Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study

Antibody microarrays have the potential to revolutionize protein diagnostics. The major problems in the fabrication of antibody arrays, however, concern the reproducibility and homogeneity of the attachment of the proteins on the solid substrate. We here compare the DNA-directed immobilization (DDI) method with two conventional strategies for immobilization of antibodies on glass substrates. DDI is based on the self-assembly of semisynthetic DNA-streptavidin conjugates which converts an array of DNA oligomers into an antibody microarray. DDI was compared with direct spotting of antibodies on chemically activated glass slides and with immobilization of biotinylated antibodies on streptavidin-coated slides. The immobilized antibodies were used as capture reagents in a two-sided (sandwich) immunoassay for the quantification of rabbit IgG as a model antigen. Detection limits down to 0.001nM (150 pg/mL) were attained with all three array formats; however, DDI and direct spotting of the antibodies led to the highest fluorescence intensities. DDI led to the best spot homogeneity and intra- and interexperimental reproducibility. Moreover, DDI allowed highly economical use of antibody materials; that is, at least 100-fold less antibody is needed for preparing an array by DDI instead of by direct spotting. Taking into account the greater versatility and convenience of handling of the self-assembly approach, this study demonstrates that DDI is an advantageous alternative for generating versatile and robust protein arrays.     

Functionalization of covalent DNA-streptavidin conjugates by means of biotinylated modulator components.

Covalent DNA-streptavidin conjugates are versatile biomolecular coupling reagents, since they have binding capacity for both a complementary nucleic acid and four molecules of biotin. The DNA-streptavidin hybrid molecules have been investigated for their capabilities to bind two different types of biotinylated components. Thus, (i) a functional biomolecule, e.g., a single-stranded DNA fragment or an enzyme and (ii) low-molecular weight biotin derivatives ("modulators") were coupled stepwise with the hybrid molecules. Modulators were D-biotin, aminobiotin, and biotin-fluorescein conjugate as well as a lysine-rich 10mer peptide, containing a biotin and a fluorescein substituent. These modulators were chosen to affected the hybridization properties of the DNA-streptavidin conjugates. As investigated by surface-plasmon resonance and microplate solid-phase hybridization measurements, D-biotin, biotin-fluorescein, and aminobiotin decreased the efficiency of hybridization with complementary, surface-bound oligonucleotides to a varying extent. The basic peptide increased the conjugate's hybridization efficiency. Moreover, it was demonstrated in two examples how modulators can be utilized as additional functional domains of streptavidin-based conjugates. First, fluorescein-containing modulators were used as hapten groups, allowing a sensitive detection by means of specific antibodies directed against the modulator. Second, the biotinylated peptide was used as a carrier molecule to attach multiple fluorogenic lanthanide-chelate groups to the streptavidin conjugate, enabling its sensitive detection by time-resolved fluorometry. The applicability of this kind of bioconjugation strategy to generate sensor-probes for gene detection assays was demonstrated.     

Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA-protein conjugates

An assay for very sensitive antigen detection is described which takes advantage of the self- assembly capabilities of semi-synthetic conjugates of DNA and proteins. The general scheme of this assay is similar to a two-sided (sandwich) enzyme-linked immunoassay (ELISA); however, covalent single-stranded DNA-streptavidin (STV) conjugates, capable of hybridizing to complementary surface-bound DNA oligomers, are utilized for the effective immobilization of either capture antibodies or antigens, rather than the chemi- or physisorption usually applied in ELISA. Immuno-PCR (IPCR) is employed as a method for signal generation, utilizing oligomeric reagents obtained by self-assembly of STV, biotinylated DNA and antibodies. In three different model systems, detecting human IgG, rabbit IgG or carcinoembryonic antigen, this combination allowed one to increase the sensitivity of the analogous ELISA approximately 1000-fold. For example, <0.1 amol/ micro l (15 pg/ml) of rabbit IgG was detectable. The immunoassay can be carried out in a single step by tagging the analyte with both reagents for capture and read-out simultaneously, thereby significantly reducing handling time and costs of analysis. Moreover, as the spatial selectivity of target immobilization is determined by the specificity of DNA base pairing, the assay is particularly suited for miniaturized microfluidics and lab-on-a-chip devices.     

Immobilization of histidine-tagged proteins on gold surfaces using chelator thioalkanes

The reversible, oriented immobilization of proteins on solid surfaces is a prerequisite for the investigation of molecular interactions and the controlled formation of supramolecular assemblies. This paper describes a generally applicable method using a synthetic chelator thioalkane that can be self-assembled on gold surfaces. The reversible binding of an anti-lysozyme F(ab) fragment modified with a C-terminal hexahistidine extension was monitored and the apparent dissociation constants determined using surface plasmon resonance. Infra-red spectroscopy demonstrated that the secondary structure of the protein was unaffected by the immobilization process. The retention of functionality of the immobilized protein was also successfully demonstrated. Given the mild reaction conditions and reversibility, this method of oriented immobilization of proteins opens possibilities for the development of biosensors.     

Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system

Biotinylation is useful for the detection, purification and immobilization of proteins. It is performed by chemical modification, although position-specific and quantitative biotinylation is rarely achieved. We developed a position-specific biotinylation method using biotinylated non-natural amino acids. We showed that biotinylated p-aminophenylalanine derivatives were incorporated into a protein more efficiently than biotinylated lysine derivatives in a cell-free translation system. In addition, the biotinylated p-aminophenylalanines overcame the serious position-dependency observed for biotinylated lysines. The present method will be useful for detection and purification of proteins along with comprehensive exploration of surface-exposed residues and oriented immobilization of proteins.     

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.     

Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins

Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.     

Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.     

Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates

Chaperonin 10 protein from Rattus norvegicus (Rat cpn10) has been reported to bind chaperonin 60 from Escherichia coli (GroEL) in an ATP-dependent manner. Chemically synthesized Rat cpn10 was immobilized in a defined orientation to agarose-bound monomeric avidin using a reversible biotinylated affinity label ( 1 ), attached to the N^α-terminal residue. The resulting affinity chromatographic matrix was then used to isolate binding proteins from a crude cell lysate. Following affinity separation the bound ligand and ligate was released by treatment with organic base. Rat cpn10 was prepared using a highly effective synthetic protocol involving HBTU/HOBt activation and capping with N-(2-chlorobenzyloxycarbonyloxy) succinimide to terminate unreacted amino groups. The biotinylated Fmoc-based molecule ( 1 ) was introduced specifically onto the N^α-terminal amino acid as the succinimidyl carbonate, before final cleavage and deprotection of side-chain protecting groups using a low-TFMSA/high-HF procedure. Crude biotinylated Rat cpn10 (Rat cpn10+ 1 ) was immobilized on monomeric avidin with a binding efficiency of approximately 75% and unlabelled truncated/capped impurities eluted off the column with buffer. The biotinylated Rat cpn10–avidin affinity matrix was then used to isolate GroEL from a crude cell lysate. The identity of the purified protein was confirmed by SDS–PAGE and binding to a specific anti-GroEL monoclonal antibody (MoAb). These results extend the applicability of the biotinylated label ( 1 ), providing a reversible non-covalent anchor for immobilization of peptide and protein ligands, thus simplifying isolation of ligates and enabling recovery of synthetic material under mild conditions. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Immunodetection of biotinylated lymphocyte-surface proteins by enhanced chemiluminescence: a nonradioactive method for cell-surface protein analysis.

Biotinylation and radioiodination have been compared for labeling lymphocyte-surface proteins and the labeled proteins symmetrically immunoprecipitated with antibodies recognizing major lymphocyte markers such as the murine Thy-1, CD25 (the alpha subunit of the interleukin-2 receptor), CD45, and human CD2 glycoproteins. The detection of biotinylated proteins by enhanced chemiluminescence after transfer to nitrocellulose was found to be fast and as efficient as the detection of iodinated proteins by autoradiography. The vectoriality of cell-surface biotinylation was ascertained by two-dimensional electrophoresis of the cellular extract in which the major cytoplasmic proteins were not found biotinylated. This nonradioactive labeling procedure offers a convenient and efficient alternative to radiolabeling of cell surfaces for the biochemical analysis of extracellular domains of membrane proteins.     

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.     

Design,production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.     

Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy

Cell surface multivalent ligands, such as proteoglycans and mucins, are often tethered by a single attachment point. In vitro, however, it is difficult to immobilize multivalent ligands at single sites due to their heterogeneity. Moreover, multivalent ligands often lack a single group with reactivity orthogonal to other functionality in the ligand. Biophysical analyses of multivalent ligand-receptor interactions would benefit from the availability of strategies for uniform immobilization of multivalent ligands. To this end, we report the design and synthesis of a multivalent ligand that has a single terminal orthogonal functional group and we demonstrate that this material can be selectively immobilized onto a surface suitable for surface plasmon resonance (SPR) experiments. The polymeric ligand we generated displays multiple copies of 3,6-disulfogalactose, and it can bind to the cell adhesion molecules P- and L-selectin. Using SPR measurements, we found that surfaces displaying our multivalent ligands bind specifically to P- and L-selectin. The affinities of P- and L-selectin for surfaces displaying the multivalent ligand are five- to sixfold better than the affinities for a surface modified with the corresponding monovalent ligand. In addition to binding soluble proteins, surfaces bearing immobilized polymers bound to cells displaying L-selectin. Cell binding was confirmed by visualizing adherent cells by fluorescence microscopy. Together, our results indicate that synthetic surfaces can be created by selective immobilization of multivalent ligands and that these surfaces are capable of binding soluble and cell-surface-associated receptors with high affinity.     

Electrochemically controllable conjugation of proteins on surfaces

The rational design of surfaces for immobilization of proteins is essential to a variety of biological and medical applications ranging from molecular diagnostics to advanced platforms for fundamental studies of molecular and cell biology. We have developed an advanced electrochemically based approach for site-selective and reaction-controlled immobilization of proteins on surfaces. When a molecular monolayer of 4-nitrothiophenol on gold electrode surfaces is reduced electrochemically in a selective fashion at its nitro groups, to afford amino groups by potentiometric scans, the amine can be employed to orchestrate the immobilization of proteins to the surface. This protein immobilization strategy could allow one to fabricate intricate protein structures on surfaces for addressing fundamental and applied problems in biology and medicine.     

An investigation of antibody immobilization methods employing organosilanes on planar ZnO surfaces for biosensor applications

One critical aspect for the development of label-free immunosensors is the employment of highly uniform and repeatable antibody immobilization techniques. In this study, we investigated the use of two different silane molecules (3-glycidyloxypropyl)trimethoxysilane (GPS), and (3-mercaptopropyl)trimethoxysilane (MTS) for the immobilization of fluorescently labeled IgG antibodies on planar ZnO surfaces. The chemical modification of the surfaces was investigated using water contact angle measurements, AFM, and fluorescence microscopy. The results of the water contact angle measurements indicate increased surface hydrophobicity after treatment with GPS and MTS as compared to the control. Surface modification was further verified through AFM measurements which demonstrate an increased surface roughness and particle height after treatment with antibodies. The results of the fluorescence studies indicate that the immobilization protocol employing MTS produced 21% higher fluorescence on average with greater uniformity than the GPS-based protocol, which indicates a higher overall density in antibody coverage on the surface of the ZnO. Acoustic sensor tests were employed to confirm the functionality of sensors treated with the MTS protocol. The results indicate that the immobilization protocol imparts sensitivity and specificity to the ZnO-based devices.     

Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (K_d ~ 10^-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.     

Membrane proteins of Aedes aegypti larvae bind toxins Cry4B and Cry11A of Bacillus thuringiensis ssp. israelensis

Proteins of molecular weight 65 and 62 kD and having affinity for toxins Cry4B and Cry11A produced by Bacillus thuringiensis ssp. israelensis have been isolated from brush border membranes of Aedes aegypti larvae using affinity chromatography. Using a ligand blotting technique, we show that the binding of these proteins to the biotinylated toxins is reversible and that the two toxins compete for binding to the two proteins. These proteins are likely to be Cry4B and Cry11A toxin receptors in gut epithelial cells of Aedes aegypti larvae.     

Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate

Biotinylated proteins are widely used as a molecular tool in biotechnological applications. In this paper, we demonstrated that biotinylated proteins after electrophoresis were detected directly in gels using an avidin-fluorescein conjugate with a fluorescence image analyzer. Upon analysis of the purified and chemically biotinylated protein, the sensitivity of this method was almost equal to that of silver staining. Chemically biotinylated proteins of Escherichia coli cell surfaces could also be specifically detected with our method. Furthermore, recombinant proteins fused with the biotin acceptor domain and biotinylated enzymatically in vivo were also detected in a lysate of E. coli specifically. The sensitivity and specificity of our method are high, and the procedure is simple. Therefore, our method would benefit detection of biotinylated proteins via gel electrophoresis and also various fields of study using avidin-biotin technology.     

Biotinylation of heat shock protein 70 induces RANTES production in HEK293 cells in a CD40-independent pathway

Biotinylated proteins and peptides have been used as popular ligands for characterization of cell surface receptors by a variety of methods including flow cytometry. The number and the location of biotin moieties incorporated could alter the structural and physicochemical properties of ligands, although biotin is thought to be such a small molecule (244Da) that it is capable of being conjugated to most proteins without affecting their activity. Here, we demonstrate that the biotinylated HSP70 molecule via primary amines bound to epithelium-like HEK 293 cells in a saturable manner whereas the unlabeled counterparts of HSP70 other than mouse Hsp72 do not. This binding was not competed by either HSP70 or the biotin entity itself. Interestingly, the biotinylated HSP70 also elicited the production of CC-chemokine RANTES independent of CD40 signaling. This response occurred regardless of sequence diversity of HSP70 derived from different species, and neither the biotinylated ovalbumin nor the unlabeled HSP70 cross-linked with a biotinylated protein stimulated a significant level of RANTES production which was induced by biotinylated HSP70 itself. Our findings suggest that modification of HSP70 such as biotinylation may function as a biological alarm signal in the innate immune system.