Single chain fragment variable recombinant antibody as a template for Fc sensors

A label free immunosensor for detection of Fc receptors expressed on cell surface was developed and characterized using a Quartz Crystal Microbalance (QCM) transducer. Taking advantage of the characteristics of single chain fragment variable (scFv) recombinant antibody and the multivalency of an antibody, the engineered recombinant scFv was immobilized onto preformed functionalized self-assembled monolayers (SAMs) template surface. The monomeric scFv can bind with the CH1 region of any rabbit IgG to form a highly oriented IgG layer with its Fc portion pointing toward a solution phase. This results in a highly oriented Fc sensor that can be used to study the thermodynamics and kinetics of binding between the Fc portion of immunoglobulin and the cell surface Fc receptor (FcR), an important area of the immune system. The Fc sensor was used to study the binding between Staphylococcus aureus and the Fc receptor on macrophage. Parallel characterization of cell surface Fc receptors in the same samples by ELISA was also performed.     

Tailored magnetic nanoparticles for direct and sensitive detection of biomolecules in biological samples

We developed nanoparticles with tailored magnetic properties for direct and sensitive detection of biomolecules in biological samples in a single step. Thermally blocked nanoparticles obtained by thermal hydrolysis, functionalized with specific ligands, are mixed with sample solutions, and the variation of the magnetic relaxation due to surface binding is used to detect the presence of biomolecules. The binding significantly increases the hydrodynamic volume of nanoparticles, thus changing their Brownian relaxation frequency which is measured by a specifically developed AC susceptometer. The system was tested for the presence of Brucella antibodies, a dangerous pathogen causing brucellosis with severe effects both on humans and animals, in serum samples from infected cows and the surface of the nanoparticles was functionalized with lipopolysaccharides (LPS) from Brucella abortus. The hydrodynamic volume of LPS-functionalized particles increased by 25-35% as a result of the binding of the antibodies, measured by changes in the susceptibility in an alternating magnetic field. The method has shown high sensitivity, with detection limit of 0.05 microg x mL(-1) of antibody in the biological samples without any pretreatment. This magnetic-based assay is very sensitive, cost-efficient, and versatile, giving a direct indication whether the animal is infected or not, making it suitable for point-of-care applications. The functionalization of tailored magnetic nanoparticles can be modified to suit numerous homogeneous assays for a wide range of applications.     

Grouping of ferritin and gold nanoparticles conjugated to pRNA of the phage phi29 DNA-packaging motor

The bacteriophage phi29 DNA-packaging motor, which translocates and compresses the DNA genome of the phage into its procapsid during virion assembly, involves an essential ring formed by the packaging RNA (pRNA). We attached electron-dense nanoparticles to pRNA by hybridizing a DNA oligonucleotide with a biotin or thiol modification to a 3'-extension of core pRNA, and by coupling streptavidin and biotinylated ferritin, or 5 nm and 10 nm colloidal gold particles, to these modifications. The pRNA conjugates bound to RNA-free phi29 procapsids, and such nanoparticle-bearing procapsids were isolated by ultracentrifugation and analyzed. Electron microscopy showed that ferritin and gold particles were specifically attached to the side of the phi29 procapsid harboring the connector, which is the pRNA binding site. The pRNA-ferritin conjugates bound to procapsids with the same efficiency as pRNA which lacked the high molecular mass label. However, full DNA packaging efficiency in an in vitro phage assembly assay was only reached after the label of such isolated procapsid-pRNA complexes had been released with RNase H. The results provide approaches to assembled ferritin or gold-containing nano-complexes via pRNA mediated assembly.     

Immunoenzymometric assay for a small molecule,11-deoxycortisol, with attomole-range sensitivity employing an scFv-enzyme fusion protein and anti-idiotype antibodies

To overcome the sensitivity limit in immunoassays for small molecules (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten molecules. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chemically linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amounts of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse beta-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic separation using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized alpha-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels determined for healthy subjects were validated as reliable.     

Simultaneous use of time-resolved fluorescence and anti-stokes photoluminescence in a bioaffinity assay

A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates are measured from a single microtitration well without any disturbance from these label technologies to each other. Up-converting phosphor (UPC-phosphor) bioconjugate was produced by grinding the commercial, micrometer-sized UPC-phosphors to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. Experiments were carried out in standard 96-well microtitration plates to determine detection limits, linearity, and cross-talk of UPC-phosphor and europium chelate. In numbers of molecules the lower limits of detection for UPC-phosphor were roughly 3 x 10(3) particles in solution and 1 x 10(4) particles in solid phase, and for europium label same values were 9 x 10(6) and 9 x 10(7) molecules. Linearity of detection was for UPC-phosphor 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase and for europium label over 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase. The cross-talk between the two labels was practically nonexistent. In this study we show that up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with long-lifetime lanthanide chelates.     

Engineering peptide linkers for scFv immunosensors

Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs). Six arginines, which were separated by glycine or serine as spacer, were incorporated in the peptide linker to form a 15-mer peptide linker (RGRGRGRGRSRGGGS). The polycationic arginine peptide was engineered into the A10B scFv-RG3 to favor its adsorption at anionic charged template surface (11-mercaptoundecanoic acid (MUA) and poly(sodium 4-styrenesulfonate (PSS))). This new approach was compared with the other engineered scFv constructs. Our results demonstrated that the anionic charged SAM template facilitated the oriented immobilization of scFvs on the SAM template surface as well as reduced the possibility of protein denaturation when directly immobilized on the solid surface. A 42-fold improvement of detection limits using MUA/A10B scFv-RG3 (less than 0.2 nM experimentally determined) was achieved compared to A10B Fab antibody and a 5-fold improvement was observed compared to A10B scFv that was engineered with a cysteine in the linker sequence. Using protein A-coated gold nanoparticles, a picomolar experimental detection limit was achieved. With 20 amino acids to choose from, engineered recombinant scFv in combination with SAM technology and nanoparticle mass amplification provide an emerging strategy for the development of highly sensitive and specific scFv immunosensors.     

Synthesis and characterization of europium(III) nanoparticles for time-resolved fluoroimmunoassay of prostate-specific antigen

Recent advances in the fabrication and bioconjugation of nanometre-sized lanthanide(III) chelate particles have led to robust high specific activity labels. This paper describes the synthesis and characterization of lanthanide(III) nanoparticle labels and the use of a nanoparticle in a bioaffinity assay system. Two europium(III) nanoparticles were prepared using an extremely simple, inexpensive and fast agglomeration strategy. A?silica-stabilized nanoparticle was synthesized from hydrophobic tris(dibenzoylmethane)-mono(phenanthroline) and tris(dibenzoylmethane)-mono(5-aminophenanthroline) europium(III) chelates in aqueous solution. In addition, a naphthoyl trifluoroacetone:tri-n-octylphosphineoxide:sodium dodecyl sulfate europium(III) complex was agglomerated in water. The particle sizes ranged from 62 to 140?nm in diameter. The silica-stabilized particle was further coated with a monoclonal antibody. The analytical performance of the bioconjugated nanoparticle label was evaluated in a model sandwich immunoassay of prostate-specific antigen. The detection limit of human prostate-specific antigen was 28?ng?l(-1), 850?fM, in a microtiter plate format using time-resolved fluorometry. The coefficient of variation ranged from 1 to 9%. The novel nanoparticle label improves the specific activity of existing lanthanide(III) nanoparticle labels and simplifies the preparation route. In addition, prepared high-density nanoparticle labels using lanthanide(III) chelates or other specific fluorochromes have potential applications in a number of other fields.     

Ferritin Protein Regulates the Degradation of Iron Oxide Nanoparticles

Proteins implicated in iron homeostasis are assumed to be also involved in the cellular processing of iron oxide nanoparticles. In this work, the role of an endogenous iron storage protein—namely the ferritin—is examined in the remediation and biodegradation of magnetic iron oxide nanoparticles. Previous in vivo studies suggest the intracellular transfer of the iron ions released during the degradation of nanoparticles to endogenous protein cages within lysosomal compartments. Here, the capacity of ferritin cages to accommodate and store the degradation products of nanoparticles is investigated in vitro in the physiological acidic environment of the lysosomes. Moreover, it is questioned whether ferritin proteins can play an active role in the degradation of the nanoparticles. The magnetic, colloidal, and structural follow‐up of iron oxide nanoparticles and proteins in lysosome‐like medium confirms the efficient remediation of potentially harmful iron ions generated by nanoparticles within ferritins. The presence of ferritins, however, delays the degradation of particles due to a complex colloidal behavior of the mixture in acidic medium. This study exemplifies the important implications of intracellular proteins in processes of degradation and metabolization of iron oxide nanoparticles.     

Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging

Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single‐chain anti‐EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR‐expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR‐QDs or ScFvEGFR‐IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular‐targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR‐targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.     

Protein oriented ligation on nanoparticles exploiting O6-alkylguanine-DNA transferase (SNAP) genetically encoded fusion

A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the site-specific covalent conjugation of targeting peptides to nanoparticles. An scFv mutant selective for HER2 tumor antigen is chosen as the targeting ligand. SNAP-scFv is immobilized on magnetofluorescent nanoparticles and its targeting efficiency against HER2-positive cells is assessed by flow cytometry and immunofluorescence.     

Single-chain fragment variable antibody piezoimmunosensors

In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled monolayer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW approximately 27000) and the ease with which they can be modified using genetic engineering, scFv's have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples.     

Nuclear localization of HIV-1 tat functionalized gold nanoparticles

The impermeable nature of the cell plasma membrane limits the therapeutic uses of many macromolecules and there is therefore a growing effort to circumvent this problem by designing strategies for targeted intracellular delivery. During the last decade several cell penetrating peptides, such as the HIV-1 tat peptide, have been shown to traverse the cell membrane, where integral protein transduction domains (PTDs) are responsible for their cellular uptake, and to reach the nucleus while retaining biological activity. It has since been discovered that PTDs can enable the cellular delivery of conjugated biomolecules and even nanoparticles, but nuclear delivery has remained problematic. This present study focuses on the development of water soluble, biocompatible gold nanoparticles of differing size functionalized with the HIV-1 tat PTD with the aim of producing nuclear targeting agents. The particles were subsequently tested in vitro with a human fibroblast cell line, with results demonstrating successful nanoparticle transfer across the plasma membrane, with 5 nm particles achieving nuclear entry while larger 30 nm particles are retained in the cytoplasm, suggesting entry is blocked via nuclear pores dimensions.     

Ultrasensitive microarray detection of short RNA sequences with enzymatically modified nanoparticles and surface plasmon resonance imaging measurements

A novel multiplexed method for short RNA detection that employs an enzymatic capture reaction onto DNA-modified silica nanoparticles (SiNPs) followed by nanoparticle-enhanced surface plasmon resonance imaging (SPRI) is demonstrated. SiNPs functionalized with 5'-phosphorylated single stranded DNA (ssDNA) are used with T4 RNA ligase to capture various short 20-24 base single-stranded RNA (ssRNA) oligonucleotides from a target solution. The ssRNA-modified SiNPs are collected from the target solution, specifically adsorbed onto a cDNA microarray and then detected with SPRI. The use of DNA-modified SiNPs to capture ssRNA for profiling has several advantages as compared to a planar SPRI surface bioaffinity adsorption format: (i) the target solution is exposed to a larger total surface area for the RNA ligation reaction; (ii) the SiNPs enhance the diffusion rate of the ssRNA to the surface; (iii) the SiNPs can be collected, washed, and preconcentrated prior to detection; and (iv) the ssRNA-modified SiNPs give an enhanced SPRI signal upon hybridization adsorption to the microarray. Our initial measurements demonstrate that this detection method can be used to detect multiple ssRNA sequences at concentrations as low as 100 fM in 500 μL.     

Gold-coated magnetic particles for solid-phase immunoassays: enhancing immobilized antibody binding efficiency and analytical performance

The preparation and characterization of gold-coated magnetic particles are described for use as more efficient solid-phase materials in immunoassay development. A thin gold coating on commercial tosylated magnetic polystyrene particles (4.5 microm) is achieved via an electroless plating method involving initial reaction of the particles with Sn(II), followed by redox deposition of Ag0, that serves as a catalytic site for the subsequent reduction of Na3Au(SO3)2 in the presence of formaldehyde to yield the adhered gold layer. Scanning electron microscopy, energy-dispersive X-ray analysis, and X-ray photoelectron spectroscopy indicate the presence of the desired Au0 outer layer. To characterize the improved yield of antibody binding sites on such gold-coated phases, the modified particles are reacted with the free thiols of Fab' fragments of an anti-alkaline phosphatase (ALP) antibody to orient all the antigenic binding sites in a favorable direction. After equilibration with ALP, the amount of ALP bound to the surface of such particles is nearly 2.5-fold greater than on non-gold-coated particles possessing the same amount of immobilized anti-ALP Fab', but oriented randomly on the surface. The new gold-coated magnetic particles are further used as a solid phase for developing a sandwich-type enzyme immunoassay to detect C-reactive protein (CRP) using horseradish peroxidase as the enzyme label. The gold-coated magnetic particles with anti-CRP monoclonal Fab' reagents provide assays with enhanced assay slope (1.8-fold), lower nonspecific adsorption, and a detection limit improvement of nearly 10-fold (0.14 vs 1.9 ng/mL) compared to the same Fab' anti-CRP immobilized on the initial tosylated polystyrene magnetic particles. The improved assay performance is attributed to the more favorable binding orientation of the self-assembled monolayer of Fab' fragments on the gold-coated particles compared to the random orientation on the non-gold-coated surfaces.     

Triple bioaffinity mass spectrometry concept for thyroid transporter ligands

For the analysis of thyroid transporter ligands, a triple bioaffinity mass spectrometry (BioMS) concept was developed, with the aim at three different analytical objectives: rapid screening of any ligand, confirmation of known ligands in accordance with legislative requirements, and identification of emerging yet unknown ligands. These three purposes share the same biorecognition element, recombinant thyroid transport protein transthyretin (rTTR), and dedicated modes of liquid chromatography-mass spectrometry (LC-MS). For screening, a rapid and radiolabel-free competitive inhibition MS binding assay was developed with fast ultrahigh performance-liquid chromatography-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS) as the readout system. It uses the nonradioactive stable isotopic thyroid hormone (13)C(6)-l-thyroxine as the label of which the binding to rTTR is inhibited by any ligand such as thyroid drugs and thyroid endocrine disrupting chemicals (EDCs). To this end, rTTR is either used in solution or immobilized on paramagnetic microbeads. The concentration-dependent inhibition of the label by the natural thyroid hormone l-thyroxine (T4), as a model analyte, is demonstrated in water at part-per-trillion and in urine at part-per-billion level. For confirmation of identity of known ligands, rTTR was used for bioaffinity purification for confirmation of naturally present free T4 in urine. As a demonstrator for identification of unknown ligands, the same rTTR was used again but in combination with nano-UPLC-quadrupole time-of-flight-MS (nLC-Q-TOF-MS) and urine samples spiked with the model "unknown" EDCs triclosan and tetrabromobisphenol-A. This study highlights the potential of BioMS using one affinity system, both for rapid screening and for confirmation and identification of known and unknown emerging thyroid EDCs.     

Preparation of highly stable oligo(ethylene glycol) derivatives-functionalized gold nanoparticles and their application in LSPR-based detection of PSA/ACT complex

A sandwich immunoassay for PSA/ACT complex detection based on gold nanoparticle aggregation using two probes was developed. The functionalized colloidal gold nanoparticles (AuNPs) showed highly stable not only in the presence of high ionic strength but also in a wide pH range. The functionalized AuNPs were tagged with PSA/ACT complex monoclonal antibody and goat PSA polyclonal antibody and served as the probes to induce aggregation of the colloidal particles. As a result, PSA/ACT complex was detected at concentrations as low as 1 ng/ml. This is the first time that a new aggregation sandwich-immunoassay technique using two gold probes has been used, and the results are generally applicable to other LSPR-based immunoassays.     

Mesoporous Silica Nanoparticles Act as a Self‐Adjuvant for Ovalbumin Model Antigen in Mice

Immunization to the model protein antigen ovalbumin (OVA) is investigated using MCM‐41 mesoporous silica nanoparticles as a novel vaccine delivery vehicle and adjuvant system in mice. The effects of amino surface functionalization and adsorption time on OVA adsorption to nanoparticles are assessed. Amino‐functionalized MCM‐41 (AM‐41) shows an effect on the amount of OVA binding, with 2.5‐fold increase in binding capacity (72 mg OVA/g AM‐41) compared to nonfunctionalized MCM‐41 (29 mg OVA/g MCM‐41). Immunization studies in mice with a 10 μg dose of OVA adsorbed to AM‐41 elicits both antibody and cell‐mediated immune responses following three subcutaneous injections. Immunizations at a lower 2 μg dose of OVA adsorbed to AM‐41 particles results in an antibody response but not cell‐mediated immunity. The level of antibody responses following immunization with nanoformulations containing either 2 μg or 10 μg of OVA are only slightly lower than that in mice which receive 50 μg OVA adjuvanted with QuilA, a crude mixture of saponins extracted from the bark of the Quillaja saponaria Molina tree. This is a significant result, since it demonstrates that AM‐41 nanoparticles are self‐adjuvanting and elicit immune responses at reduced antigen doses in vivo compared to a conventional delivery system. Importantly, there are no local or systemic negative effects in animals injected with AM‐41. Histopathological studies of a range of tissue organs show no changes in histopathology of the animals receiving nanoparticles over a six week period. These results establish the biocompatible MCM‐41 silica nanoparticles as a new method for vaccine delivery which incorporates a self‐adjuvant effect.     

Influence of solution and gas phase processes on protein-carbohydrate binding affinities determined by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry

The influence of solution pH, analyte concentration and in-source dissociation on the measurement of the association constant for a single chain variable fragment of a monoclonal antibody (scFv) and its native trisaccharide ligand by nanoelectrospray-Fourier transform ion cyclotron resonance mass spectrometery has been systematically investigated. From the results of this study, experimental conditions that preserve the original distribution of bound and unbound protein in solution into the gas phase, such that the nanoES mass spectrum provides a quantitative measure of the solution composition, were identified. These include the use of short spray durations (<10 min) to minimize pH changes, equimolar concentrations of protein and ligand to minimize the formation of nonspecific complexes, and short accumulation times (<2 s) in the hexapole of the ion source to avoid collisional heating and dissociation of the gaseous complex. Application of this methodology to the scFv and a series of carbohydrate ligands yields results that are in agreement with values previously determined by isothermal titration calorimetry. Competitive binding experiments performed on solutions containing the scFv and a mixture of carbohydrate ligands were also found to yield accurate association constants.     

Surface-imprinted core-shell nanoparticles for sorbent assays

In this paper, we present a general protocol for the making of surface-imprinted core-shell nanoparticles via surface reversible addition-fragmentation chain-transfer (RAFT) polymerization using RAFT agent functionalized model silica nanoparticles as the chain-transfer agent. In this protocol, trichloro(4-chloromethylphenyl)silane was immobilized on the surface of SiO2 nanoparticles, forming chloromethylphenyl functionalized silica (silica-Cl). RAFT agent functionalized silica was subsequently produced by substitute reaction of silica-Cl with PhC(S)SMgBr. The grafting copolymerization of 4-vinylpyridine and ethylene glycol dimethacrylate using surface RAFT polymerization and in the presence of 2,4-dichlorophenoxyacetic acid as the template led to the formation of surface-imprinted core-shell nanoparticles. The resulting surface-imprinted core-shell nanoparticles bind the original template 2,4-D with an appreciable selectivity over structurally related compounds. The potential use of the surface-imprinted core-shell nanoparticles as the recognition element in the competitive fluorescent binding assay for 2,4-D was also demonstrated.