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.     

Feasibility of protein A for the oriented immobilization of immunoglobulin on silicon surface for a biosensor with imaging ellipsometry

The feasibility of using protein A to immobilize antibody on silicon surface for a biosensor with imaging ellipsometry was presented in this study. The amount of human IgG bound with anti-IgG immobilized by the protein A on silicon surface was much more than that bound with anti-IgG immobilized by physical adsorption. The result indicated that the protein A could be used to immobilize antibody molecules in a highly oriented manner and maintain antibody molecular functional configuration on the silicon surface. High reproducibility of the amount of antibody immobilization and homogenous antibody adsorption layer on surfaces could be obtained by this immobilization method. Imaging ellipsometry has been proven to be a fast and reliable detection method and sensitive enough to detect small changes in a molecular monolayer level. The combination of imaging ellipsometry and surface modification with protein A has the potential to be further developed into an efficient immunoassay protein chip.     

Protein fractionation using fast flow immobilized metal chelate affinity membranes

A new group-specific affinity membrane using metal chelates as ligands and inorganic glass hollow fiber microfiltration membranes as support matrices is developed and tested. The study focused on developing the optimum activation and coupling procedures to bind the chelating agent (iminodiacetic acid, IDA) to the surface of the microporous glass hollow fiber membrane and testing the resultant affinity membrane. Starting with three different glass surfaces, five modification reactions were evaluated. All the modified "active surfaces" were first tested for their protein adsorptive properties in batch mode with suspended microporous glass grains using model proteins with known binding characteristics with Cu-IDA systems. The metal loading capacities of the surfaces exhibiting favorable fractionation were then measured by atomic absorption spectroscopy.The results were compared with the results obtained with a commercial material used in immobilized metal affinity column chromatography. The protein binding characteristics of the hollow fiber affinity membranes were also evaluated under conditions of convective flow. This was performed by flowing single solute protein solutions through the microporous membrane at different flow rates. These results were then used to estimate the optimum loading and elution times for the process. A mathematical model incorporating radial diffusion was solved using a finite difference discretization method. Comparison between model predictions and experimental results was performed for four different proteins at one flow rate. These results suggested that the kinetics of adsorption was concentration dependent. Finally, the hollow fiber affinity membranes were challenged with two component mixtures to test their ability to fractionate mixed protein solutions. Efficient separation and good purity were obtained.The results presented here represent the development of a new fast flow affinity membrane process-immobilized metal affinity membranes (IMAM). (c) 1994 John Wiley & Sons, Inc.     

Review: Multipoint binding and heterogeneity in immobilized metal affinity chromatography

Studies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal-coordinating groups on the protein (histidines and deporotonated amines), m in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple-site interactions is a significant in crease in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functioal groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temikin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. (c) 1995 John Wiley & Sons, Inc.     

Plasma modified surfaces for covalent immobilization of functional biomolecules in the absence of chemical linkers: towards better biosensors and a new generation of medical implants

Plasma modification and plasma polymer deposition are valuable technologies for the preparation of surfaces for the covalent binding of biomolecules for applications such as biosensors, medical prostheses, and diagnostic devices as well as surfaces for enzyme-mediated reactions. Covalency is conveniently tested by the ability of the surface to retain the attached molecules after vigorous washing with sodium dodecyl sulphate (SDS). Covalency is indicated if the fraction of protein retained lies above the curve characteristic of physisorption. Confidence in covalency is strengthened when the washing protocol is aggressive enough to remove all adsorbed protein from a control significantly more hydrophobic than the test surface. The use of linker chemistry to space the molecules from the surface is in some cases beneficial. However, the use of linker chemistry is not necessary to retain molecular function for long periods when the polymer surface is modified by energetic bombardment. The energetic bombardment retains hydrophilicity of the surface by crosslinking the subsurface, and this appears to facilitate retention of protein function. Energetic bombardment also increases the functional life of molecules immobilized and then freeze dried on plasma-modified surfaces. Analysis of the surfaces shows that the covalent binding mechanism is related to the presence of free radicals on the surface and in the subsurface regions. The unpaired electrons associated with the radicals appear to be mobile within the modified region and can diffuse to the surface to take part in binding interactions. Proactive implantable devices can make use of these principles of covalent attachment by seeding the surface of an implant with a biomolecule that elicits the desired interaction with cells and prevents undesirable responses.     

Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces

We present a generic immunoassay platform that uses enhanced total internal reflection fluorescence in the proximity of silver island films (SIFs), a surface coating consisting of metal (silver) particles. This platform is used with a model immunoassay where a protein antigen, rabbit immunoglobulin G, was immobilized on the SIF-coated glass surface. The signal from a fluorescent dye-labeled anti-rabbit antibody binding to the surface antigen was detected; different color dyes have been tested. Close placement of the fluorophore to surface-bound silver nanostructures results in dramatic signal enhancement (up to 40-fold) on the SIFs as compared with the glass slides. Use of the total internal reflection mode of excitation has significant advantages (over classic front-face excitation) for practical assay development. The limited evanescent wave excitation volume makes it possible to minimize the background signal and use the immunoassay with no need for any washing steps.     

Assisted assignment of ligands corresponding to unknown electron density

A semi-automated computational procedure to assist in the identification of bound ligands from unknown electron density has been developed. The atomic surface surrounding the density blob is compared to a library of three-dimensional ligand binding surfaces extracted from the Protein Data Bank (PDB). Ligands corresponding to surfaces which share physicochemical texture and geometric shape similarities are considered for assignment. The method is benchmarked against a set of well represented ligands from the PDB, in which we show that we can identify the correct ligand based on the corresponding binding surface. Finally, we apply the method during model building and refinement stages from structural genomics targets in which unknown density blobs were discovered. A semi-automated computational method is described which aims to assist crystallographers with assigning the identity of a ligand corresponding to unknown electron density. Using shape and physicochemical similarity assessments between the protein surface surrounding the density and a database of known ligand binding surfaces, a plausible list of candidate ligands are identified for consideration. The method is validated against highly observed ligands from the Protein Data Bank and results are shown from its use in a high-throughput structural genomics pipeline.     

UNDERSTANDING INTERCELLULAR INTERACTIONS AND CELL ADHESION: LESSONS FROM STUDIES ON PROTEIN—METAL INTERACTIONS

To understand cell—cell interactions and the interactions of cells to non-biological materials, studies on binding forces between cellular proteins and between proteins and non-biological material such as metal surfaces are essential. The adsorption of proteins to solid—water interfaces is a multifactorial and a multistep process. First steps are determined by long-range interactions where surface properties such as hydrophobicity, distribution of charged groups, ion concentrations and pH play important roles. In later steps structural rearrangements in the protein molecule and dehydration effects become more important making the adsorption process often irreversible. In the following we demonstrate that protein A and tubulin have a specific type of interaction to metal surfaces probably as an intermediate step in the adsorption process. The proteins were attached to the tip of a microfabricated cantilever in such a way that only one molecule interacts with the surface. By recording force—distance curves with an atomic force microscope the adhesion forces of single molecules binding to gold, titanium and indium—tinoxid surfaces were measured.     

Metal particle-enhanced fluorescent immunoassays on metal mirrors

We present fluoroimmunoassays on plain metal-coated surfaces (metal mirrors) enhanced by metal nanoparticles (silver island films [SIFs]). Metal mirrors (aluminum, gold, or silver protected with a thin silica layer) were coated with SIFs, and an immunoassay (model assay for rabbit immunoglobulin G or myoglobin immunoassay) was performed on this surface using fluorescently labeled antibodies. Our results showed that SIFs alone (on glass surface not coated with metal) enhance the immunoassay signal approximately 3- to 10-fold. Using a metal mirror instead of glass as support for SIFs results in up to 50-fold signal enhancement.     

A surface modification strategy on silicon nitride for developing biosensors

A surface modification strategy for the use of giant magnetoresistive materials in the detection of protein-protein interactions is developed. This modification strategy is based on silanization of semiconductive materials. A native silicon nitride surface was treated with concentrated hydrofluoric acid to improve surface homogeneity. Nano-strip was used to oxidize silicon nitride to form a hydrophilic layer. Aminopropyltriethoxysilane was subsequently used to functionalize the treated surfaces to form amine groups, which were further activated with glutaraldehyde to introduce a layer of aldehyde groups. The effectiveness of this modification strategy was validated by chemiluminescence immunoassays of purified 6x His-HrpW of Pseudomonas syringae pv. tomato DC3000 and human transferrin. Signals with intensities related to concentrations of these two immobilized model proteins were observed. The modified surface was also validated by a more complex system: intercellular proteins secreted by DC3000. HrpW in these protein mixtures was successfully recognized by anti-HrpW antibodies when mixed proteins were immobilized onto activated surfaces. This surface modification strategy provides a platform onto which proteins can be directly immobilized for biosensor and protein array applications.     

Magnetic acoustic resonance immunoassay (MARIA): a multifrequency acoustic approach for the non-labelled detection of biomolecular interactions

A unique sensing platform, comprising an electromagnetic field detector and an acoustic resonator, has been used as a wireless system for remote sensing of biorecognition events. The MARS (Magnetic Acoustic Resonator Sensor) technique has proven useful for detecting the formation of protein multilayers derived from specific binding phenomena. The technique enables multifrequency analysis, without the need of electrodes attached to the sensing element, and also facilitates the in situ surface modification of the substrate for antibody attachment. The MARS sensor was utilized as the platform on which a standard immunoassay was carried out. Two different conditions for the attachment of the first antibody to the quartz surface were tested: (i) Adsorption of the antibody onto the surface of a bare quartz disc; (ii) covalent immobilization of the antibody to a chemically modified quartz surface. Both methods can be successfully utilized for the 'label-less' detection of the biorecognition event between goat IgG and anti-goat IgG by analysis of the multifrequency spectrum. Covalent attachment of the primary antibody results in a more efficient immobilization, with higher surface density, and a consistently enhanced response for the binding of the secondary antibody. This approach will be of interest to life scientists and biochemists that require high performance assay methodologies that do not use chemical labels.     

Modification of a disulfide in the hinge region of rabbit IgG and study of the interaction of polyvalent ferritin antigen, protein A, and anti-IgG]

Since immunoglobulins are used in a vast variety of immunoassays, the problem of obtaining antibodies with enhanced antigen-binding activity is of great importance. In order to discriminate between putative approaches to activating antibody modification, some functional characteristics of rabbit IgG modified at the hinge disulfide by three reagents: iodoacetamide, N-ethylmaleimide, and 2.2'-dipyridyl disulfide, have been studied. As can be judged from gel-permeation chromatography data, the molecular sizes of modified rabbit IgG were slightly increased in comparison with the native protein. Using enzyme immunoassay, it was shown that modification by each of the above reagents results in the same degree of activation of the antibody binding to the protein polyvalent antigen-human ferritin, due to the increase in segmental flexibility, i.e., Fab motion around the Fo fragment of IgG. The type of concentration dependencies of antigen binding suggest that another determinant stimulating the antigen binding in addition to the increase in segmental flexibility, can be attributed to intra- or interdomain flexibility of domains constituting the Fab fragments. Using protein A and anti-IgG as conformational probes for the antibody Fo fragment, the conformation and conformational dynamics of both CD2 domain epitopes and the switch region between the CD2 and CH3 domains have been shown to be essentially unaffected by modification.     

Estimation of amino acid residue substitution rates at local spatial regions and application in protein function inference: a Bayesian Monte Carlo approach

The amino acid sequences of proteins provide rich information for inferring distant phylogenetic relationships and for predicting protein functions. Estimating the rate matrix of residue substitutions from amino acid sequences is also important because the rate matrix can be used to develop scoring matrices for sequence alignment. Here we use a continuous time Markov process to model the substitution rates of residues and develop a Bayesian Markov chain Monte Carlo method for rate estimation. We validate our method using simulated artificial protein sequences. Because different local regions such as binding surfaces and the protein interior core experience different selection pressures due to functional or stability constraints, we use our method to estimate the substitution rates of local regions. Our results show that the substitution rates are very different for residues in the buried core and residues on the solvent-exposed surfaces. In addition, the rest of the proteins on the binding surfaces also have very different substitution rates from residues. Based on these findings, we further develop a method for protein function prediction by surface matching using scoring matrices derived from estimated substitution rates for residues located on the binding surfaces. We show with examples that our method is effective in identifying functionally related proteins that have overall low sequence identity, a task known to be very challenging.     

Chemiluminescence-based detection and comparison of protein amounts adsorbed on differently modified silica surfaces

The biological consequences of protein adsorption on biomaterial surfaces are considered to be of utmost importance for their biocompatibility. A new method based on amino group-labeling coupled to a chemiluminescence reaction for direct determination of proteins adsorbed on material surfaces was employed. This method was used to explore the effects of surface chemistry and surface roughness on protein adsorption in a silicon oxide model system. Corundum sandblasting was applied to silicon wafers to create roughened surfaces while immobilization of fluorocarbon-, hydrocarbon-, and poly(ethylene glycol)-containing silanes produced surfaces of varying wettability. The adsorption behavior of two complex body fluids, human serum and saliva, and of two purified components, human serum albumin and fibronectin, was strongly influenced by the surface parameters. A general tendency to higher amounts of adsorbed protein was found on roughened surfaces and modification with poly(ethylene glycol) or with fluorocarbon moieties reduced protein adsorption. The values obtained with the new method could be confirmed by a colorimetric determination of protein amounts adsorbed on identically modified silica beads and were in accordance with those previously reported utilizing established methods for protein quantification. The presented method, which was methodically simple to perform and allowed the simultaneous measurement of a large number of samples, may be of future value for high-throughput surveying of the protein adsorption characteristics of biomaterials.     

Ion binding to cytochrome c

This paper is a further study of ion binding to protein surfaces and builds on the studies of the binding of [Cr(CN)6]3- and [Fe(edta)(H2O)]- previously reported [Williams et al. (1982) FEBS Lett. 15, 293-299; Eley et al. (1982) Eur. J. Biochem. 124, 295-303]. In the present paper the binding of polyaminocarboxylate complexes of gadolinium have been studied. Eight ion-binding sites have been identified on the surface of cytochrome c. These exhibit different binding specificities which, in some cases, are not full understood. However it is clear that simple outer-sphere interactions are not the sole determining factor for the association of metal ion complexes with proteins. The NMR paramagnetic difference spectrum method has been shown to be good at locating binding sites and revealing qualitative differences in their relative affinities for a range of complex types. However the use of relaxation probes is not a good method for the quantitative determination of binding constants; for this, isostructural shift probes must be sought.     

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.     

A streptavidin surface on planar glass substrates for the detection of biomolecular interaction

Based on the requirements of biomolecular interaction analysis on direct optical transducers, a streptavidin surface is examined. A general protocol was developed allowing the immobilization of biotinylated compounds using the rife biotin-streptavidin system. This type of surface modification can be applied to all biosensors using glass surfaces as sensor devices. Reflectometric interference spectroscopy (RIfS), a label-free, direct optical method was used to demonstrate the quality of the transducer surfaces. The surface modification is based on an aminofunctionalized polyethylene glycol layer covalently bound to the silica surface of the transducer and shows very little nonspecific binding. Biotin molecules can be easily coupled on such layers. Streptavidin followed by a biotinylated estrone derivative was immobilized by incubation of the biotinylated transducer surface. For the streptavidin layer we obtained interference signals corresponding to a protein monolayer. Finally, using a surface prepared as described above, biomolecular interaction experiments with an antibody against estrone were carried out to show the quality of the transducer surface. With RIfS all of the affinity-based surface modifications can be detected online and time resolved.     

A high-capacity streptavidin-coated microtitration plate

A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). The maximum immobilization capacity of the modified plate was up to 2.5 times higher than that of the control plate. The higher binding capacity was especially emphasized with small-size molecules. The modified high capacity plate increased the linear ranges of the immunoassays and thus delayed the high-dose hook effect. At high antigen concentrations the signal increased up to 59%, and at the conventional linear ranges of the assays, the increase was up to 29%. We conclude that the modified coating method will be valuable for the future miniaturized systems, where high immobilization capacity is needed at limited areas.     

Evaluation of the Biological Sampling Kit (BiSKit) for large-area surface sampling

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m2) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 +/- 5.8 CFU/m2 for wet sampling and 100.5 +/- 10.2 CFU/m2 for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.