Site-directed biotinylation of antibodies for controlled immobilization on solid surfaces

Site-directed biotinylation of antibodies at the hinge region was developed to immobilize antibodies in an oriented manner via biotin-streptavidin linkage. When intact antibody was biotinylated with maleimide-activated biotin after reduction, the reaction preferentially occurred at the sulfhydryl groups between the C(H1) and the C(L) domains and, provided that the reagent concentration exceeded a certain level, at those between the C(H2) and the C(H2) domains at the hinge. Based on this result, we devised an approach in which free maleimide was added to compete with the activated biotin for the preferential sites between the C(H1) and the C(L) domains. Since the smaller molecular size of free maleimide made it more accessible for the reaction than biotin, maleimide bound to the groups between the C(H1) and the C(L) domains first and thus conceded the groups between the C(H2) and the C(H2) domains to biotin under optimal conditions. In an alternative approach, selective biotinylation at the hinge was also achieved by reacting activated biotin with F(ab')(2) fragment prepared by enzymatic cleavage. This result indicated that, when free of Fc, the hinge structure, which contains the functional groups, of the fragment was open, allowing easy access to the biotin derivative from the aqueous medium. Both site-directed biotinylation preparations were tested as capture antibodies in sandwich-type immunoassays and compared to whole antibody randomly biotinylated at amino groups on the molecule. Preparations of both the intact antibody and the F(ab')(2) showed consistently enhanced detection capabilities that were 2.6 and 20 times that of the control, respectively.     

A new heterobifunctional reagent for immobilization of biomolecules on glass surface

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.     

Coordination complexes as molecular glue for immobilization of antibodies on cyclic olefin copolymer surfaces

A novel metal-based chelating method has been used to provide an order of magnitude increase in immunoassay performance on cyclic olefin copolymer (COC) plastics compared with passive binding. COCs are hydrophobic, and without surface modification they are often unsuitable for applications where protein adhesion is desired. When interacting with the bare plastic, the majority of the bound proteins will be denatured and become nonfunctional. Many of the surface modification techniques reported to date require costly equipment setup or the use of harsh reaction conditions. Here, we have successfully demonstrated the use of a simple and quick metal chelation method to increase the sensitivity, activity, and efficiency of protein binding to COC surfaces. A detailed analysis of the COC surfaces after activation with the metal complexes is presented, and the immunoassay performance was studied using three different antibody pairs.     

N-(3-Trifluoroethanesulfonyloxypropyl)anthraquinone- 2-carboxamide: a new heterobifunctional reagent for immobilization of biomolecules on a variety of polymer surfaces

A new heterobifunctional reagent, N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide (NTPAC) has been developed, useful for making bioconjugates and immobilization of biomolecules, viz., oligonucleotides, peptides, proteins, etc., on a variety of carbon-containing solid surfaces. Its trifluoroethanesulfonate ester group reacts with aminoalkyl or mercaptoalkyl functions present in biomolecules, and the anthraquinone structure reacts with a variety of carbon-containing polymers under ultraviolet irradiation (365 nm). The reagent has been used in two ways. First, the reagent, NTPAC, was first brought in contact with the above said supports and exposed to long wavelength ultraviolet light (365 nm), thereby generating active trifluoroethanesulfonate ester functions on the support, which subsequently react with appropriate mercaptoalkyl- or aminoalkyl-containing biomolecules to fix them on the supports. In another route, the proposed reagent was allowed to react first with proteins or 5'-aminoalkyl- or mercaptoalkyl-modified oligonucleotides to form the appropriate biomolecule-anthraquinone conjugate, which was then brought in contact with a variety of carbon-containing polymers, viz., modified controlled pore glass (CPG), modified glass microslides, cross-linked polystyrene, nylon, cross-linked polysaccharides, polypropylene (PP), polyethylene (PE), etc., and exposed to long wavelength ultraviolet light (365 nm), resulting in immobilization of the conjugates on the support. Both of the routes work satisfactorily and we could successfully immobilize a number of enzymes and modified oligonucleotides on a variety of supports.     

Immobilization of immunoglobulins on silica surfaces. Kinetics of immobilization and influence of ionic strength.

The kinetics of, and the influence of ionic strength on, the immobilization of rabbit immunoglobulin G (IgG) on different types of well-characterized silica surfaces were investigated. Adsorptive immobilization was compared with covalent attachment via thiol-disulphide exchange reactions. The amount of immobilized IgG on five different types of silica surfaces as a function of IgG concentration, at two different ionic strengths, was determined. The IgG-solid-surface interaction involved different types of interaction forces, depending on the surface chemistry of the solid surface. The solid-surface chemistry is an important parameter determining the immobilized amount of IgG. When conditions for covalent attachment of IgG to the surfaces were fulfilled, the IgG showed high affinity and the immobilized amount of IgG showed a fast saturation. Changes in ionic strength showed no significant influence on the kinetics of immobilization on these surfaces. The amount of covalently attached IgG was partially ionic-strength-dependent, indicating that adsorptive interactions were involved. The results are of fundamental interest for the development of new immunosensors based on surface-concentration-measuring devices.     

Peptide-modified surfaces for enzyme immobilization

Background

Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity.

Methodology/Principal Findings

A method is presented for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to β-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation.

Conclusions/Significance

A simple method for immobilizing enzymes through specific interactions with peptides anchored on surfaces has been developed. This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.     

Oriented covalent immobilization of antibodies on physically inert and hydrophilic support surfaces through their glycosidic chains

Immobilization of antibodies by their oxidized sugar chain on aminated supports is a very efficient methodology to have a properly oriented antibody. However, these supports may behave as anionic exchangers, producing the unspecific adsorption of other proteins and reducing the selectivity of the system. To overcome this problem, we have proposed two solutions based in tailor-made support surfaces to immobilize antihorseradish peroxidase (HRP). The first solution was the use of supports having a very low amount of amino groups. These amino groups need to be very reactive with the aldehyde groups generated in the protein sugar chains to be efficient. Using supports having 7 micromol EDA/g (e.g., ethylenediamine modified glyoxyl-agarose), the antibody may be immobilized, keeping over 90% of the anti-HRP functionality. Second, by mixing amino groups and carboxylic groups, a neutral surface of the support has been generated. Again, this support has been unable to adsorb proteins while oxidized anti-HRP could be immobilized, giving functional anti-HRP antibodies. Both preparations retained 100% functionality after 2 months of storage at 4 degrees C. This way, the tailoring of the support surfaces has permitted solving some limitations of the immobilization of sugar-chain oxidized antibodies on primary amino supports.     

抗体在二氧化硅表面固定及活性测定

应用分子自组装技术,通过表面羟基化、氨基硅烷化和对苯二甲醛组装,在二氧化硅表面衍生活泼的醛基基团。利用抗体的氨基和醛基发生还原胺化反应将抗体固定在二氧化硅表面,通过抗原抗体反应定性检测固定抗体的生物活性。结果显示应用这种方法能够有效将抗体固定在二氧化硅表面,并保持抗体生物活性,该固定方法在蛋白质检测、分析和蛋白质芯片中有广泛应用价值。     

Oligonucleotide immobilization on micropatterned streptavidin surfaces

We describe a simple procedure for photolithographic patterning of streptavidin on silicon substrates. Long wavelength UV (365 nm) light was used to direct the covalent attachment of photoactivatable biotin onto silylated silicon wafers. Fluorescently labeled streptavidin was found to bind only in areas exposed to the light. We used this procedure to selectively pattern streptavidin inside microwells etched in silicon, and we investigated the binding characteristics of biotinylated oligonucleotides of lengths, n = 16, 54 and 99 bases. The binding curves were found to fit the functional form of the Langmuir isotherm, with binding saturation proportional to n^–3/4.     

Efficient immobilization technique for enhancement of cellobiose dehydrogenase activity on silica gel

In this study, cellobiose dehydrogenase (CDH) of Phanerochaete chrysosporium ATCC 32629 was immobilized on silica gel for the further application of CDH in the saccharification process of biomass. To prevent the loss of enzyme activity during enzyme immobilization, the pretreatment of CDH was performed by various pretreatment materials before immobilization. When pretreated enzymes were used in immobilization, the activities of immobilized CDH were higher than non-pretreated CDH even in same amounts of immobilized protein. The specific activity of pretreated immobilized CDH with lactose was about two times higher than that of non-pretreated immobilized CDH. Moreover, the pretreated immobilized CDH showed better reusability than non-pretreated immobilized CDH, with 67.3% of its original activity being retained after 9 reuses.     

A streptavidin mutant useful for directed immobilization on solid surfaces.

A streptavidin mutant has been designed and produced that allows the specific, covalent immobilization of streptavidin on solid surfaces. This streptavidin mutant was constructed by fusing a six-residue sequence, containing a single cysteine, to the carboxyl terminus of streptavidin. Because this mutant has no other cysteine residues, the reactive sulfhydryl group of the cysteine residue serves as a unique immobilization site for conjugation using sulfhydryl chemistry. This streptavidin mutant was efficiently immobilized on maleimide-coated solid surfaces via its unique immobilization site. Characterization of the immobilized streptavidin mutant for the ability to bind to biotinylated macromolecules and the dissociation rates of bound biotin showed that the biotin-binding properties of this mutant were minimally affected by immobilization on solid surfaces. This streptavidin could be readily incorporated into a wide variety of solid-phase diagnostic tests and biomedical assays. This could enhance the performance of streptavidin-based solid-phase assay systems.     

Reversible immobilization of antibodies on magnetic beads.

A streptavidin-biotin system was utilized to prepare an antibody-polyadenylic acid conjugate which was subsequently attached to commercially available magnetic beads, Dynabeads oligo(dT)25. Biotinylated polyadenylic acid was combined with streptavidin and the resulting polyadenylic acid-streptavidin was conjugated with an antibody-biotin derivative. The immobilized antibody-polyadenylic acid conjugate was separated from the reaction mixture by hybridization with complementary oligonucleotide immobilized on the surface of Dynabeads oligo(dT)25. The immobilized antibody-polyadenylic acid can be released from the carrier, utilizing low-ionic-strength buffers. The system is intended to be utilized in cell sorting, using immobilized antibodies against cell surface antigens. Dissociation of antibody-containing conjugate from magnetic beads is essential for the isolation of viable cells via positive cell sorting.     

Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces

We used electron-beam lithography to fabricate chemical nanostructures, i.e. amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of approximately 2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of approximately 1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to approximately 2. The S/B-ratio of the fluorescence signals could be further increased to approximately 4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes.     

Plasma polymerized epoxide functional surfaces for DNA probe immobilization

The development of functional surfaces for the immobilization of DNA probe is crucial for a successful design of a DNA sensor. In this report, epoxide functional thin films were achieved simply by pulsed plasma polymerization (PP) of glycidyl methacrylate (GMA) at low duty cycle. The presence of epoxide groups in the resulting ppGMA films was confirmed by Fourier transform infrared spectroscopy. The ppGMA coatings were found to be resistant to the non-specific adsorption of DNA strands, while the epoxide groups obtained could react with amine-modified DNA probes in a mild basic environment without any activation steps. A DNA sensor was made, and was successfully employed to distinguish different DNA sequences with one base pair mismatch as seen by surface plasmon enhanced fluorescence spectroscopy (SPFS). The regeneration of the present DNA sensor was also discussed. This result suggests that surface modification with ppGMA films is very promising for the fabrication of various DNA sensors.     

Live diatom silica immobilization of multimeric and redox-active enzymes

Living organisms are adept in forming inorganic materials (biominerals) with unique structures and properties that exceed the capabilities of engineered materials. Biomimetic materials syntheses are being developed that aim at replicating the advantageous properties of biominerals in vitro and endow them with additional functionalities. Recently, proof-of-concept was provided for an alternative approach that allows for the production of biomineral-based functional materials in vivo. In this approach, the cellular machinery for the biosynthesis of nano-/micropatterned SiO₂ (silica) structures in diatoms was genetically engineered to incorporate a monomeric, cofactor-independent (“simple”) enzyme, HabB, into diatom silica. In the present work, it is demonstrated that this approach is also applicable for enzymes with “complex” activity requirements, including oligomerization, metal ions, organic redox cofactors, and posttranslational modifications. Functional expression of the enzymes β-glucuronidase, glucose oxidase, galactose oxidase, and horseradish peroxidase in the diatom Thalassiosira pseudonana was accomplished, and 66 to 78% of the expressed enzymes were stably incorporated into the biosilica. The in vivo incorporated enzymes represent approximately 0.1% (wt/wt) of the diatom biosilica and are stabilized against denaturation and proteolytic degradation. Furthermore, it is demonstrated that the gene construct for in vivo immobilization of glucose oxidase can be utilized as the first negative selection marker for diatom genetic engineering.     

Polyclonal anti-chymotrypsin antibodies suitable for oriented immobilization of chymotrypsin

Summary Polyclonal antibodies obtained from the serum of pig immunized by DIP-chymotrypsin were separated into two fractions, anti-chymotrypsin IgG I and anti-chymotrypsin IgG II, by the use of chromatography on biospecific adsorbents prepared by chymotrypsin (CHT) immobilization in different ways. IgG I, which did not decrease the proteolytic activity of CHT, was obtained by biospecific affinity chromatography on a column of Sepharose with CHT attached through an immobilized polyvalent inhibitor, antilysin (AL). IgG II was isolated from the fraction unretarded on the column of CHT-AL-Sepharose by chromatography on a column with CHT directly attached to AH-Sepharose activated by glutaraldehyde. IgG II strongly decreased the proteolytic activity of CHT. Comparison of the proteolytic activity of CHT covalently bound to AH-Sepharose with that of CHT noncovalently intercepted by biospecific sorption to Sepharose with attached anti-CHT-IgG I showed a great advantage of the immobilization of CHT by oriented adsorption.     

Prostaglandin-H-synthase: stability and activity during immobilization on silica gel]

Prostaglandin H synthase was isolated in the form of microsomes from sheep vesicular glands and immobilized on silica gel. This system of prostaglandin synthesis was activated by calcium ions and stabilized by adrenaline. Microsomes immobilized in the presence of adrenaline and calcium ions were stable upon storage at 4 degrees C. After two months, their activity was 80% of the initial activity. Immobilized microsomes were able to catalyze several cycles of prostaglandin E2 synthesis with no substantial loss of activity: after eight utilization cycles, they retained 66% of their initial enzymic activity.     

A method for the non-covalent immobilization of heparin to surfaces

The interaction of heparan sulfate (HS) with specific proteins facilitates a wide range of fundamental biological processes including cellular proliferation and differentiation, tissue homeostasis, and viral pathogenesis. This multiplicity of function arises through sequence diversity within the HS chain. Heparin, which is very similar in structure to the sulfated regions of HS, is an excellent model for studying HS-protein interactions. The development of high-throughput enzyme-linked immunosorbent-like assays using surface-immobilized heparin has been hindered by the inability of this glycosaminoglycan to adhere to microtiter surfaces. Here we report the passive noncovalent adsorption of heparin onto microtiter wells following their treatment by plasma polymerization; there was no detectable binding of functional heparin onto untreated plates. Heparin immobilized in this way was able to interact with four different heparin-binding proteins tested, i.e., TSG-6, chemokines IL-8 and KC, and complement factor H. Heparin preparations ranging in size from high molecular weight to a defined decasaccharide could be adsorbed onto these plates in a functionally active form. Since plasma polymerization is possible for virtually any surface, this technique is likely to be of general use in the identification and characterization of heparin/HS-binding proteins in a wide range of applications.     

High-throughput optimization of surfaces for antibody immobilization using metal complexes

Using a high-throughput surface discovery approach, we have generated a 1600-member library of metal-containing surfaces and screened them for antibody binding potential. The surface library assembly involved graft modification of argon plasma-treated polyvinylidenedifluoride (PVDF) membranes with alternating maleic anhydride-styrene copolymer followed by anhydride ring opening with a range of secondary amines and microarray contact printing of transition metal complexes. The microarrays of metal-containing surfaces were then tested for their antibody binding capacity by incubation with a biotinylated mouse antibody in a chemiluminescence assay. A total of 11 leads were identified from the first screen, constituting a "hit" rate of 0.7%. A smaller 135-member surface library was then synthesized and screened to optimize existing hits and generate additional leads. To demonstrate the applicability of these surfaces to other formats, high-binding surface leads were then transferred onto Luminex beads for use in a bead flow cytometric immunoassay. The novel one-step antibody coupling process increased assay sensitivity of a Luminex tumor necrosis factor immunoassay. These high-binding surfaces do not require prior incorporation of polyhistidine tags or posttreatments such as oxidation to achieve essentially irreversible binding of immunoglobulin G.     

Covalent immobilization of antibodies on finally inert support surfaces through their surface regions having the highest densities in carboxyl groups

The correct immobilization of antibodies is one of the most critical steps in the preparation of immunosensors and immunochromatography matrices. In addition, the final support has to be chemical and physically inert to avoid the unspecific adsorption of proteins that can reduce the sensitivity of the biosensor or the purification achieved by the chromatography. The solution to both problems is one of the major challenges in the field. Here, we have presented two different novel and simple alternatives to have the unmodified antibody anionically exchanged to a support, further covalently immobilized with more than 90% of the antibodies bonded to the support by the four subunits, retaining a high functionality and giving a final "inert" surface. The first solution was the use of supports having a low superficial density of amino groups activated with glutaraldehyde. Here, the inertness was achieved by the use of a very low density of amino groups, unable to adsorb proteins at 100 mM sodium phosphate, while immobilization proceeds mainly via a first adsorption of the antibody and a further reaction with the glutaraldehyde groups. The second solution implies the design of a novel support (amino-epoxy). This support again produces a first ionic exchange of the antibody on the support and a further reaction with the epoxy groups, but because the epoxy groups can be finally blocked with aspartic groups (annulling the charge), the initial density of amino-epoxy groups can be as high as possible. Both systems permitted the correct and oriented immobilization of IgG. The immobilized antibody showed high-functionality (65-75%) and a final inert support surface. This immobilized antibody (antiperoxidase) was able to capture fully specifically HRP contaminating a protein crude extract from E. coli.