Ultrabright fluorescein-labeled antibodies near silver metallic surfaces

Fluorescein-labeled antibodies are widely used in clinical assays and fluorescence microscopy. The fluorescent signal per labeled antibody is limited by fluorescein self-quenching, which occurs when the antibody is heavily labeled with multiple fluoresceins. We examined immunoglobulin G (IgG) when labeled with 0.7 to about 30 fluoresceins per antibody molecule. The extent of self-quenching was decreased, and the signal increased, when the labeled antibody was in close proximity to metallic silver particles. Time-resolved measurements showed that the intensity increase was due in part to a silver-induced increase in the radiative decay rate. These results suggest the use of labeled antibodies conjugated to silver particles as ultrabright probes for imaging or analytical applications.     

Synthesis and evaluation of near-infrared (NIR) dye-herceptin conjugates as photoacoustic computed tomography (PCT) probes for HER2 expression in breast cancer

We are evaluating PCT imaging in conjunction with NIR dye labeled Herceptin antibody for noninvasive assessment of HER2 expression in tumors. Herceptin was labeled with Alexa Fluor-750 amine reactive dye for characterization of photoacoustic and fluorescence signals. Measurements were performed in solution and after incubation in cultured cell lines that were positive or negative in expression of HER2. The dye to antibody ratio was controlled to achieve a broad range of degree of labeling (DOL = 2 to 15). Photoacoustic signal intensity of Herceptin-dye conjugates in solution increased with increases over the entire DOL range studied. In contrast, fluorescence exhibited significant quenching for higher DOL. In vitro PCT imaging of the labeled HER2 (+) and HER2 (-) cells revealed the targeting specificity of the NIR dye labeled Herceptin. In HER2 (+) cells lines, photoacoustic signal intensity gradually increased with increasing DOL and with increasing number of cells. These results demonstrate that PCT-based measurement of HER2 receptor binding using NIR dye labeled Herceptin is feasible. The absence of a quenching effect with increased DOL advantages this method over traditional methods based on fluorescence measurement.     

Multi-wavelength immunoassays using surface plasmon-coupled emission

We describe a new method for multi-wavelength immunoassays using surface plasmon-coupled emission (SPCE). This phenomenon is coupling of excited fluorophores with a nearby thin metal film, in our case silver, resulting in strongly directional emission into the underlying glass substrate. The angle at which the radiation propagate through the prism depends on the surface plasmon angle for the relevant wavelength. These angles depend on emission wavelength, allowing measurement of multiple analytes using multiple emission wavelengths. We demonstrated this possibility using antibodies labeled with either Rhodamine Red-X or AlexaFluor 647. These antibodies were directed against an antigen protein bound to the silver surface. The emission from each labeled antibody occurred at a different angle on the glass prism, allowing independent measurement of surface binding of each antibody. This method of SPCE immunoassays can be readily extended to 4 or more wavelengths.     

Anisotropy-based sensing with reference fluorophores

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.     

Oligomeric fluorescent labels for DNA

In an effort to find fluorescent labels that have large Stokes shifts and increased emission intensity, a strategy for fluorescence labeling of DNA was explored in which multiple individual fluorophores are incorporated at adjacent positions at the end of a DNA probe. To encourage close interactions, hydrocarbon and heterocycle fluorophores were substituted at C-1 of deoxyribose, replacing the DNA base. The C-glycosides studied contained the well-known fluorophores terphenyl, pyrene, and terthiophene. For comparison, a commercial fluorescein-dU nucleotide was examined. Oligomeric labels containing up to five fluorophores were tested. Interestingly, all four dyes behaved differently on multiple substitution. Fluorescein displayed strong self-quenching properties, with the quantum yield dropping severalfold with each additional substitution and with a constant, small Stokes shift. In contrast, pyrene showed increases in quantum yield on addition of more than one fluorophore and yielded efficient long-wavelength emission on multiple substitution, with Stokes shifts of >130 nm. Oligomeric terphenyl labels gave a small progressive red shift in absorption and a marked red shift in emission wavelength and showed a strong increase in brightness with more monomers. Finally, terthiophene oligomers showed self-quenching combined with increasing Stokes shifts. Overall, the results suggest that some oligomeric fluorescent labels exhibit properties not available in common fluorescein class (or other commercial) labels, such as large Stokes shifts and increasing brightness with increasing substitution.     

Simulation of structure, orientation, and energy transfer between AlexaFluor molecules attached to MscL

Measurements of time-resolved fluorescence anisotropy and fluorescence resonance energy transfer are finding many applications in the study of biological macromolecules as they enable structural properties of the host molecules to be determined in their natural environment. A difficulty in interpreting these experiments is that they both require knowledge of the relative orientation of the fluorophores, a property that is almost impossible to measure. Here we conduct simulations of AlexaFluor488 and AlexaFluor568 attached to two sites on the membrane channel MscL to provide an alternative mechanism for determining the likely configurations and orientational freedom of the fluorophores, as well as the most likely value of the orientation factor κ² for energy transfer between them. The fluorophores are relatively mobile, and are found to be more so when immersed in bulk water than when they interact with the lipid membrane. The fluorophores never insert deeply into the lipid, despite their hydrophobic linkers and aromatic headgroup structures. Properties such as the fluorescence anisotropy decay can be predicted from simulations of the fluorophores in bulk water that closely match experimental data. In contrast, when the fluorophores were attached to the large MscL protein it was difficult to sample all the possible configurations of the fluorophores due to the computational time required. While this approach is likely to provide useful data on solvent-accessible fluorophores attached to small proteins, simulations lasting >50 ns or the use of biasing forces are required to accurately predict orientation factors for use in energy transfer experiments on larger membrane-bound proteins.     

Detection of receptor trimers on the cell surface by flow cytometric fluorescence energy homotransfer measurements

Fluorescence energy homotransfer offers a powerful tool for the investigation of the state of oligomerization of cell surface receptors on a cell-by-cell basis by measuring the polarized components of fluorescence intensity of cells labeled with fluorescently stained antibodies. Here we describe homotransfer-based methods for the flow cytometric detection and analysis of hetero- and homo-associations of cell surface receptors. Homotransfer efficiencies for two- and three-body energy transfer interactions are defined and their frequency distribution curves are computed from the fluorescence anisotropy distributions of multiple-labeled cells. The fractions of receptors involved in homo-clustering is calculated based on the dependence of the fluorescence anisotropy on the surface concentration of the fluorescently stained antibodies. A homotransfer analysis of the homo- and hetero-clustering of the MHCI and MHCII glycoproteins, the cytokine receptor IL-2Ralpha, transferrin receptor and the receptor-type tyrosine phosphatase CD45 on JY B and Kit-225-K6 T cells is presented. We investigated how various factors such as the type of dye, rotational mobility of the dye and dye-targeting antibody, as well as the wavelength of the exciting light affect the homotransfer. We show that the homotransfer technique combined with the high statistical resolution of flow cytometry is an effective tool for detecting different oligomeric states of receptors by using fluorophores having restricted rotational mobility on the time scale of fluorescence.     

Determination of the orientational distribution and orientation factor for transfer between membrane-bound fluorophores using a confocal microscope

Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound fluorophores. We demonstrate how information about the orientation of fluorophores can be used to calculate the orientation factor kappa(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.     

A fluorescent imaging method for analyzing the biodistribution of therapeutic monoclonal antibodies that can distinguish intact antibodies from their breakdown products

Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies.     

Integrated fluorescence and transmission electron microscopy

Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.     

The citrate carrier CitS probed by single-molecule fluorescence spectroscopy

A prominent region of the Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitS-sC398, CitS-sC414) were selectively labeled with the thiol-reactive fluorophores AlexaFluor 546/568 C(5) maleimide (AF(546), AF(568)). While both single-cysteine mutants were catalytically active citrate carriers, labeling with the fluorophore was only tolerated at C398. Upon citrate addition to the functional protein fluorophore conjugate CitS-sC398-AF(546), complete fluorescence quenching of the majority of molecules was observed, indicating a citrate-induced conformational change of the fluorophore-containing domain of CitS. This quenching was specific for the physiological substrate citrate and therefore most likely reflecting a conformational change in the citrate transport mechanism. Single-molecule studies with dual-labeled CitS-sC398-AF(546/568) and dual-color detection provided strong evidence for a homodimeric association of CitS.     

Structural dynamics of a lytic peptide interacting with a supported lipid bilayer

The interaction of a melittin mutant with a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)-supported lipid bilayer was studied with the use of time-resolved evanescent wave-induced fluorescence spectroscopy (TREWIFS) and evanescent wave-induced time-resolved fluorescence anisotropy measurements (EW-TRAMs). The mutant peptide was labeled at position K14 with AlexaFluor 430 and retained the lytic activity characteristic of native melittin. The fluorescence decay kinetics of the conjugate was found to be biexponential with a short-lived component, τ₁, due to photoinduced electron transfer between AlexaFluor 430 and proximal side chains within or between the peptides. The longer-lived component, τ₂, was sensitive to the polarity of the microenvironment at or near the K14 position of the peptide. Upon interaction with a DPPC-supported bilayer, the proportional contribution of τ₁ increased, indicating a conformational change of the peptide. The values of τ₁ and τ₂ indicate that the AlexaFluor 430 probe experienced an environment with an equivalent polarity no less than that of methanol. EW-TRAMs data from the melittin mutant revealed hindered rotational motions of the AlexaFluor 430 probe both in the plane and perpendicular to the plane of the supported lipid bilayer. The data indicate a highly ordered and polar environment near the center of the melittin helix consistent with the formation of a toroidal pore.     

Characterization of antibody binding to three cancer-related antigens using flow cytometry and cell tracking velocimetry

Proper antibody labeling is a fundamental step in the positive selection/isolation of rare cancer cells using immunomagnetic cell separation technology. Using either a two-step or single-step labeling protocol, we examined a combination of six different antibodies specific for three different antigens (epithelial specific antigen, epithelial membrane antigen, and HER-2/Neu) on two different breast cancer cell lines (HCC1954 and MCF-7). When a two-step labeling protocol was used (i.e., anti-surface marker-fluoroscein-isothiocyanate [FITC] [primary Ab], anti-FITC magnetic colloid [secondary Ab]) saturation of the primary antibody was determined using fluorescence intensity measurements from flow cytometry (FCM). The saturation of the secondary antibody (or saturation of a single-step labeling) was determined using magnetophoretic mobility measurements from cell tracking velocimetry (CTV). When the maximum magnetophoretic mobility was the primary objective, our results demonstrate that the quantities necessary for antibody saturation with respect to fluorescence intensity were generally higher than those recommended by the manufacturer. The results demonstrate that magnetophoretic mobility varies depending on the types of cell lines, primary antibodies, and concentration of secondary magnetic colloid-conjugated antibody. It is concluded that saturation studies are a vital preparatory step in any separation method involving antibody labeling, especially those that require the specificity of rare cell detection.     

Development of a novel FRET immunosensor technique

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.     

High affinity of the cell-penetrating peptide HIV-1 Tat-PTD for DNA

During cellular uptake of fluorescently labeled cell-penetrating peptides (CPPs), intense fluorescent signals are commonly observed in the nucleus of the cell, suggesting intracellular CPP relocation and potential binding to the genome of the host. We therefore investigated the interaction of the CPP HIV-1 Tat(47-57) with double-stranded DNA, and we also tested whether the fluorescence intensity of the labeled CPP allows for linear predictions of its intracellular concentration. Using isothermal titration calorimetry, we observe that the CPP has a high affinity for salmon sperm DNA as characterized by a microscopic dissociation constant of 126 nM. The binding is exothermic, with a reaction enthalpy of -4.63 kcal/mol CPP (28 degrees C). The dissociation constant and reaction enthalpy decrease further at higher temperatures. The affinity of the CPP for DNA is thus 1-2 magnitudes higher than for extracellular heparan sulfate, the likely mediator of the CPP uptake. Accordingly, the high affinity for DNA confers stability to extracellular transport complexes of CPP and DNA but potentially affects the regulation and molecular organization of the host's genome after nuclear uptake. Moreover, the CPP leads to the condensation of DNA as evidenced by the pronounced increase in light-scattering intensity. The fluorescence quantum yield of the FITC-labeled CPP decreases considerably at concentrations > 5 micromol/L, at pH < 7, and upon binding to DNA and glycosaminoglycans. This change in fluorescence quantum yield impedes the microscopic identification of uptake routes and the comparison of uptake efficiency of different CPPs, especially if the accumulation in subcellular compartments (self-quenching and pH difference) and transitory binding partners (quenching and condensation) is unknown.     

Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles.

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric microscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and alpha-actinin bands of skeletal muscle tissue and also actin and alpha-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM.     

Surface plasmon resonance-enhanced fluorescence implementation of a single-step competition assay: demonstration of fatty acid measurement using an anti-fatty acid monoclonal antibody and a Cy5-labeled fatty acid

The development of a single-step, separation-free method for measurement of low concentrations of fatty acid using a surface plasmon resonance-enhanced fluorescence competition assay with a surface-bound antibody is described. The assay behavior was unexpectedly complex. A nonlinear coverage-dependent self-quenching of emission from surface-bound fluorescent label was deduced from the response kinetics and attributed to a surface plasmon-mediated energy transfer between adsorbed fluorophores, modified by the effects of plasmon interference. Principles of assay design to avoid complications from such effects are discussed. An anti-fatty acid mouse monoclonal antibody reacting to the alkyl chain was prepared and supported on a gold chip at a spacing appropriate for surface-plasmon field-enhanced fluorescence spectroscopy (SPEFS), by applying successively a self-assembled biotinylated monolayer, then streptavidin, then biotinylated protein A, and then the antibody, which was crosslinked to the protein A. Synthesis of a fluorescently (Cy5) tagged C-11 fatty acid is reported. SPEFS was used to follow the kinetics of the binding of the labeled fatty acid to the antibody, and to implement a competition assay with free fatty acid (undecanoic acid), sensitive at the 1 μM scale, a sensitivity limit caused by the low affinity of antibodies for free fatty acids, rather than the SPEFS technique itself. Free fatty acid concentration in human serum is in the range 0.1-1 mM, suggesting that this measurement approach could be applied in a clinical diagnostic context. Finally, a predictive, theoretical model of fatty acid binding was developed that accounted for the observed “overshoot” kinetics.