Joseph P. Skinner

Using nonfluorescent Förster resonance energy transfer acceptors in protein binding studies.

The purpose of this article is to highlight the versatility of nonfluorescent Förster resonance energy transfer (FRET) acceptors in determination of protein equilibrium dissociation constants and kinetic rates. Using a nonfluorescent acceptor eliminates the necessity to spectrally isolate the donor fluorescence when performing binding titrations covering a broad range of reagent concentrations. Moreover, random distribution of the donor and acceptor chromophores on the surface of proteins increases the probability of FRET occurring on their interaction. Three high-affinity antibodies are presented in this study as characteristic protein systems. Monoclonal antibody (mAb) 106.3 binds brain natriuretic peptide (BNP)5-13(C10A) and full-length BNP1-32 with the dissociation constants 0.26+/-0.01 and 0.05+/-0.02 nM, respectively, which was confirmed by kinetic measurements. For anti-hCG (human chorionic gonadotropin) mAb 8F11, studied at two incorporation ratios (IRs=1.9 and 3.8) of the nonfluorescent FRET acceptor, K(D) values of 0.04+/-0.02 and 0.059(-0.004)(+0.006) nM, respectively, were obtained. Likewise, the binding of goat anti-hamster immunoglobulin G (IgG) antibody was not affected by conjugation and yielded K(D) values of 1.26+/-0.04, 1.25+/-0.05, and 1.14+/-0.04 nM at IRs of 1.7, 4.7, and 8.1, respectively. We conclude that this FRET-based method offers high sensitivity, practical simplicity, and versatility in protein binding studies.

Affinity assisted selection of antibodies for Point of Care TSH immunoassay with limited wash.

BACKGROUND Molecular binding characteristics of several thyroid stimulating hormone (TSH) antibodies were determined for the TSH antigen, along with its closely related endogenous interfering hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). METHODS This data was compared to the same antibodies used in the low wash sandwich ELISA immunoassay system, the Point of Care i-STAT® immunoassay. From this information we developed binding criteria useful in the low wash i-STAT® immunoassay to permit good signal generation from TSH and low cross-reactivity from its interfering hormones. For the TSH Assay we have developed characteristics that enable antibody selection in the i-STAT® immunoassay cartridge. Our antibody screening approach used a dot blot approach as a first screen to select for the most useful antibodies. We then compared a FRET (Förster Resonance Energy Transfer) and electrochemical cartridge approach to determine the appropriate antibody combinations. RESULTS Both methods generated similar data, but the FRET method was not capable of differentiating the antibody with the best characteristics as a capture antibody or a detection conjugate in a sandwich ELISA assay. Finally, we performed binding characterizations of the antibodies using each of the above mentioned glycoproteins. CONCLUSIONS We found that we need sub-picomolar detection of TSH, and at least 100 fold or higher values for the cross-reacting species.

Determining Antibody Stoichiometry using Time-Integrated Fluorescence Cumulant Analysis

We applied fluorescence fluctuation spectroscopy to resolve the binding heterogeneity of fluorescently labeled ligand derived from brain natriuretic peptide (BNP), a widely used diagnostic marker of heart failure, to a corresponding monoclonal antibody. This system includes three species: (1) free ligand molecules, (2) antibody with a single site occupied, and (3) antibody with both sites occupied. The method we used, time-integrated fluorescence cumulant analysis (TIFCA), utilizes cumulants of fluorescence fluctuations to resolve subpopulations of multiple fluorescent species freely diffusing in a solution. The values of the cumulants depend on the concentration, molecular brightness and diffusion time of the fluorescent molecules. The number of molecules in each species reflects the antibody affinity. We apply TIFCA to successfully establish the stoichiometry of the system, estimate affinity, and identify the presence of an inactive fraction of antigen in a single titration experiment.

Rapid single-molecule imaging in cyclic olefin copolymer channels

Rapid preparation of high quality capture surfaces is a major challenge for surface‐based single‐molecule protein binding assays. Here we introduce a simple method to activate microfluidic chambers made from cyclic olefin copolymer for single‐molecule imaging with total internal reflection fluorescence microscopy. We describe a surface coating protocol and demonstrate single‐molecule imaging in off‐the‐shelf microfluidic parts that can be activated for binding assays within a few minutes. As the first example, biotinylated protein directly captured on the neutravidin‐coated surface was detected using fluorescently labeled antibody. We then showed detection of a fusion construct containing green fluorescence protein and verified its single fluorophore behavior by observing stepwise photobleaching events. Finally, a target protein was identified in the crude cell lysate using antibody–sandwich complex formation. In all experiments, controls were completed to ensure that nonspecific binding to the surface was minimal. Based on our results, we conclude that the simple surface preparation described in this paper enables single‐molecule imaging assays without time‐consuming coating procedures. Microsc. Res. Tech. 78:309–316, 2015.

Characterization of Fluorescent 3DNA Dendrimers with Fcs and Single Molecule Imaging

3DNA dendrimers are uniform three-dimensional DNA structures commercially available from Genisphere, Hatfield, PA. The base unit is made by two strands of DNA that are complementary only in the central region, while the four end regions can be annealed with other base units of different sequences. Thus, dendrimers can grow in layers with finite control. At two layers, the dendrimer has 36 free arms which are available for attaching multiple labels of interest. In our study, eight arm extensions were incorporated into the dendrimer. Each of these arms is capable of annealing to three independent short oligomers labeled with a fluorophore. Using fluorescence correlation spectroscopy, we monitor changes of the dendrimer brightness and calculate the number of the labeled oligonucleotides incorporated into the dendrimers in a titration experiment. We confirmed that the incorporation of the labeled oligomers into the dendrimer is close to the expected value and the fluorescence of the labeled nucleotides is not quenched. Single molecule images of the dendrimers show high homogeneity and multiple bleaching steps. Therefore, 3DNA dendrimers can serve as good scaffolds for advanced fluorescence imaging.

Thermodynamically Driven Blinking for Super-Resolution Microscopy

Super-resolution microscopy enables imaging of structures smaller than the diffraction limit, defined by Abbes law. Over the last years, a continuously increasing toolset of physical methods, fluorescent probes and labeling strategies has emerged, making this imaging approach accessible to a broad range of applications. Single-molecule based stochastic methods, as STORM, PALM and their derivates require the least complex instrumentation, which gained these methods fast growing popularity among scientist of very diverse backgrounds. However, the relative simplicity of the instrumentation comes with a trade-off in probe requirements. These methods rely on the partial suppression of the detectable on-state, which can be achieved by photo-activation of an initially dark state or reversible switching between a bright (on) and a dark (off) state. Most synthetic dyes allow for the latter activation scheme, but require special treatment in order to do so. Chemical additives, as reducing reagents and oxygen scavenger, complex illumination schemes, molecular scaffolds, high irradiation intensities or a combination of the above is usually required to optimize the ratio between on- and off-state that enables a maximal densely labeling of the structure of interest. Here, we report a rhodamine derivate, which converts between a bright and a dark state in response to pH changes. At pH7, an average of >1% is fluorescent and tumbles between the two states on the molecular level. The resulting blinking occurs on timescales of up to several seconds and can therefore be exploited for e.g. STORM without further sample treatment or complex illumination schemes. Its ease-of-use and its outstanding photo-stability and brightness render this dye an excellent tool for super-resolution imaging techniques based on stochastic read-out.

Use of Cyclic Olefin Polymer in Single Molecule Total Internal Reflection Fluorescence Microscopy

Single molecule imaging is an established technique for many researchers in the biophysical sciences. Often, a fluorescent molecule bound near a glass or quartz surface is excited using total internal reflection fluorescence (TIRF) microscopy. TIRF excitation selectively excites fluorophores at the surface of a well or channel, thereby eliminating background fluorescence from bulk solution. However, nonspecific binding of fluorescent probes and fluorescently labeled molecules can become a limiting factor for protein association experiments. Extensive cleaning is required when using a glass or quartz substrate and the substrate is often coated with polymers such as poly-ethylene-glycol (PEG) to reduce non-specific binding. To eliminate the extensive wash and coating procedure steps we use a plastic cyclic olefin copolymer (COC) substrate that is extremely hydrophobic and can be passively activated to perform specific protein-protein interaction experiments in a very simple manner. Furthermore, COC has optical properties similar to glass making it amenable to TIRF microscopy. We first show simple processes to activate the surface in under a minute while rejecting fluorescent labeled antibody at concentrations of 1 nM. This is confirmed using objective-based TIRF microscopy and off-the-shelf COC microfluidic channels. We also show sufficient signal to noise to observe green fluorescent protein at the single molecule level using TIRF excitation. Finally, activated surfaces are used to perform a protein association experiment by detecting a sandwich of two antibodies bound to a single antigen molecule. We believe fluidic devices made of COC polymer will eliminate many of the preparation steps employed to treat other substrates allowing for easier and significantly faster protein association experiments.

Studying Antibody–Antigen Interactions with Fluorescence Fluctuation Spectroscopy

Antibodies are excellent binding proteins that have found numerous applications in biological research, biotechnology, and medicine. Characterization of their ligand binding properties has long been, and continues to be, the focus of many researchers. Antibodies are also perfect test systems which can be used for the evaluation of newly introduced biophysical techniques. Working with many different antibodies, we continuously implement the growing arsenal of methods offered by fluorescence fluctuation spectroscopy (FFS) and apply them for antibody research. In this chapter, we will describe applications of FFS for antibody binding characterization and also provide examples how studying of antibodies helps to develop and enhance the tool set offered by FFS technology. In addition to traditional affinity evaluations, we will describe how resolving molecular populations enables determinations of the binding stoichiometry and provides further information about the system. Even though all our examples include antibodies, the same experimental procedures can also serve well for characterizing various proteins and other ligand binding systems.

Potential and Limitation of Microparticle-Based Immunoassays: A Thermodynamic and Kinetic Study

The fundamental significance of immunoassays in diagnostics and research drives the ongoing quest for improving assay formats, sensitivity and instrument technology. Early on, the advantage of immobilizing antibodies to a surface to allow for separation of bound and unbound reagent was realized in the immunometric (sandwich) assay format. Today, this approach is established in most immunoassays as performed using ELISA (enzyme linked immunosorbent assay), GCSPR (grating-couples surface plasmon resonance) and microparticle-based chemiluminescence. However, antibody immobilization to a surface hampers reaction kinetics and imposes mass transport limitation and sterical hindrance. Theoretical considerations indicate that these effects can be minimized by using a spherical surface underlining why recently developed ultra-high-sensitivity techniques rely on the utilization of microparticles. In the present study, we investigate the potential and limitation of microparticle-based immunoassays in respect to impact of mass transport and surface immobilization on the intrinsic thermodynamic and kinetic properties of antibody-analyte interaction.

Understanding High Definition Immunoassays (HDIA): Studies of Binding Kinetics on Microparticles

Fluorescence imaging provides both spatial and temporal information about target molecules in biological systems. We have proposed to use imaging as a detection technique in microparticle based immunoassays. It has advantage over traditional approaches which measure only total signal but do not make use of the spatial or temporal information embedded in the system. The method can be readily adapted for quick assay prototyping and high throughput screening on any conventional fluorescence imaging system.In microparticle immunoassays, analytes are captured with antibody coated microparticles and subsequently detected using second antibody labeled with a reporter group. In HDIA, fluorescence images of the microparticles are examined pixel-by-pixel to extract binding information only from the microparticles, thus minimizing irrelevant signals from solution and vessel surfaces. Our model systems include sandwich based Troponin and Bcl-xl (B-cell lymphoma-extra large) protein assays, as well as homogeneous competitive Methotrexate immunoassay.Performance of the HDIA is dominated by binding kinetics of the microparticles. Depending on the microparticle number, concentration of the binding sites on microparticles, sample volume, sample concentration and the geometry of the reaction vessel, the binding kinetics can be either reaction limited or diffusion limited. We will present the effects of these factors on the binding kinetics of HDIA.