Low Unspecific Binding Research Articles

Synthesis, Characterization, and Application of Muscarinergic M3 Receptor Ligands Linked to Fluorescent Dyes.

Through the linkage of two muscarinergic M3 receptor ligands to fluorescent tetramethylrhodamine- and cyanine-5-type dyes, two novel tool compounds, OFH5503 and OFH611, have been developed. Based on the suitable binding properties and kinetics related to the M3 subtype, both ligand-dye conjugates were found to be useful tools to determine binding affinities via flow cytometric measurements. In addition, confocal microscopy underlined the comparably low unspecific binding and the applicability for studying M3 receptor expression in cells. Along with the proven usefulness regarding studies on the M3 subtype, the conjugates OFH5503 and OFH611 could, due to their high affinity to the M1 receptor, evolve as even more versatile tools in the field of research on muscarinergic receptors.

Density of Conjugated Antibody Determines the Extent of Fc Receptor Dependent Capture of Nanoparticles by Liver Sinusoidal Endothelial Cells.

Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB-antibody conjugates by in vivo imaging and flow cytometry. We observed that PB-antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB-antibody conjugates were selectively captured in the liver via Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab')2 fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab')2 antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.

New potent A1 adenosine receptor radioligands for positron emission tomography

8-Cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX) is meanwhile an accepted receptor ligand to examine the A1 adenosine receptor (A1AR) in humans by positron emission tomography (PET). A major drawback of this compound is its rather fast metabolic degradation in vivo.Therefore two new xanthine derivatives, namely 8-cyclobutyl-1-cyclopropymethyl-3-(3-fluoropropyl)xanthine (CBCPM; 5) and 1-cyclopropylmethyl-3-(3-fluoropropyl)-8-(1-methylcyclobutyl)xanthine (CPMMCB; 6) were designed and synthesized as potential alternatives to CPFPX. In membrane binding studies both compounds showed nanomolar affinity for the A1AR. In vitro autoradiographic studies of [18F]5 and [18F]6, using rat brain slices, showed the expected accumulation in regions known to have a high adenosine A1 receptor expression while exhibiting the necessary low unspecific binding. However, in vitro metabolite studies using human liver microsomes revealed a comparable metabolic degradation rate for both new xanthine derivatives and CPFPX.

A Novel Method of Immobilizing Antigens on Gold Electrode for Immunosensing

An antigen modified gold electrode based on a self-assembled lipoic acid monolayer has been developed. The contact angle measurement and cyclic voltammetry confirm the formation of a dense self-assembled monolayer on gold from 100 mM lipoic acid in ethanol. The electrochemical behavior shows a stable activity in a range of potential from -0.2 V to 0.65 V vs. Ag/AgCl. By applying a potential of 1 V vs. Ag/AgCl the monolayer is completely removed by oxidative desorption and a clean gold surface re-established. This allows for an easy renewal of the gold surface and recycling of modified immunosensor chips. This lipoic acid monolayer was covalently functionalized with a small molecule antigen synthesized from progesterone-3-(O-carboxymethyl)oxime and 4,7,10-trioxa-1,13 tridecanediamine to form a well ordered, low unspecific binding, antigen layer for an antibody-antigen interaction study.The effectiveness of antigen - antibody binding reaction was demonstrated by fluorescence imaging using a fluorescence-labeled anti-progesterone antibody as the binding partner. The new approach represents a way of improving the surface chemistry of electrochemical, surface-plasmon resonance and QCM-based immunosensors for which reusability, simplicity and sensitivity in flow-injection mode are required.

Selective immobilization and detection of DNA on biopolymer supports for the design of microarrays

DNA immobilization for the manufacturing of microarrays requires sufficient probe density, low unspecific binding and high interaction efficiency with complementary strands that are detected from solutions. Many of these important parameters are affected by the surface chemistry and the blocking steps conducted during DNA spotting and hybridization. This work describes an alternative method to selectively immobilize probes and to detect DNA on biocompatible, hydrophilic cellulose coated supports with low unspecific binding, high selectivity and appropriate sensitivity. It takes advantage of a relatively selective adsorption of water soluble polysaccharides on a solid cellulose matrix. Single strands of DNA were conjugated to this soluble polysaccharide and subsequently micro-spotted on solid cellulose thin films that were coated on glass and polymer slides. This resulted in adsorptively bound DNA-probes that were used to detect complementary, labelled DNA strands with different lengths and sequences by hybridization. The interaction of the DNA-conjugates with cellulose surfaces and the selectivity of hybridization were investigated by a quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence scanning. The method of non-covalent immobilization of DNA probes on an uncharged, non-reactive, hydrophilic support lowers the unspecific binding and the number of handling steps required to conduct the experiments for the detection of DNA on microarrays. Simultaneously selectivity, hybridization efficiency and detection limits are maintained.

2D crystalline protein layers as immobilization matrices for the development of DNA microarrays

There is a growing demand for functional layers for the immobilization of (bio)molecules on different kinds of substrates in the field of biosensors, microarrays, and lab-on-a-chip development. These functional coatings should have the ability to specifically bind (bio)molecules with a high binding efficiency, while showing low unspecific binding during the following assay. In this paper we present rSbpA surface layer proteins (S-layer proteins) as a versatile immobilization layer for the development of DNA microarrays. S-layer proteins show the ability to reassemble into two-dimensional arrays on solid surfaces and their functional groups, such as carboxylic groups, are repeated with the periodicity of the lattice, allowing for immobilization of other (bio)molecules. Different fluorescently labeled amino functionalized DNA oligomers were covalently linked to the S-layer matrices to allow the characterization of DNA binding on S-layers. Hybridization and dissociation of DNA-oligomers were studied on S-layer coated slides, revealing low levels of unspecific adsorption of DNA on S-layer based immobilization matrices. In the following the principle was transferred to a DNA microarray design showing successful spotting and hybridization on whole microarray slides. Besides common laser scanning for fluorescence detection, S-layer based microarrays were evaluated with a compact, low cost platform for direct fluorescence imaging based on surface plasmon enhanced fluorescence excitation. It could be shown that S-layer protein layers are promising as immobilization matrices for the development of biosensors and microarrays.

Chemical tools selectively target components of the PKA system

BackgroundIn the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. Here we describe the design, synthesis and characterisation of specifically tailored cAMP analogs which can be utilised as a tool for affinity enrichment and purification as well as for proteomics based analyses of cAMP binding proteins.ResultsTwo sets of chemical binders were developed based on the phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and -antagonists, respectively. These compounds were tested via direct surface plasmon resonance (SPR) analyses for their binding properties to PKA R-subunits and holoenzyme. Furthermore, these analogs were used in an affinity purification approach to analyse their binding and elution properties for the enrichment and improvement of cAMP binding proteins exemplified by the PKA R-subunits. As determined by SPR, all tested Sp-analogs provide valuable tools for affinity chromatography. However, Sp-8-AEA-cAMPS displayed (i) superior enrichment properties while maintaining low unspecific binding to other proteins in crude cell lysates, (ii) allowing mild elution conditions and (iii) providing the capability to efficiently purify all four isoforms of active PKA R-subunit in milligram quantities within 8 h. In a chemical proteomics approach both sets of binders, Rp- and Sp-cAMPS derivatives, can be employed. Whereas Sp-8-AEA-cAMPS preferentially binds free R-subunit, Rp-AHDAA-cAMPS, displaying antagonist properties, not only binds to the free PKA R-subunits but also to the intact PKA holoenzyme both from recombinant and endogenous sources.ConclusionIn summary, all tested cAMP analogs were useful for their respective application as an affinity reagent which can enhance purification of cAMP binding proteins. Sp-8-AEA-cAMPS was considered the most efficient analog since Sp-8-AHA-cAMPS and Sp-2-AHA-cAMPS, demonstrated incomplete elution from the matrix, as well as retaining notable amounts of bound protein contaminants. Furthermore it could be demonstrated that an affinity resin based on Rp-8-AHDAA-cAMPS provides a valuable tool for chemical proteomics approaches.

Imaging SPR for detection of local electrochemical processes on patterned surfaces

The central theme of this thesis is the use of imaging Surface Plasmon Resonance (iSPR) as a tool in the characterization of surfaces with laterally varying properties. Within the scope of this work, an instrument for iSPR analysis was designed and built. SPR is a very sensitive technique for monitoring changes in optical properties in the immediate vicinity of a sensor surface, which is very useful in biosensing and surface science research. We have employed SPR in the Kretschmann configuration, wherein surface plasmons are excited by means of an evanescent field arising from total internal reflection from the backside of the sensor surface. In iSPR, the signal is the reflectivity of TM-polarized light which is measured using an imaging detector, typically a CCD camera. Advantages of this technique include extreme surface sensitivity and, because detection is done from the backside, compatibility with complex samples. In addition, SPR is a non-labeling technique, and in imaging mode, a lateral resolution in the µm range can be attained. The imaging SPR instrument could be operated in either wavelength interrogation mode or in intensity mode. In the former case, the objective is to find the SPR wave-length, λSPR, which is the wavelength at which the reflected intensity is at a minimum. In intensity mode, a snapshot of the intensity reflectance is taken at a fixed wavelength hand incidence angle. In biosensor science, the use of an imaging technique offers a major advantage by enabling parallelization and thereby increasing throughput. We have, for example, used iSPR in biochemical interaction analysis to monitor immobilization and specific binding to protein and synthetic polypeptide micro arrays. The primary interest has been the study of soft matter surfaces that possess properties interesting in the field of biomimetics or for applications in biosensing. Specifically, the surfaces studied in this thesis include patterned self-assembled monolayers of thiolates on gold, a graft polymerized poly(ethylene glycol) (PEG) based hydrogel, a dextran hydrogel, and a polyelectrolyte charge gradient. Our results show that the PEG-based hydrogel is very well suited for use as a platform in protein immobilization in an array format, owing to the very low unspecific binding. In addition, well defined microarray templates were designed by patterning of hydrophobic barriers on dextran and monolayer surfaces. A polypeptide affinity microarray was further designed and immobilized on such a patterned monolayer substrate, in order to demonstrate the potential of analyte quantification with high sensitivity over a large dynamic range. Furthermore, iSPR was combined with electrochemistry to enable laterally resolved studies of electrochemical surface reactions. Using this combination, the electrochemical properties of surfaces patterned with self assembled monolayers can be studied in parallel, with a spatial resolution in the µm regime. We have also employed electrochemistry and iSPR for the investigation of potential and current density gradients on bipolar electrodes. The imaging SPR instrument could be operated in either wavelength interrogation mode or in intensity mode. In the former case, the objective is to find the SPR wave-length, λSPR, which is the wavelength at which the reflected intensity is at a minimum. In intensity mode, a snapshot of the intensity reflectance is taken at a fixed wavelength hand incidence angle.In biosensor science, the use of an imaging technique offers a major advantage by enabling parallelization and thereby increasing throughput. We have, for example, used iSPR in biochemical interaction analysis to monitor immobilization and specific binding to protein and synthetic polypeptide micro arrays. The primary interest has been the study of soft matter surfaces that possess properties interesting in the field of biomimetics or for applications in biosensing. Specifically, the surfaces studied in this thesis include patterned self-assembled monolayers of thiolates on gold, a graft polymerized poly(ethylene glycol) (PEG) based hydrogel, a dextran hydrogel, and a polyelectrolyte charge gradient. Our results show that the PEG-based hydrogel is very well suited for use as a platform in protein immobilization in an array format, owing to the very low unspecific binding. In addition, well defined microarray templates were designed by patterning of hydrophobic barriers on dextran and monolayer surfaces. A polypeptide affinity microarray was further designed and immobilized on such a patterned monolayer substrate, in order to demonstrate the potential of analyte quantification with high sensitivity over a large dynamic range.Furthermore, iSPR was combined with electrochemistry to enable laterally resolved studies of electrochemical surface reactions. Using this combination, the electrochemical properties of surfaces patterned with self assembled monolayers can be studied in parallel, with a spatial resolution in the µm regime. We have also employed electrochemistry and iSPR for the investigation of potential and current density gradients on bipolar electrodes.

A novel tool for the detection of allergic sensitization combining protein microarrays with human basophils

Protein microarray (PM) is a powerful alternative to costly or labour-intensive diagnostic for the large-scale detection of allergen-specific IgE. In this study, we established a proof-of-concept that coupling the diversity of protein array with the biological output of basophilic cells is a feasible proposition. Human basophils purified from the peripheral blood of healthy donors were stripped, re-sensitized with the serum or IgE preparation to be tested, and incubated with manually spotted protein array chips (FAST slides). The basophilic cell lines KU-812 and RBL-703/21 likewise sensitized were compared with peripheral blood basophils by the same approach. Purified basophils or other basophilic cells were incubated with FAST slides for various periods of time, washed, and cell binding was visualized by light microscopy. Basophil activation, indicating the effective cross-linking of IgE by allergens, was monitored via up-regulation of basophil activation surface marker (CD 63). Purified stripped peripheral basophils, re-sensitized with the serum of a grass pollen-allergic patient, displayed strong binding to anti-IgE antibody and grass pollen extract with relatively low unspecific binding. Similar results were obtained with RBL-703/21, which may be a good replacement for peripheral basophils to avoid the costly, cumbersome and time-consuming basophil purification. Our data suggest that coupling the diversity of a PM approach with the potential functionality and biological activity of a cell-based test is feasible and may result in a new system to detect allergic sensitization.

Lipopolysaccharide removal by a peptide-functionalized surface

Five peptides: BPI 85–109; CAP18 106–137; endotoxin inhibitor (EI); GQ33 and GQ33C, derived from lipopolysaccharide (LPS)-binding molecules were investigated for LPS-binding ability with a view to a potential use in extracorporeal therapy. The surface plasmon resonance technique (SPR) was used to monitor the interaction between LPS in solution and the surface-immobilized peptides. The peptides were covalently bound to a model dextran surface via inherent amino groups or via terminally introduced cysteine residues. The results showed that the binding efficacy and binding stability of the peptides varied greatly. The CAP18 106–137 peptide, which exhibited the highest binding efficacy and binding stability, was also immobilized on a poly(ethylene imine)–poly(ethylene glycol) (PEI–PEG) surface through maleimide-terminal PEG. The binding efficacy of the CAP18 106–137 peptide was not significantly affected by the different immobilization methods used in the attachment to a dextran or a PEI–PEG surface. LPS bound selectively to CAP18 106–137 and showed very low unspecific binding to the PEI–PEG surface layer. The EI peptide proved to have a reasonably good binding capacity but a less stable interaction with LPS. The other peptides exhibited much poorer binding efficacy. We believe that the results presented in this work can be of practical value for the development of extracorporeal treatment of patients suffering from septic shock.

Demonstration of immunoglobulin G with affinity for dopamine in cerebrospinal fluid from psychotic patients

Using an enzyme-linked immunosorbent assay, significantly raised concentrations of immunoglobulin G with affinity for the neurotransmitter dopamine were demonstrated in cerebrospinal fluid psychotic patients. We have varied the antigen presentation in order to find a conjugate with low unspecific binding. The conjugation of dopamine to carbodiimide-activated poly- L-glutamic acid and that to activated succinimide ester of biotin are described. The use of glutaraldehyde conjugation is not recommended because of the risk of formation of tetrahydroisoquinolines. A strong correlation ( r = 0.94, P < 0.001) between the results obtained with dopamine conjugated to poly- L-glutamic acid and dopamine conjugated to biotin was observed. Forty-two human cerebrospinal fluid samples from 20

A highly sensitive and rapid radioimmunoassay for the determination of arginine8-vasopressin.

A highly sensitive radioimmunossay for arginine8-vasopressin (argipressin; INN) measurement was developed using Amberlite XAD 2 resin columns to extract arginine8-vasopressin from acidified human plasma. Arginine8-vasopressin was determined by a rapid radioimmunoassay method (2 x 20 h) using a specific antibody and 125I-labelled antigen. The bound fraction was separated by adsorption of the free fraction onto bovine serum albumin-coated charcoal; this resulted in low unspecific binding of less than 2%. Recovery experiments in the physiological range resulted in a mean (+/- SEM) recovery of 88 +/- 3%. The radioimmunoassay consistently yielded a detection limit of 0.3 ng/l (ED90) and a mean 50% binding intercept (ED50) of 3.5 ng/l. Arginine8-vasopressin immunoreactivity was characterized by reverse-phase high performance liquid chromatography, which confirmed the specificity of the assay. Serial plasma dilution curves paralleled the standard curve. The intra- and inter-assay variations were 9.4% and 15%, respectively. Arginine8-vasopressin concentrations in healthy subjects were determined in normal hydration status (2.2 +/- 0.3 ng/l; n = 11), as well as during suppression by water immersion (1.5 +/- 0.2 ng/l; n = 11) or by water loading (1.6 +/- 0.2 ng/l; n = 8). Thus, this assay allows for a sensitive, accurate and rapid quantification of plasma arginine8-vasopressin concentrations.

Total Versus Unspecific Binding: Standardization and Optimization of Receptor-Ligand Binding Assays

Intra- and interassay imprecision was evaluated in 3 in vitro ligand binding assays using different types of filtering devices, glass fiber filters and variations in methodology. In the [3H]-spiroperidol test, the already low unspecific binding seemed to be dependent on the type of filters employed, whereas in the [3H]-ketanserin- and [3H]-GR 65630-binding tests, differences were within the range of the normal variabilities. However, in the latter test, which is problematic owing to the high unspecific binding of [3H]-GR 65630, it was found that although the percentage of specific binding was fairly constant on different sheets, large differences in the absolute amounts of total and unspecific binding were observed on consecutive sheets in the same experiment. Thus, it is critically important to filter samples for total and unspecific binding together on the same filter sheet for the calculation of specific binding. Under these precautions, highly reproducible results for IC50-values in the screening of potential 5HT3-receptor ligands were obtained in spite of using rat entorhinal cortex, a relatively large area with suboptimal 5HT3-receptor density. In contrast, when using rat area postrema, which is optimal with respect to receptor density, more than 10 times the number of rats is necessary for a competition experiment due to the small size of this brain part. Since IC50-values for both areas compare favorably, entorhinal cortex should be used for ethical reasons and to minimize costs.

Optimal Conditions for the Preparation of Ferritin-labeled Antibodies Defined by Binding to Their Antigen in an ELISA

An ELISA with covalently fixed antigens was used to measure and define ferritin-labeled antibodies. In the presented model, RIG covalently fixed to glass tubes served as antigen. Twenty ferritin-labeled SWaRIG conjugates were prepared with different molar ratios of antibody: ferritin: glutaraldehyde. At various dilutions, their ability to react with the antigen was compared. The amount of FRT in the reactive conjugates was measured using alkaline phosphatase-labeled antibodies against ferritin. The covalent binding of antigens to glass surfaces resulted in a low unspecific binding as shown previously. Besides, these glass tubes with antigens immobilized on their inner surface could be used more than once. This aspect of it renders this test particularly suitable for systems where rare or expensive antigens are used. The range of O.D. values reflecting the amount of reactive SWaRIG/FRT detected in 20 different conjugates (dil 1:1000) was spread over a good range which allowed a specification of optimal conditions for FRT labeling of antibodies. It is concluded that in addition to an electron microscopic evaluation of the conjugates, this assay may be very helpful in defining optimal conditions for the coupling procedure.