Biomedical subjects

Larry A Sklar

Publications and source records attributed to Larry A Sklar.

8 recordsLinked to original sources

PubMed2002

Biomolecular recognition on well-characterized beads packed in microfluidic channels.

We describe a new approach for the analysis of biomolecular recognition in microfluidic channels. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer (FRET). We establish a model system that detects the FLAG epitope. The assay can potentially detect subfemtomole quantities of antibody with a high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter-to-submicroliter volumes of analyte fluid. Kinetic and equilibrium constants for the reaction of this receptor-ligand pair are obtained through modeling of kinetic responses of the affinity microcolumn and are consistent with those obtained by flow cytometry. Because of the correlation between kinetic and equilibrium data obtained for the microcolumns, quantitative analysis can be done prior to the steady-state end point of the recognition reaction. This method has the promise of combining the utility of affinity chromatography with the advantage of direct, quantitative, and real-time analysis and the cost-effectiveness of microanalytical devices. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes and molecular assembly and microsystem-based multianalyte determinations.

Antibodies, Monoclonal↗

PubMed2002

Nozzle design parameters and their effects on rapid sample delivery in flow cytometry.

BACKGROUND: Rapid kinetic and high throughput flow cytometry are emerging as valuable tools in biotechnology research applications ranging from mechanistic analysis of molecular assemblies to high throughput screening. Many of these new applications have been made possible by improved sample delivery capabilities, focusing increased attention on fluidic issues associated with rapid sample delivery. METHODS: Using basic fluidic premises, we derived a model that predicted the effect of nozzle parameters during rapid sample delivery. We tested the model using the rapid mix flow cytometer and modifications were made to the equipment to optimize performance. RESULTS: The model predicted that shorter nozzles with wide exit orifices decrease the delay before initial particle analysis and the fluidic stabilization time. Experimental results confirmed this prediction and model-based modifications allowed analysis of particles within 55 ms or 600 ms after mixing, with or without electronic gating, respectively. CONCLUSIONS: The model along with modifications to commercial equipment will allow rapid mix flow cytometry to analyze reactions in time frames threefold shorter than previously possible. The model allows for nozzle design predictions that should allow for analysis in the millisecond time frame. Furthermore, these findings are general for all rapid delivery applications, including high throughput flow cytometry.

Automation↗

PubMed2002

Arrestin variants display differential binding characteristics for the phosphorylated N-formyl peptide receptor carboxyl terminus.

The phosphorylation-dependent binding of arrestins to cytoplasmic domains of G protein-coupled receptors (GPCRs) is thought to be a crucial step in receptor desensitization. In some GPCR systems, arrestins have also been demonstrated to be involved in receptor internalization, resensitization, and the activation of signaling cascades. The objective of the current study was to examine binding interactions of members of the arrestin family with the formyl peptide receptor (FPR), a member of the GPCR family of receptors. Peptides representing the unphosphorylated and phosphorylated carboxyl terminus of the FPR were synthesized and bound to polystyrene beads via a biotin/streptavidin interaction. Using fluorescein-conjugated arrestins, binding interactions between arrestins and the bead-bound FPR carboxyl terminus were analyzed by flow cytometry. Arrestin-2 and arrestin-3 bound to the FPR carboxyl-terminal peptide in a phosphorylation-dependent manner, with K(d) values in the micromolar range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin-2-(1--382) and arrestin-3-(1--393), truncated mutant forms of arrestin that display phosphorylation-independent binding to intact receptors, were also observed to bind the bead-bound FPR terminus in a phosphorylation-dependent manner, but with much greater affinity than the full-length arrestins, yielding K(d) values in the 5--50 nm range. Two additional arrestin mutants, which are full-length but display phosphorylation-independent binding to intact GPCRs, were evaluated for their binding affinity to the FPR carboxyl terminus. Whereas the single point mutant, arrestin-2 R169E, displayed an affinity similar to that of the full-length arrestins, the triple point mutant, arrestin-2 I386A/V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxyl terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.

Amino Acid Sequence↗

PubMed2002

A quantitative approach for studying IgE-FcepsilonRI aggregation.

Aggregation of cell surface receptors is a ubiquitous means of initiating signal transduction in many cellular systems. In this manuscript, we describe a combined theoretical and experimental approach based on multiparameter flow cytometry for measuring the time course of ligand induced aggregation of IgE-FcepsilonRI on RBL cells. By fluorescently labeling both the ligand and surface IgE (sIgE), we have developed an assay that permits us to simultaneously measure both occupancy of sIgE combining sites and association of antigen with the cell surface. This allows for a direct calculation of the degree of receptor aggregation present on the cell. By employing new mixing technologies developed for flow cytometry, we are able to look at aggregation in the sub second time domain. To extend our work, we have synthesized a new set of chemically well defined ligands (of valences 1-3) to use as probes in our studies. We show that the magnitude of the cellular response is dramatically increased as the valence of our ligand is raised from two to three.

Animals↗

PubMed2002

Suspension array technology: evolution of the flat-array paradigm.

Suspension arrays of microspheres analyzed using flow cytometry offer a new approach to multiplexed assays for large-scale screening applications. By optically encoding micron-sized polymer particles, suspension microarrays can be created to enable highly multiplexed analysis of complex samples. Each element in the array is comprised of a subpopulation of particles with distinct optical properties and each array element bears a different surface receptor. Nucleic acids, proteins, lipids or carbohydrates can serve as receptors to support the analysis of a wide range of biomolecular assemblies, and applications in genomic and proteomic research are being developed. Coupled with recent innovations for rapid serial analysis of samples, molecular analysis with microsphere arrays holds significant potential as a general analysis platform for both research and clinical applications.

Directed Molecular Evolution↗

PubMed2002

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.

Biosensing Techniques↗

PubMed2002

Regulation of human basophil adhesion to endothelium under flow conditions: Different very late antigen 4 regulation on umbilical cord blood-derived and peripheral blood basophils.

BACKGROUND: Although soluble mediators released by basophils in tissue sites contribute to the chronic injury that occurs in hypersensitivity diseases, only limited information is available about how circulating basophils are recruited to tissues. In particular, the interaction of basophils with endothelium under conditions that mimic physiologic flow has not been explored. OBJECTIVE: We sought to identify adhesion molecules regulating the attachment of human basophils to IL-4-activated human umbilical vein endothelial cells (HUVECs) under flow conditions. METHODS: A parallel-plate flow chamber and blocking mAbs were used to define the adhesion molecules involved in the interactions of peripheral blood basophils (PBBs) and cord blood-derived basophils (CBDBs) with IL-4-activated HUVECs and with Chinese hamster ovary (CHO) cell transfectants expressing specific adhesion molecules. A fluorescent ligand specific for very late antigen 4 (VLA-4) was used to directly examine the VLA-4 affinity state of basophils. RESULTS: Flowing PBBs and CBDBs attached to activated HUVECs and to CHO cells expressing P- or E-selectin. However, only CBDBs attached to vascular cell adhesion molecule 1 (VCAM-1)-transfected CHO cells under flow conditions. The attachment of CBDBs to CHO cells was blocked by mAbs directed against E-selectin, P-selectin, and VCAM-1, whereas attachment of PBBs was blocked by E-selectin and P-selectin mAbs. Activating VLA-4 with Mn(2+) on PBBs resulted in adhesion to the VCAM-1-transfected CHO cells, indicating that VLA-4 activity on PBBs can be regulated, at least in part, through affinity changes. The Mn(2+)-induced upregulation of basophil VLA-4 affinity was demonstrated directly by using a fluorescent ligand for VLA-4 and flow cytometry. CONCLUSIONS: The interaction of human CBDBs and PBBs with endothelium under flow conditions is mediated in part by both P- and E-selectin. VLA-4 additionally contributes to the adhesion of flowing CBDBs. However, the affinity of VLA-4 is too low to support the adhesion under flow conditions of unstimulated PBBs.

Animals↗

PubMed2001

Flow cytometric analysis of ligand-receptor interactions and molecular assemblies.

Flow cytometers make homogeneous real-time measurements of ligand-receptor interactions and, simultaneously, the physiological responses of cells. Their multiparameter capabilities are also useful in resolving multicomponent assemblies or in developing multiplexed assays. Recent advances suggest that these approaches can be extended in several important ways. Sample delivery in the millisecond time domain is applicable to the analysis of complex binding kinetics and reaction mechanisms. The homogeneous discrimination of free components and particle-based assemblies can be extended into the micromolar concentration range. Measurements can be made of molecular assemblies among proteins, DNA, RNA, lipids, and carbohydrates on beads. The topography and assembly of components within cells can be evaluated with resonance energy transfer. Temperature dependence can be evaluated with Peltier temperature control. Many assembly endpoints can be assessed through new tools for high-throughput flow cytometry using plate-based assay formats and small volume samples.

Animals↗