Qiaoqiao Ruan

Molecular Brightness Characterization of EGFP In Vivo by Fluorescence Fluctuation Spectroscopy

We characterize the molecular properties of autofluorescence and transiently expressed EGFP in the nucleus and in the cytoplasm of HeLa cells by fluorescence correlation spectroscopy (FCS) and by photon counting histogram (PCH) analysis. PCH has been characterized and applied in vitro, but its potential for in vivo studies needs to be explored. Thus, this study mainly focuses on the characterization of PCH analysis in vivo. The strength of PCH lies in its ability to distinguish biomolecules by their molecular brightness value. Because the concept of molecular brightness is crucial for PCH analysis, we study the molecular brightness of EGFP and determine the statistical accuracy of its measurement under in vivo conditions. We started by characterizing the influence of autofluorescence on EGFP measurements. We found a molecular brightness of EGFP that is a factor of 10 higher than the brightness of the autofluorescence. Moment analysis demonstrates that the contribution of autofluorescence to fluorescence fluctuation experiments is negligible at EGFP concentrations of one protein per excitation volume. The molecular brightness of EGFP measured in the nucleus, the cytoplasm, and in vitro are identical and our study demonstrates that molecular brightness is a very stable and predictable quantity for cellular measurements. In addition to PCH, we also analyzed the autocorrelation function of EGFP. The diffusion coefficient of EGFP is a factor of 3 lower in vivo than compared to in vitro, and a simple diffusion process describes the autocorrelation function. We found that in the nucleus the fluorescence intensity is stable as a function of time, while measurements in the cytoplasm display fluorescence intensity drifts that complicate the data analysis. We introduce and discuss an analysis method that minimizes the influence of the intensity drifts on PCH analysis. This method allows us to recover the correct molecular brightness of EGFP even in the presence of drifts of the fluorescence intensity signal. We found the molecular brightness of EGFP to be a very robust parameter, and anticipate the use of PCH analysis for the study of oligomerization processes in vivo.

Cellular Characterization of Adenylate Kinase and Its Isoform:Two-Photon Excitation Fluorescence Imaging and Fluorescence Correlation Spectroscopy

Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the mobility of the target protein in live cells. This technology offers advantages in studying protein interactions and function in the cell.

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.

Rapid determination of antigenic epitopes in human NGAL using NMR.

The recent remarkable rise in biomedical applications of antibodies and their recombinant constructs has shifted the interest in determination of antigenic epitopes in target proteins from the areas of protein science and molecular immunology to the vast fields of modern biotechnology. In this article, we demonstrated that measuring binding induced changes in two-dimensional NMR spectra enables rapid determination of antibody binding footprints on target protein antigens. Such epitopes recognized by six high-affinity monoclonal murine antibodies (mAbs) against human neutrophil gelatinase-associated lipocalin (NGAL) were determined by measuring chemical shifts or broadening of peaks in (1)H-(15)N-TROSY HSQC and (1)H-(13)C HSQC spectra of isotope-labeled NGAL occurring upon its binding to the antibodies. Locations of the epitopes defined by the NMR studies are in good agreement with the results of antibody binding pairing observed by dual-color fluorescence cross-correlation spectroscopy. In all six cases, the antibodies recognize conformational epitopes in regions of relatively rigid structure on the protein. None of the antibodies interact with the more flexible funnel-like opening of the NGAL calyx. All determined epitope areas in NGAL reflect the dimensions of respective antibody binding surface (paratopes) and contain amino acid residues that provide strong interactions. This NMR-based approach offers comprehensive information on antigenic epitopes and can be applied to numerous protein targets of diagnostic or therapeutic interest.

Application of fluorescence correlation spectroscopy to hapten-antibody binding.

Two-photon fluorescence correlation spectroscopy 2P-FCS has received a large amount of attention over the past ten years as a technique that can monitor the concentration, the dynamics, and the interactions of molecules with single molecule sensitivity. In this chapter, we explain how 2P-FCS is carried out for a specific ligand-binding problem. We briefly outline considerations for proper instrument design and instrument calibration. General theory of autocorrelation analysis is explained and straightforward equations are given to analyze simple binding data. Specific concerns in the analytical methods related to IgG, such as the presence of two equivalent sites and fractional quenching of the bound hapten-fluorophore conjugate, are explored and equations are described to account for these issues. We apply these equations to data on two antibody-hapten pairs: antidigoxin IgG with fluorescein-digoxin and antidigitoxin IgG with Alexa488-digitoxin. Digoxin and digitoxin are important cardio glycoside drugs, toxic at higher levels, and their blood concentrations must be monitored carefully. Clearly, concentration assays based on IgG rely on accurate knowledge of the hapten-IgG binding strengths. The protocols for measuring and determining the dissociation constants for both IgG-hapten pairs are outlined and discussed.

Crystal structure and thermodynamic analysis of diagnostic mAb 106.3 complexed with BNP 5-13 (C10A)

B‐type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high‐affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5‐13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three‐dimensional structure by X‐ray diffraction to 2.1 Å resolution. The structure reveals the detailed interactions that five of the complementarity‐determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, ΔG = −54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion‐limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine‐scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides. Proteins 2009.

Simplified confocal microscope for counting particles at low concentrations

We describe a compact scanning confocal fluorescence microscope capable of detecting particles concentrations less than 100 particles∕ml in ~15 min. The system mechanically moves a cuvette containing ~3 ml of sample. A relatively large confocal volume is observed within the cuvette using a 1 mm pinhole in front of a detection PMT. Due to the motion of the sample, particles traverse the confocal volume quickly, and analysis by pattern recognition qualifies spikes in the emission intensity data and counts them as events. We show linearity of detection as a function of concentration and also characterize statistical behavior of the instrument. We calculate a detection sensitivity of the system using 3 μm fluorescent microspheres to be 5 particles/ml. Furthermore, to demonstrate biological application, we performed a dilution series to quantify stained E. coli and yeast cells. We counted E. coli cells at a concentration as low as 30 cells∕ml in 10 min/sample.

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.

Water channel in the binding site of a high affinity anti-methotrexate antibody.

In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

Supporting immunoassay design with biophysical tools

In this article, we demonstrate how the application of biophysical tools facilitates the design of robust immunoassays. The binding characteristics of the reagents used in an immunoassay determine the assay response to the analyte concentrations. We applied several biophysical methods to obtain pertinent equilibrium and kinetic coefficients and used this information in the design of a microparticle-based immunoassay for detection of neutrophil gelatinase-associated lipocalin (NGAL), which is a new diagnostic marker of acute kidney injury (AKI). We characterized the conformational stability of recombinant human NGAL and the solution phase binding properties of six monoclonal antibodies. A preferred antibody pair was selected on the basis of the affinities of the antibodies and their sandwich pairing capabilities. One of the antibodies was coated on magnetic microparticles, and the second antibody was conjugated with a reporter group. The apparent kinetic rates of the immobilized and conjugated antibodies were measured and used to compute the assay calibration plot for the target concentration range of the analyte at specific incubation times. The experimental assay results were found to be in good agreement with the computed data, confirming that applying biophysical tools provides a solid foundation for immunoassay design and optimization.