Non-specific Background Binding Research Articles

Continuous flow of reagents interferes with the kinetics and equilibrium of antibody-antigen binding in microfluidic heterogeneous immunoassays

Though fundamentally important for the design and development of quantitative point-of-care diagnostics devices, the effect of fluid flow in heterogeneous immunoassays remains unclear. We present the first experimental study using actual microfluidic strips showing both kinetics and equilibrium of antibody-analyte binding are dependent on flow rate, Q of reagents/sample. We have passively adsorbed an antibody in the inner surface of 212 ± 12 µm, 10-bore microcapillary film and pumped continuously a second (antigen) molecule in the range Q = 10–1000 µL/min. Sections of the film with 40 mm in length were then sacrificed over time enabling ELISA quantitation of the extent of antibody-antigen binding on each strip; other microfluidic technologies would require sacrificing the whole device, making it laborious and expensive. Absorbance data fitted to a kinetic model enabled determining Kon, Koff and affinity constant Keq = Kon/Koff, confirming the impact of Q. With limiting antigen, a 4-fold reduction in Kon and 3-fold in Keq was observed by shifting antigen incubation from stagnant to continuous Q = 10 µL/min. Flow caused up to 2-fold increment in the limit of detection (LoD) and assay time, suggesting a direct physical interference of fluid flow on the molecular binding and non-specific (background) binding, presumably through the effect of shear flow, which could include re-orientation of biomolecules. Although the biophysics behind such flow effect is yet to be understood, this work will help speeding up the development of protocols for development and implementation of microfluidic immunoassay technology for high-performance point-of-care (POC) testing.

Mesial temporal tau in amyloid-\u03b2-negative cognitively normal older persons

BackgroundTau deposition in the mesial temporal lobe (MTL) in the absence of amyloid-β (Aβ−) occurs with aging. The tau PET tracer 18F-MK6240 has low non-specific background binding so is well suited to exploration of early-stage tau deposition. The aim of this study was to investigate the associations between MTL tau, age, hippocampal volume (HV), cognition, and neocortical tau in Aβ− cognitively unimpaired (CU) individuals.MethodsOne hundred and ninety-nine Aβ− participants (Centiloid < 25) who were CU underwent 18F-MK6240 PET at age 75 ± 5.2 years. Tau standardized uptake value ratio (SUVR) was estimated in mesial temporal (Me), temporoparietal (Te), and rest of the neocortex (R) regions and four Me sub-regions. Tau SUVR were analyzed as continuous variables and compared between high and low MTL SUVR groups.ResultsIn this cohort with a stable clinical classification of CU for a mean of 5.3 years prior to and at the time of tau PET, MTL tau was visually observed in 9% of the participants and was limited to Braak stages I–II. MTL tau was correlated with age (r = 0.24, p < 0.001). Age contributed to the variance in cognitive scores but MTL tau did not. MTL tau was not greater with subjective memory complaint, nor was there a correlation between MTL tau and Aβ Centiloid value, but high tau was associated with smaller HV. Participants with MTL tau had higher tau SUVR in the neocortex but this was driven by the cerebellar reference region and was not present when using white matter normalization.ConclusionsIn an Aβ− CU cohort, tau tracer binding in the mesial temporal lobe was age-related and associated with smaller hippocampi, but not with subjective or objective cognitive impairment.

A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting.

Cell-free nucleic acids (cfNAs) such as short non-coding microRNA (miRNA) and circulating tumor DNA (ctDNA) that reside in bodily fluids have emerged as potential cancer biomarkers. Methods for the rapid, highly specific, and sensitive monitoring of cfNAs in biofluids have, therefore, become increasingly attractive as clinical diagnosis tools. As a next generation technology, we provide a practical guide for an amplification-free, single molecule Förster resonance energy transfer (smFRET)-based kinetic fingerprinting approach termed intramolecular single molecule recognition through equilibrium Poisson sampling, or iSiMREPS, for the rapid detection and counting of miRNA and mutant ctDNA with virtually unlimited specificity and single molecule sensitivity. iSiMREPS utilizes a pair of fluorescent detection probes, wherein one probe immobilizes the target molecules on the surface, and the other probe transiently and reversibly binds to the target to generate characteristic time-resolved fingerprints as smFRET signal that are detected in a total internal reflection fluorescence microscope. Analysis of these kinetic fingerprints enables near-perfect discrimination between specific binding to target molecules and nonspecific background binding. By accelerating kinetic fingerprinting using the denaturant formamide and reducing background signals by removing target-less probes from the surface via toehold-mediated strand displacement, iSiMREPS has been demonstrated to count miR-141 and EGFR exon 19 deletion ctDNA molecules with a limit of detection (LOD) of ~1 and 3 fM, respectively, as well as mutant allele fractions as low as 0.0001%, during a standard acquisition time of only ~10s per field of view. In this review, we provide a detailed roadmap for implementing iSiMREPS more broadly in research and clinical diagnostics, combining rapid analysis, high specificity, and high sensitivity.

Acquisition count dependence of the specific binding ratio in 123I-FP-CIT SPECT.

In the [123I]FP-CIT single-photon emission computed tomography (SPECT) examination, the specific binding ratio (SBR), calculated from the ratio of the striatal specific to extra-striatal background non-specific binding in the brain, is now commonly used as a quantitative index of parkinsonian syndrome. The purpose of this study was to examine the influence of count reduction on the SBR and to clarify the reliability of SBR values in patients with shorter scan times. A striatum phantom was used in a phantom study, with the radioactivity concentration adjusted so that the right striatum:left striatum:brain parenchyma ratio was 8:4:1. Changes in SBR values and image quality, expressed as the % coefficient of variation (%CV) and normalized mean squared error (NMSE), with decreasing acquisition counts were evaluated. In the clinical study, 106 patients (73.1 ± 9.6years) with suspected parkinsonian syndrome underwent [123I]FP-CIT SPECT, and SBR values from normal 30min acquisitions (fullSBR) and half-count acquisitions (halfSBR) were compared. SBR values were calculated using the Tossici-Bolt (SBRTB) and a fully automatic count-based (SBRcb) methods. In the phantom study, image quality decreased with a reduction of acquisition counts. The %CV and NMSE decreased by up to 52.5% and 81.5%, respectively. SBR values decreased slightly as acquisition counts decreased. In the clinical study, the mean values of halfSBR were lower than those of fullSBR, and they were significantly different except for SBRTB without attenuation correction. halfSBR and fullSBR values correlated well, with halfSBR values 1-8% lower than fullSBR. The accuracy of diagnosis did not decrease even after acquisition counts were reduced by half. This study demonstrated that SBR values decrease as a function of reduced acquisition counts. Since halfSBR and fullSBR showed excellent correlation, it is suggested that fullSBR can be estimated from halfSBR using a calibration formula when scan times are reduced.

A zwitterionic near-infrared fluorophore for real-time ureter identification during laparoscopic abdominopelvic surgery

Iatrogenic injury of the ureters is a feared complication of abdominal surgery. Zwitterionic near-infrared fluorophores are molecules with geometrically-balanced, electrically-neutral surface charge, which leads to renal-exclusive clearance and ultralow non-specific background binding. Such molecules could solve the ureter mapping problem by providing real-time anatomic and functional imaging, even through intact peritoneum. Here we present the first-in-human experience of this chemical class, as well as the efficacy study in patients undergoing laparoscopic abdominopelvic surgery. The zwitterionic near-infrared fluorophore ZW800-1 is safe, has pharmacokinetic properties consistent with an ideal blood pool agent, and rapid elimination into urine after a single low-dose intravenous injection. Visualization of structure and function of the ureters starts within minutes after ZW800-1 injection and lasts several hours. Zwitterionic near-infrared fluorophores add value during laparoscopic abdominopelvic surgeries and could potentially decrease iatrogenic urethral injury. Moreover, ZW800-1 is engineered for one-step covalent conjugatability, creating possibilities for developing novel targeted ligands.

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.

Effect of the nonspecific binding in differential impedance biosensing.

The detection limits of impedance biosensors are dictated by the presence of background nonspecific binding, yet almost all the detection limits reported in the literature are determined using a clean buffer solution without confronting this real challenge. In this work, the authors employed the simplest "differential" impedance biosensor, composed of poly-l-lysine-polyethylene glycol-biotin-coated gold electrodes for the detection of streptavidin in the presence of 0.1% fetal calf serum, and studied the effect of the nonspecific binding on the performance of the differential impedance biosensing. The lowest streptavidin concentration detected by the system (5 μg/ml) was 1 order of magnitude higher (worse) than that from a previously demonstrated impedance biosensor where avidin was detected in the absence of background proteins. Interestingly, the origin of the differential signal was not due to the electrochemical properties of streptavidin itself but was that of the serum, where the coverage of the electrode by streptavidin indirectly modulated the electrical signal by suppressing the accessibility of the serum to the electrode.

Generation of iPSC-Derived Human Megakaryocytes for Flow Cytometric Detection of Platelet-Specific Alloantibodies

Antibodies that form against human platelet alloantigens (HPAs) are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, and multitransfusion platelet refractoriness. Some of HPAs are relatively rare in the population, and difficult to obtain for purposes of transfusion therapy and diagnostic testing. In addition, HPA alloantisera often contain antibodies against human leucocyte antigen (HLA) class I, thereby limiting antibody detection to glycoprotein (GP)-specific assays such as the modified antigen capture enzyme-linked immunosorbent assay (MACE) and the monoclonal immobilization of platelet antigen (MAIPA), which are tedious and require solubilization of platelet GPs that may cause the loss of epitopes. In this study we aimed to generate gene-edited, HPA-specific, megakaryocytes (MKs) derived from human induced pluripotent stem cells (iPSCs) that could be used for simple flow cytometric detection of specific HPA alloantibodies present in patient sera. The HPA-3a/HPA-3b alloantigen system, also known as Baka/Bakb, is caused by a single T13809G nucleotide substitution in the ITGA2B gene, resulting in an Ile874Ser amino acid polymorphism near the C terminus of the integrin αIIb subunit (GPIIb). Here we targeted HPA-3 system because alloantibodies targeting HPA-3 are often hard to detect with current detection methods, in part due to the requirement for cell type-specific glycosylation. To prevent interference of anti-A or anti-B antibodies in patient sera, a blood type O iPSC line (OT1-1) was generated from human peripheral blood mononuclear cells derived from a healthy donor using integration-free episomal vectors. The gene for β2 microglobulin (B2M) was first ablated from the OT1-1 iPS cell line using CRISPR/Cas9 to prevent binding of HLA class I alloantibodies. The resulting B2M knockout (B2MKO) cells were then additionally gene edited to convert the endogenous HPA-3a alloantigenic epitope present on B2MKO OT1-1 cells to HPA-3b. Two different guide RNAs targeting sequences that flank exon 26 of the ITGA2B gene were designed such that the entire exon harboring the HPA-3 polymorphic site was removed. A plasmid harboring a template replacing exon 26 with the G13809 mutation, flanked by 600 bp homology arms, was cotransfected into the B2MKO OT1-1 iPSCs together with the two CRISPR/Cas9 guide RNA constructs. iPSC clones containing the desired targeted T13809G mutation were identified by a diagnostic MfeI digestion specific for the G13089-bearing HPA-3b allele. Sequence analysis confirmed conversion of T13089 to G in these HPA-3b clones. Flow cytometric analysis showed the HPA-3a iPSCs, when differentiated into CD41+/CD42b+ MKs, specifically reacted with HPA-3a, but not HPA-3b, patient sera, while the HPA-3b iPSC-derived MKs lost reactivity with HPA-3a patient serum, and gained the reactivity with HPA-3b patient sera. Taken together, we have established genetically modified iPSC-derived MKs expressing specific HPAs that are suitable for simple flow cytometry-based detection of HPA alloantibodies in patient sera, with low non-specific background binding. This system provides intact antigens on the cell surface with carbohydrate moieties that likely mimic those found on human platelets, thus facilitating the detection of HPA alloantibodies that are normally hard to detect with current methods. Application of this strategy to genetically edit this and other clinically-important HPAs holds great potential for producing Designer Platelets for diagnostic, investigative and ultimately therapeutic use. DisclosuresNo relevant conflicts of interest to declare.

ImmunoCAP cellulose displays cross-reactive carbohydrate determinant (CCD) epitopes and can cause false-positive test results in patients with high anti-CCD IgE antibody levels

Cross-reactive carbohydrate determinants (CCDs) in plants and insect venoms are a common cause of irrelevant positive test results during invitro allergy diagnosis. We observed that some CCD-positive sera show nonspecific IgE binding even with CCD-free recombinant allergens when using the Phadia ImmunoCAP platform. We investigated whether cellulose used as an allergen carrier in ImmunoCAP harbors residual N-glycans, causing nonspecific background binding in CCD-positive sera. IgE binding to 6 samples of blank ImmunoCAPs coupled to either streptavidin (SA-CAP-1 or 2) or nonallergenic maltose-binding protein (MBP; MBP-CAP-1 to 4) and binding to a panel of 4 recombinant allergens were compared in CCD-positive sera before and after inhibition with a CCD inhibitor (MUXF3-human serum albumin). Of 52 CCD-positive sera (bromelain, 1.01-59.6 kilounits of antigen per liter [kUA/L]) tested on SA-CAP-1, 35 (67%) showed IgE binding of greater than 0.35 kUA/L (0.41-4.22 kUA/L). Among those with anti-CCD IgE levels of greater than 7.0 kUA/L, 90% (26/29) were positive. IgE binding to SA-CAP-1 correlated with IgE binding to bromelain (r=0.68) and was completely abolished by serum preincubation with the CCD inhibitor (n=15). Binding scores with SA-CAP-2 and MBP-CAP-1 to MBP-CAP-4 were generally lower but strongly correlated with those of SA-CAP-1 and bromelain. IgE reactivity of 10 CCD-positive sera (14.0-52.5 kUA/L) with the recombinant allergens rPhl p 12, rFel d 1, rAra h 2, and rPru p 3 was positive to at least 1 allergen in 8 of 10 (0.36-1.63 kUA/L) and borderline in 2 of 10 (0.21-0.25 kUA/L). Binding correlated with antibody binding to bromelain (r=0.61) and to all blank ImmunoCAPs (r>0.90) and could be completely blocked by the CCD inhibitor. Overall, mean background binding to cellulose CCDs corresponded to 2% to 3% of the reactivity seen with bromelain. Cellulose used as a solid-phase allergen carrier can contain varying amounts of CCDs sufficient to cause false-positive test results up to 2 kUA/L with nonglycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

Abstract B02: Complex molecular-scale dynamics of HER2 receptor transport in live SKBR3 breast cancer cells revealed by nanoscale quantum dot imaging

Abstract The dynamic behavior of proteins in the spatial and temporal context of a cell is critical for dictating downstream signaling and subsequent cellular effects. Human epidermal growth factor receptor 2 (HER2) is a ligand-less receptor tyrosine kinase whose heterodimerization with other HER receptors (EGFR, HER3, HER4) activates potent downstream signaling to modulate cell proliferation and survival. HER2 represents a major therapeutic target as its overexpression is prevalent in a major subset of invasive breast cancers. Yet while current understanding of HER2 signaling entails a broad sequence of signaling events, the spatiotemporal dynamics of how these signaling mechanisms are executed remains undefined. Here, we describe the molecular-scale motions that underlie HER2 signaling behavior in live cells using a bright and photostable anti-HER2 quantum dot (QD) probe designed and validated to track the receptor for long durations with high spatiotemporal resolution. Methods: HER2 was detected using an anti-HER2 affibody that was fused to a fluorogen activating peptide (FAP) which bound with high affinity to a fluorogen (Malachite Green)-QD complex. HER2-QDs were imaged in live unstimulated and stimulated human SKBR3 breast cancer cells. Cells were serum-starved overnight and: 1) received no treatment during the course of imaging (unstimulated) or 2) were chronically treated with the HER3 and HER4 specific ligand, heregulin-beta1 (HRG) during the course of imaging (stimulated). HRG was administered globally across all cells or locally to a specific cellular region. Trajectory information was analyzed using custom single particle tracking MATLAB software to measure key biophysical parameters. Results: The anti-HER2-QD probe showed robust and increased signal-to-noise compared to conventional organic dyes. Validation was measured by sensitive quantitation of single probes in low and high HER2-expressing cell lines with background non-specific binding of less than five probes per cell. HER2 motions were complex and composed of a heterogeneous composite of diffusive, constrained, and stationary motions. These motions were observed in the trajectories of HER2 receptors traveling along cellular protrusions, cell body, and along protrusions between adjacent cells. HRG stimulation produced morphological changes (e.g. membrane ruffling, protrusion growth) as well as changes in HER2 dynamics. HRG-stimulated cells demonstrated an increase in HER2 mobility (as measured by diffusion constant), increased speed (as measured by instantaneous velocity), and wider exploratory area (as measured by maximum trajectory distance) than in unstimulated cells. Interestingly, increased receptor mobility did not correlate strongly with a wider exploratory area, and more mobile receptors showed more constrained motion. Conclusions: In contrast to the simple diffusive motions previously reported for members of major classes of receptors (e.g. GPCRs, tyrosine kinase receptors), our sensitive measurements of HER2 receptor motion demonstrate complex molecular dynamics in both unstimulated and stimulated cells. Ligand stimulation induces changes in cell morphology such as membrane ruffling and protrusion growth that accompany changes in HER2 mobility and range of motion. The diversity of receptor dynamics suggest a heterogeneous environment in which HER2 engages. Future experiments include understanding the dynamic interplay between HER2 with the cytoskeleton and downstream signaling with HER2-targetting therapies using real-time measurements of HER2 molecular motions. Citation Format: Wai Yan Lam, Yi Wang, Mark J. Olah, Keith A. Lidke, Marcel Bruchez, Joe Gray, Tania Q. Vu. Complex molecular-scale dynamics of HER2 receptor transport in live SKBR3 breast cancer cells revealed by nanoscale quantum dot imaging. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B02.

P040 : THREE METHODS TO IDENTIFY CYTOTOXIC ANTIBODIES; ONE METHOD IS NOT ENOUGH

Background Donor HLA specific complement dependent cytotoxic (CDC) antibodies (abs) have been associated with hyperacute rejection and are a detriment to solid organ transplantation. The Luminex single antigen (LSA)-C1q binding assay has been introduced to detect CDC+ abs. In a study of 190 patients (pts) sera tested by CDC and LSA-C1q, LSA-C1q was more sensitive than CDC in detecting 85% of class I and 75% of class II abs. 15–25% of abs detected by CDC were negative by the LSA-C1q assay. Aim: The aim of our study was to determine the appropriate methods to identify unacceptable antigens (UAs) for the virtual crossmatch by comparing the ability of a third assay, LSA-C3d binding, to detect CDC+ abs not detected by LSA-C1q. Methods Sera from 25 pts from the original cohort of 190 pts with positive LSA, CDC and negative C1q assay results were tested by the LSA-C3d assay. Results In 59% of the cases, the results of LSA-C3d were also negative and consistent with C1q data. 19% of the abs detected by CDC that were LSA-C1q neg were LSA-C3d pos. 9% of the abs detected by CDC were C1q pos and C3d neg. 13% of the abs showed no correlative patterns for CDC, LSA-C1q and LSA-C3d. The LSA-C3d assay appears to give false pos results for class I abs to rare alleles and HLA-C locus antigens. Both solid phase assays have non-specific background binding issues for some sera that make interpretation difficult. Conclusions In most cases LSA-C1q and LSA-C3d results are consistent with each other and identify more cytotoxic abs than the CDC assay. Although both solid phase assays are more sensitive than CDC in detecting the majority of cytotoxic pos abs. In 15–20% of the cases, the CDC assay is required to resolve the discrepancies and high background issues. A systematic analysis of LSA, CDC, LSA-C1q, and LSA-C3d results is needed to identify clinically relevant abs for the assignment of UAS.

Improving the biodistribution of PSMA-targeting tracers with a highly negatively charged linker

Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer and in tumor vasculature. Small molecule based inhibitors of PSMA have promised to provide sensitive detection of primary and metastatic prostate tumors. Although significant progress has been made, many of the radiolabeled imaging agents exhibit non-specific background binding. Prevailing tracer designs focus on high affinity urea-based inhibitors with strategically placed hydrophobic patches that interact favorably with the substrate tunnel of PSMA. We hypothesized that a novel PSMA inhibitor design incorporating highly negatively charged linkers may minimize non-specific binding and decrease overall background. Through iterative redesign, we generated a series of PSMA inhibitors with highly negatively charged linkers that connect to urea inhibitors and bulky radionuclide chelates. We then performed in vivo imaging and biodistribution studies with the radiolabeled tracers. The tracers derived from our iterative redesign have affinities for PSMA comparable to the "parent" urea ligand Cys-C(O)-Glu. Using a fluorine-18 labeled PSMA targeting tracer, we found that these highly negatively charged molecules exhibit rapid renal excretion with minimal non-specific binding. The biodistribution data at 2 hr showed 4.6%ID/g PC3-PIP tumor uptake with spleen, liver, bone, and blood background levels of 0.1%, 0.17%, 0.1%, and 0.04%, respectively. Placement of multiple negative charges in the linker region of PSMA tracers significantly reduced the non-specific background binding without significant reduction of binding affinity. This increased tumor/background contrast in positron emission tomography promises to provide more sensitive tumor detection while decreasing the overall radiation exposure to patients.

Absolute quantification of protein copy number using a single-molecule-sensitive microarray

We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of <30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6 × 10(6) molecules of analyte in the analysis chamber and a dynamic range of >5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of >3 orders of magnitude. We show that these protocols can be used to calibrate data retrospectively to determine the absolute protein copy number at the single cell level in two human cancer cell lines.

High-Throughput and High-Intensive Biosensor Microarray Fabrication by Selective Dewetting on a Wettability Controlled Substrate

In a microarray biosensor, the surface characteristic for reducing background nonspecific binding is a critical aspect for achieving the high sensitivity and accurate detection. In this paper, we proposed a new high-intensity and reduced nonspecific binding microarray fabrication method with a selective dewetting process on a mixed self-assembled monolayer (SAM), consisting of methyl-terminated and methoxy-[poly(ethylene glycol) (PEG)]-terminated silanes. The combination of hydrophobic and hydrophilic properties of the mixed SAM background layer provided the hydrophobicity that allowed the selective dewetting of the protein solution at specific versus nonspecific regions and reduced the nonspecific binding owing to hydrated PEG chains in the mixed SAM. The wiping process was applied for obtaining high throughput and reducing protein usage. Finally, the background non-specific binding was significantly reduced by 78% with a selective dewetting method compared with the standard method using a cover slip. Furthermore, the peak intensity was increased by 20% and the protein usage was reduced versus the standard method.

Influence of Target Concentration and Background Binding on In Vitro Selection of Affinity Reagents

Nucleic acid-based aptamers possess many useful features that make them a promising alternative to antibodies and other affinity reagents, including well-established chemical synthesis, reversible folding, thermal stability and low cost. However, the selection process typically used to generate aptamers (SELEX) often requires significant resources and can fail to yield aptamers with sufficient affinity and specificity. A number of seminal theoretical models and numerical simulations have been reported in the literature offering insights into experimental factors that govern the effectiveness of the selection process. Though useful, these previous models have not considered the full spectrum of experimental factors or the potential impact of tuning these parameters at each round over the course of a multi-round selection process. We have developed an improved mathematical model to address this important question, and report that both target concentration and the degree of non-specific background binding are critical determinants of SELEX efficiency. Although smaller target concentrations should theoretically offer superior selection outcome, we show that the level of background binding dramatically affect the target concentration that will yield maximum enrichment at each round of selection. Thus, our model enables experimentalists to determine appropriate target concentrations as a means for protocol optimization. Finally, we perform a comparative analysis of two different selection methods over multiple rounds of selection, and show that methods with inherently lower background binding offer dramatic advantages in selection efficiency.

A Highly Conserved Cytoplasmic Cysteine Residue in the α4 Nicotinic Acetylcholine Receptor Is Palmitoylated and Regulates Protein Expression

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.

Identification and Characterization of Unique Proline-rich Peptides Binding to the Mitochondrial Fission Protein hFis1

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six alpha helices [alpha1-alpha6] out of which [alpha2-alpha5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the alpha1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[alpha2-alpha6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the alpha2 and alpha3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the alpha1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of alpha1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.

Rewritable Glycochips

We describe microarraying of carbohydrates for protein screening using either disulfide bridge or Schiff base imine immobilization chemistries on plasmachemical deposited functional nanolayers. The commonly observed issue of nonspecific background binding of proteins is overcome by spotting carbohydrates through a protein-resistant overlayer yielding spatially localized interaction with a reactive functional underlayer.

AZD2184: a radioligand for sensitive detection of β‐amyloid deposits

The presence of beta-amyloid plaques in brain is a hallmark of Alzheimer's disease (AD) and serves as a biomarker for confirmation of diagnosis postmortem. Positron emission tomography (PET) radioligands such as Pittsburgh compound B ([(11)C]-2-(3-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol) (PIB) binds selectively to beta-amyloid and are promising new tools supporting the clinical diagnoses of AD. In addition, such methodology may be useful for evaluation of new drugs aiming at reduction of amyloid plaque load. The objective of this study is to develop a new amyloid selective PET radioligand with higher signal-to-background ratio when compared with existing amyloid PET ligands. The lead compound, AZD2184, (2-[6-(methylamino)pyridin-3-yl]-1,3-benzothiazol-6-ol) was found to have high affinity for amyloid fibrils in vitro (K(d): 8.4 +/- 1.0 nM). Two minutes after i.v. administration in rats, about 1% of the dose was in brain. In vitro autoradiography on cortical brain sections from amyloid-beta precursor protein/presenilin 1 (APP/PS1) mice and AD patients showed that while [(3)H]AZD2184 and [(3)H]PIB are mutually displaceable, [(3)H]AZD2184 displays a higher signal-to-background ratio primarily by virtue of lower background binding levels. The ratio of binding ability in prefrontal cortex (high plaque load) to subcortical white matter (background) was 4.5 for [(3)H]AZD2184 and 0.8 for [(3)H]PIB at 1 nM. In adjacent cortical sections from APP/PS1 mouse as well as from AD cortical tissue, [(3)H]AZD2184 and antibodies to human beta-amyloid labeled identical structures. In vivo administration of [(3)H]AZD2184 to APP/PS1 mice further showed that [(3)H]AZD2184 labels amyloid deposits with low non-specific background binding. Taken together, the pre-clinical profile of AZD2184 in relation to the reference ligand PIB, suggests that (11)C-labeled AZD2184 is a potential radioligand for PET-visualization of beta-amyloid deposits in the living human brain.