraphysiologic doses. Existing FDA-approved enzyme immunotherapies are highlighted, followed by discussion of emerging experimental strategies such as those designed to enhance antitumor immunity or resolve inflammation.

Disulfide-modified antigen for detection of celiac disease-associated anti-tissue transglutaminase autoantibodies.

A simple and rapid immunosensor for the determination of the celiac disease-related antibody, anti-tissue transglutaminase, was investigated. The antigenic protein tissue transglutaminase was chemically modified, introducing disulfide groups through different moieties of the molecule (amine, carboxylic, and hydroxyl groups), self-assembled on gold surfaces, and used for the detection of IgA and IgG autoantibodies. The modified proteins were evaluated using enzyme-linked immunosorbent assay and surface plasmon resonance, which showed that only introduction of the disulfide groups through amine moieties in the tissue transglutaminase preserved its antigenic properties. The disulfide-modified antigen was co-immobilized via chemisorption with a poly(ethylene glycol) alkanethiol on gold electrodes. The modified electrodes were then exposed to IgA anti-tissue transglutaminase antibodies and subsequently to horseradish peroxidase-labeled anti-idiotypic antibodies, achieving a detection limit of 260 ng ml-1. Immunosensor performance in the presence of complex matrixes, including clinically relevant serum reference solutions and real patient samples, was evaluated. The introduction of disulfides in the antigenic protein enabled a simple and convenient one-step surface immobilization procedure involving only spontaneous gold-thiol covalent binding. Complete amperometric assay time was 30 min.

Nature of immobilization surface affects antibody specificity to placental alkaline phosphatase.

Retention of native conformation of immobilized protein is essential for various applications including selection and detection of specific recombinant antibodies (scFvs). Placental alkaline phosphatase (PAP), an onco-fetal antigen expressed on the surface of several tumors, was immobilized on supermagnetic particles for selection of recombinant antibodies from a human phage display antibody library. The isolated antibodies were found to be cross-reactive to either of the isozymes of alkaline phosphatase, i.e., bone alkaline phosphatase (BAP) or intestinal alkaline phosphatase (IAP) and could not be used for tumor targeting. A specific anti-PAP monoclonal antibody H17E2 was tested for retention of specificity under these conditions. Binding of the antibody to magnetic beads conjugated IAP and BAP along with PAP and the ability of the two isozymes to inhibit its binding to PAP depicted the loss of isozyme specificity of the antibody. However, the antibody retained its specificity to PAP immobilized on polyvinyl chloride (PVC) surface. Enzyme activity was observed on both surfaces. This demonstrates that nature of immobilization may affect antigen-antibody binding in subtle ways, resulting in alteration of conformation of the epitopes. This may have consequences for determining the specificity of antibody binding for proteins that share a high degree of homology.

New application of indirect fluorescent antibody (IFA) technique using latex particles coupled with verotoxin 2 from Escherichia coli O157:H7 in order to determine colostral antibody titers in immunized dairy cows.

A simple and novel assay method for determining colostral and serum against soluble verotxin 2 (VT2) titers by indirect fluorescent antibody (IFA) assay using latex sensitized with VT2 was devised. The latex particles did not auto-fluoresce, and non specific reactions disappeared after washing with phosphate buffered saline containing 3 M Nacl. The highest titer measured by neutralizing test was observed at 1 day after delivery. The highest titer for each immunoglobulin class measured by enzyme-linked immunosorbent assay (ELISA) or IFA using latex sensitized with VT2 was also observed at 1 day after delivery. The changes in titer measured by each method showed similar patterns. Furthermore, the titers for IgG antibody were higher than those for IgM or IgA antibodies. Thus, the titers of bovine immune colostral antibody and each immunoglobulin class could be measured by IFA using latex sensitized with VT2.

A high-affinity recombinant antibody permits rapid and sensitive direct detection of myeloperoxidase.

Over the past 10 years, a growing field of research supporting the value of myeloperoxidase (MPO) as a prognostic indicator in acute cardiac pathophysiologies has emerged. The availability of a rapid and disposable MPO detection platform would enable research clinicians to more readily assess MPO indications for guiding therapy and also facilitate clinicians at the patient interface to readily adopt MPO testing and potentially drive more informed prognoses. Here we describe the isolation of a high-affinity avian MPO-specific recombinant antibody panel using phage display. Rapid isolation of a suitable single-chain variable fragment (scFv) antibody was facilitated using a surface plasmon resonance (SPR)-based "off-rate ranking" screening process. The selected scFv was then successfully incorporated into a rapid, simple, and sensitive one-step lateral flow immunoassay (LFIA) for the detection of MPO. This "one-step" feature of the developed assay was made possible by the scFv's strong affinity for MPO, obviating the need for sandwich signal enhancement steps. The assay's rapid performance was also further enhanced by exploiting the intrinsic enzymatic properties of MPO in its final detection. Use of the optimized LFIA facilitated the sensitive detection of MPO in MPO-depleted serum within clinically relevant reference ranges.

[Prospects for using antigenic nanosystems in the diagnosis and treatment of inflammatory rheumatic diseases].

AIM: To study whether immobilized antigenic nanosystems (ANS) may be designed on the basis of antigens of varying chemical nature to identify and to remove specific antibodies (Ab) from the blood of patients with systemic lupus erythematosus (SLE). SUBJECTS AND METHODS: Sixty patients with the diagnosis of SLE verified by the 1997 American College of Rheumatology criteria and 30 apparently healthy individuals were followed up. The levels of Ab to catalase (Cat), xanthine oxidase (XO), and cardiolipin (CL) were measured by enzyme immunoassay, by applying the respective ANS as an antigenic matrix. RESULTS: There was a significant relationship of the levels of Ab to Cat and XO to the activity of SLE. It was shown that Ab to Cat and XO could affect the functional activity of serum enzymes. The level of Ab to CL in patients with SLE was found to depend on two parameters - the intensity of the disease and the presence of antiphospholipid syndrome; acute cerebral circulatory disorder and thrombocytopenia were observed to have a significant unidirectional impact on the level of Ab to CL. Immobilized CL-based ANSs were effective in eliminating Ab to CL from the whole blood of patients with SLE, without resulting in a significant hemolysis of blood corpuscles and in a reduction of total protein concentrations. CONCLUSION: The development and introduction of preventive methods for the early diagnosis of SLE may be extended, by using ANS based on Cat, XO, and CL antigen. The designing and putting into practice novel ANS-based hemosorbents may allow immunosorption to occupy a prominent place in the pathogenetic therapy of inflammatory autoimmune diseases.

The immobilization of proteins on biodegradable polymer fibers via click chemistry.

A facile and efficient method to immobilize bioactive proteins onto polymeric substrate was established. Testis-specific protease 50 (TSP50) was immobilized on ultrafine biodegradable polymer fibers, i.e., (1) to prepare a propargyl-containing polymer P(LA90-co-MPC10) by introducing propargyl group into a cyclic carbonate monomer (5-methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one, MPC) and copolymerizing it with l-lactide; (2) to electrospin the functionalized polymer into ultrafine fibers; (3) to azidize the TSP50, and (4) to perform the click reaction between the propargyl groups on the fibers and the azido groups on the protein. The TSP50-immobilized fibers can resist non-specific protein adsorptions but preserve specific recognition and combination with anti-TSP50. ELISA tests were carried out by using HRP-goat-anti-mouse-IgG(H+L) as secondary antibody and o-phenylenediamine (OPDA)/H(2)O(2) as substrate to detect the combination of immobilized TSP50 with anti-TSP50. The results showed that anti-TSP50 can be selectively adsorbed from its solution onto the TSP50-immobilized fibers in the presence of BSA of as high as 10(4) times concentration. TSP50 immobilized on the fiber and anti-TSP50 combined to the fiber were also quantitatively determined. Anti-TSP50 can be then eluted off from the fiber when pH changes. The eluted fiber can re-combine anti-TSP50 at an efficiency of 75% compared to the original TSP50-immobilized fiber. Therefore, the TSP50-immobilized fibers can be used in the detection, separation, and purification of anti-TSP50. The "click" method can lead to a universal strategy to protein immobilization.

Peroxidase-immobilized porous silica particles for in situ formation of peroxidase-free hydrogels with attenuated immune responses.

Enzymatic crosslinking chemistry using horseradish peroxidase (HRP) has been widely utilized as an effective approach to fabricating injectable hydrogels with high efficiency under mild reaction conditions. However, their clinical applications are limited by the immunogenicity of the plant-derived enzyme. Herein we report the design, synthesis and characterization of HRP-immobilized porous silica particles (HRP-particles) and their use for in situ formation of HRP-free hydrogels. HRP was immobilized on aminopropyl-modified porous silica particles of 70-140 µm in diameter via poly(ethylene glycol) spacers of different molecular weights by reductive amination reaction. Two different HRP-free hydrogels based on dextran-tyramine and gelatin-hydroxyphenylpropionic acid (GHPA) conjugates were produced by passing a solution containing the conjugates and H2O2 through a syringe packed with HRP-particles. The storage modulus and gelation rate of both hydrogels were tunable by varying the contact time between the polymer solution and HRP-particles. Our in vitro study revealed that HRP-free GHPA hydrogel was less stimulatory to activated mouse macrophages than HRP-containing GHPA hydrogel with the same stiffness. Furthermore, HRP-free GHPA hydrogel exhibited remarkably lower levels of local and systemic inflammation than HRP-containing one upon subcutaneous injection in immunocompetent C57BL/6J mice. The attenuated immunogenicity of HRP-free GHPA hydrogels makes them an attractive platform for tissue engineering applications. STATEMENT OF SIGNIFICANCE: The immunogenicity of HRP is a significant issue for clinical application of HRP-catalyzed in situ forming hydrogels. HRP-particles are developed to overcome the safety concerns by fabricating HRP-free hydrogels. The porosity of silica particles and molecular weight of poly(ethylene glycol) spacers are discovered as important factors determining the catalytic ability of HRP-particles to induce the in situ crosslinking of polymer-phenol conjugates. Although several articles speculate the potential of HRP to trigger immune responses when administered as a part of hydrogel formulation, no literature has attempted to investigate the immunogenicity of HRP-containing hydrogels in comparison with HRP-free hydrogels. Our findings suggest that the immunogenicity issue should be carefully considered before clinical translation of HRP-containing hydrogels.

Production of glutathione-coated microtitre plates for capturing recombinant glutathione S-transferase fusion proteins as antigens in immunoassays.

Glutathione S-transferase (GST) is commonly used as a fusion partner in producing recombinant proteins and this technology is increasingly being used to produce antigens for use in immunoassays to measure antibodies. To circumvent the requirement to purify such antigens before use, we developed a method for coupling glutathione to microtitre plates so that GST-containing recombinant proteins could be purified and immobilised in one step in a suitable state for immunoassays. This procedure involves covalent linkage (using the heterobifunctional cross-linker sulphosuccinimidyl 4-(p-maleimidophenyl)butyrate) of reduced glutathione through its sulphydryl group to lysine residues of haemoglobin previously immobilised on microtitre plates. Haemoglobin was superior over other proteins tested in giving the lowest non-specific binding; in this regard it was also important to limit the amount of cross-linker used to 0.1 mM. Using glutamic acid decarboxylase as a model antigen, the new affinity capture assay was at least as good as the two-step procedure involving direct adsorption to plates of previously purified antigen; it may have the additional advantage of preserving the antigen in a more native conformation than direct adsorption. The new assay also performed as well as an assay using anti-GST antibodies adsorbed onto plates; glutathione plates, unlike anti-GST plates, will only capture recombinant proteins containing functional GST--a significant point for some recombinant expression systems in which a large proportion of the protein product is insoluble because of incorrect folding.

Small molecule-based chemical affinity system for the purification of proteins.

A new chemical affinity system is described for the purification of proteins. The Linx Affinity Purification System enables researchers to quickly and easily bind a protein ligand to a chromatographic matrix and use the resulting affinity resin to purify a second protein from crude mixtures. The entire process takes approximately 2 h.

B-cell autoepitopes on the acetylcholinesterase-homologous region of human thyroglobulin: association with Graves' disease and thyroid eye disease.

OBJECTIVE: Thyroglobulin (Tg) is a large autoantigen involved in autoimmune thyroid diseases. Tg epitopes have, so far, been identified within large peptides. In the present study, we used small synthetic peptides to finely map serological epitopes on the highly immunogenic C-terminal region of Tg. Homology of this region to acetylcholinesterase (AChE) has been implicated in the pathogenesis of thyroid eye disease (TED) through cross-reactive antibodies. METHODS: We tested total IgG purified from four pilot Graves' disease (GD) sera reactive with both Tg and AChE and from three healthy controls, for reactivity against overlapping 20mer peptides (pin synthesis) covering the sequence 2171-2748 of human Tg. Antibody-reactive peptides were subsequently synthesized by a solid-phase technique for confirmation with a large number of sera: 99 GD, 32 Hashimoto's thyroiditis (HT) and 45 healthy controls. RESULTS: Peptides TgP15, TgP26 and TgP41 (amino acids 2339-2358, 2471-2490 and 2651-2670 respectively) were found to be targets of autoantibodies on intact Tg, recognized by a statistically significant proportion of GD sera (22.2%, 35.4% and 30.3% respectively), compared with HT (0%, 15.6% and 6.3% respectively) and healthy controls (0%, 4.4% and 4.4% respectively). The majority of GD sera (56.6%) were positive for at least one of the three peptides. In GD, TgP26 reactivity was found to be associated with TED (48.6% with TED versus 25.5% without TED, P<0.05). CONCLUSION: Some epitopes on the C-terminal region of Tg are associated with GD. A subset of Tg-reactive autoantibodies, directed to this region, is associated with TED and may be involved in the development of the disease.

A new assay format for electrochemical immunosensors: polyelectrolyte-based separation on membrane carriers combined with detection of peroxidase activity by pH-sensitive field-effect transistor.

A new rapid immunotechnique combining separation of reactants by filtration through a porous membrane and potentiometric detection of the bound enzyme label by a pH-sensitive field-effect transistor is proposed. The complexes to be detected are formed by the method described earlier in (Anal. Chem. 71 (1999) 3538), including a homogeneous binding of immunoreactants and a polyanion carrier (polymethacrylate) followed by heterogeneous separation on a membrane incorporating an immobilized polycation (poly-N-vinyl-4-ethylpyridinium). The proposed technique for a sensitive detection of peroxidase label is based on the measurement of pH changes in the optimised substrate solution containing o-phenylenediamine, hydrogen peroxide and ascorbic acid. The antigens studied were herbicide atrazine and hormone testosterone. Their specific detection is realised via competitive binding of free and peroxidase-labelled antigens by antibodies integrating with a (staphylococcal protein A-polyanion) conjugate. The total analysis time is 20-25 min. The range of quantitative detection is 0.2-100 ng ml(-1) for atrazine and 5-300 ng ml(-1) for testosterone. Data scatter of replicate tests varies from 3 to 10%. Application of protein A-polyanion conjugate allows to use the proposed protocol for different antigens without additional treatment of specific antisera.

Automated microanalysis using magnetic beads with commercial capillary electrophoretic instrumentation.

The potential of a new microanalytical method using magnetic beads (MBs) and commercial capillary electrophoresis (CE) instrumentation for performing enzymatic and inhibition assays, as well as for analysis of biological molecules such as antigens, substrates, etc., has been explored. A small quantity of magnetic beads containing immobilized biomolecules was injected into a neutral hydrophilic-coated fused-silica capillary. The short plug (2-3 mm) of beads was held fixed by a magnet placed in the cartridge of the CE system, without the use of frits. The beads could be replaced after each run, eliminating the need to regenerate the solid support. Two protocols were used for analysis: sequential injection (SI) and SI followed by isotachophoretic (ITP) focusing. Alkaline phosphatase (AP) and HIV-protease were used to demonstrate the SI procedure for enzymatic and inhibition assays. The second protocol, SI/ITP, was employed to quantitate an antigen (mouse mAB) using antibodies (sheep IgG towards mouse AB) immobilized on the beads. The MB-CE method, requiring only femtomole (fmol) quantities of material, can potentially be employed in diagnostic and forensic assays, kinetic studies and searching for inhibitors, ligands, receptors, etc.

Antigenic and immunogenic properties of gamma-glutamyl-transferase preparations.

Incubation time of human kidney gamma-glutamyl-transferase (GGT) with specific antiserum and precipitate formation caused no effect on inhibition of the enzyme activity. In the obtained precipitate GGT activity was independent of the amount of antiserum and contained 30% of the initial enzyme activity. After reaction of the serum with human liver GGT or with the urine enzyme a precipitate formation and decrease in the enzyme activity was noted. In the serum incubated with GGT isolated from animal kidneys, neither inhibition nor precipitation was observed. The precipitate of bovine kidney GGT with a specific antibody independently of the amount of antibody contained 50% of activity. After treatment of bovine GGT with bromelain four protein fractions giving precipitin lines in crossed-immunoelectrophoresis were observed but only one of them preserved catalytic activity. Immunogenicity of newly obtained immobilized GGT preparations was much lower than that of the native enzyme. A distinct and different influence of antibody on the affinity of native and immobilized GGT preparations to donor and acceptors of gamma-glutamyl group was noted. The immunofluorescent technique was used for GGT localization in some bovine tissue sections.

Acrylic microspheres in vivo V: Immunological properties of immobilized asparaginase in microparticles.

L-Asparaginase was immobilized in microparticles of polyacrylamide. Such particles were then injected by intramuscular/subcutaneous, intraperitoneal, or intravenous routes into mice to investigate the immunological consequences of the immobilization. Entrapment of L-asparaginase in microparticles did not prevent the formation of antibodies in intensively treated animals. Intraperitoneal and intravenous injections of particles produced significantly higher antibody levels than soluble L-asparaginase. Antigen administered intramuscularly/subcutaneously in microparticles elicited, however, a weak immune response. Dependent on the route of administration, the particles may thus function as an adjuvant. A modified Arthus reaction in the foot pads of immunized mice indicated that antigenicity decreased when L-asparaginase was immobilized in microparticles. Injection of free L-asparaginase, intramuscularly/subcutaneously (2 x 20 IU) in the preimmunized mice produced no effects on the serum level of L-asparagine, whereas intramuscular/subcutaneous injection of L-asparaginase in microparticles produced a depression of the serum concentration. It is concluded that the intramuscular/subcutaneous injection of L-asparaginase in microparticles is the choice route of administration with respect to duration and the immunological reaction.

Immobilized earthworm fibrinolytic enzyme III-1 with carbonyldiimidazole activated-agarose.

Earthworm fibrinolytic enzyme III-1 (EFE-III-1) was prepared to couple with cross-linking agarose activated by 1,'-Carbonyl- diimidazole (CDI) in this study. Although the activity of the immobilized protease decreased to approximately 64% of the native enzyme, the activity of EFE-III-1 coupled with the resin activated by CDI was higher than that activated by cyanogen bromide (CNBr). The immobilized protease was experimentally demonstrated to hydrolyze IgG, albumin and creatine kinase, besides fibrin(ogen) and plasmin(ogen), suggesting that EFE had a broad substrate specificity.

[Noncovalent immobilization of catalase on antibodies adsorbed on carbon fabric]. / Nekovalentnaia immobilizatsiia katalazy na antitelakh sorbirovannykh ugol'noi tkan'iu.

Catalase was immobilized on an immunosorbent prepared by anticatalase adsorption on an activated carbon fabric (ACF), and its kinetic parameters were determined. The immobilized catalase activity depended on the binding capacity of anticatalase. Under the optimum conditions (6 micrograms/mg anticatalase, 5.24 nM catalase) the immobilized catalase activity was 1.5-ford higher as compared to soluble catalase. Antibodies stabilized soluble catalase, but decreased its thermostability on immobilization of immunocomplexes on ACF. Noncovalent immobilization of catalase on adsorbed antibodies opens up the way to the use of this approach for immobilization of other oligomeric enzymes.

An amperometric immunosensor for diagnosis of celiac disease based on covalent immobilization of open conformation tissue transglutaminase for determination of anti-tTG antibodies in human serum.

A new amperometric immunosensor based on the covalent immobilization of tissue transglutaminase enzyme in its open conformation (open-tTG) was developed and optimized for determination of anti-tissue transglutaminase antibodies (anti-tTG) in human serum. Experimental design allowed us to find the optimal conditions for quantification of both IgA and IgG isotypes of anti-tTG in order to assess suitability of the device for diagnostic purposes. The glassy carbon electrodic substrate was electrochemically functionalized with gold nanoparticles and subsequently derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid for the covalent anchoring of the enzyme. This step was performed under carefully controlled conditions in order to keep the open conformation of the tTG. The immunosensor showed good analytical performance with limit of detection levels (1.7 AU mL(-1) for IgA and 2.7 AU mL(-1) for IgG) below the diagnostic threshold value (3.0 AU mL(-1)) and inter-sensor reproducibility giving RSD lower than 10%. The developed sensor was validated in serum samples from pediatric patients for clinical applications, using two ELISA kits specific for the determination of anti-tTG IgA and IgG antibodies as reference methods; good recovery rates ranging from 74% to 117% were calculated.