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PubMed1984

Laboratory evaluation of five assay methods for vancomycin: bioassay, high-pressure liquid chromatography, fluorescence polarization immunoassay, radioimmunoassay, and fluorescence immunoassay.

We compared the precision and accuracy of five methods used to measure the concentration of vancomycin in serum: bioassay, high-pressure liquid chromatography, fluorescence polarization immunoassay (FPIA [TDX; Abbott Laboratories, North Chicago, Ill.]), radioimmunoassay (RIA), and fluorescence immunoassay. Based on an analysis of seven standards and of 106 patient samples, all five methods were accurate, and four (bioassay, high-pressure liquid chromatography, FPIA, and RIA) were also precise. The FPIA was the most precise and the fluorescence immunoassay was the least precise of the methods tested; intrarun coefficients of variation for these two methods were 0.9 to 3.0% versus 8.9 to 14.5%, and interrun coefficients of variation were 2.8 to 8.1% versus 12.2 to 16.2%, respectively. The RIA was inconvenient because it required an extra dilution of the specimen being tested and an additional (64 micrograms/ml) vancomycin standard for specimens with 32 to 64 micrograms of vancomycin per ml. Based on its rapid turnaround time and the stability of its standard curve, we believe that the FPIA is the best method currently available to quantitate vancomycin in the clinical laboratory.

Biological Assay↗
PubMed1988

Measurement of protein rotational motion using frequency domain polarized fluorescence depletion.

Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are examined anaerobically in a fluorescence microscope. In time domain PFD experiments a several-microsecond pulse of linearly polarized light produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. Monitoring the decay of ground state depletion with a probe beam alternatively polarized, parallel, and perpendicular to the depletion pulse permits the triplet lifetime and rotational correlation time to be resolved and evaluated. We have now explored fluorescence depletion methods in the frequency domain to see whether such measurements could provide simpler and more efficient routine measurements of protein rotational relaxation than previous time domain PFD methods. An acousto-optic modulator (AOM) modulates the intensity of a 514.5 nm argon ion laser beam and a Pockels cell (PC) rotates its plane of polarization. These devices are driven by sinusoidal or square waves in fixed frequency relation, and rigidly phase locked, one to another. The fluorescence emitted from a sample then contains various overtones and combinations of the AOM and PC frequencies. The magnitude and phase of individual fluorescence signal frequencies are measured by a lock-in amplifier using a reference also phase-locked to both the AOM and PC. Specific frequencies permit evaluation of the rotational correlation time of the macromolecule and of the fluorophore triplet state lifetime, respectively. Measurement of bovine serum albumin rotation in glycerol solutions by this method is described.

Mathematics↗
PubMed1985

Time-resolved fluorescence polarization from ordered biological assemblies.

We calculate the time dependence of the polarized fluorescence signal from fluorescent-labeled elements of a biological assembly that are rotationally diffusing in an arbitrary three-dimensional angular potential. We have formulated this calculation using the model-independent description of the angular potential wherein the angular potential is described by an expansion in a complete set of orthonormal functions with the expansion coefficients (or order parameters) determined by time-independent methods (Burghardt, T. P., 1984, Biopolymers, 23:2382-2406). We have applied the calculation to fluorescent-labeled myosin cross-bridges in relaxed muscle fibers. In a related paper we describe the experimental observation of the myosin cross-bridges rotationally diffusing in an angular potential (Burghardt, T. P., and N. L. Thompson, 1985, Biochemistry, 24:3731-3735).

Animals↗
PubMed2004

Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies: Src kinase as a model system.

High-throughput screening (HTS), a major component of lead identification, often utilizes fluorescence-based assay technologies. For example, HTS kinase assays are formatted using a variety of fluorescence-based assay technologies including, but not limited to, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP). These assays offer tremendous advantages such as a nonradioactive format, ease of automation, and excellent reproducibility. Fluorescence-based assays frequently used for lead identification can also be useful for structure activity relationship (SAR) studies during lead optimization. An important issue when assessing an assay to be used for SAR is the ability of the assay to discriminate high-affinity small molecule inhibitors (pM-nM) from low-affinity inhibitors (microM-mM). The purpose of this study was to utilize HTS-friendly assay formats for SAR by developing TR-FRET, FP, and DELFIAassays measuring Src kinase activity and to define the theoretical lower limit of small molecule inhibitor detection achievable with these assay formats. The authors show that 2 homogeneous assay formats, TR-FRET and FP, allowed for the development of Src kinase assays with a lower limit of detection of K(i) = 0.01 nM. This study indicates that assay technologies typically used for HTS can be used during lead optimization by providing quantitative measurements of compound activity critical to driving SAR studies.

Biological Assay↗
PubMedDate not supplied

Fluorescence resonance energy transfer imaging via fluorescence polarization measurement.

Fluorescence resonance energy transfer (FRET) has become a widely used spectroscopic tool for detecting molecular interactions and molecular proximity in solution, as well as in membranes. On the other hand, fluorescence polarization (FP) is a convenient measure: ratiometric and simple to execute. This work presents a novel methodology for determining energy transfer efficiency (E) via FP measurement. The methodology is based on the fact that a donor's fluorescence lifetime is shortened due to FRET and, consequently, its FP increases. As a model, the present work evaluates the E between fluorescein and rhodamine conjugated ConA attached to the receptors in the lymphocyte membrane. It shows not only that FRET imaging via FP is possible, but also that it is inexpensive, simple to perform, conveniently adaptable to the commonly used fluorescent microscopy, and readily interpretable.

Cell Line↗
PubMed1985

[Structural changes in the myelin sheath membranes exposed to anisotonic media. A fluorescence polarization study].

Extrinsic fluorescence polarization (P parallel and P perpendicular) of intact myelinated nerve fibres in anisotonic media has been investigated. Fluorescent probes of Acridine orange and Perylen were used for the experiment. Changes in fluorescent polarization of nerve fibres, associated with osmotic movement of water, arise immediately after the incubation of fibres in hypo- or hypertonic solutions. During exosmotic and endosmotic movement of water P parallel and P perpendicular and in quite a different manner. This fact demonstrates an asymmetry of structural membrane alterations. The results obtained are explained by asymmetry of swelling and shrinking of carbohydrate gel between two adjacent extracellular membrane surfaces of the myelin sheath.

Animals↗
PubMed2000

Measurement of the time course of DNA/RNA hybridization and RNA detection using fluorescence polarization analysis.

Using fluorescence polarization analysis, the time courses of hybridization between probe oligo-DNAs and target RNAs were measured. The RNAs were amplified using the DNA templates of Shiga toxin genes by NASBA (Nucleic Acid Sequence Based Amplification). Two DNA probes were designed for detecting the genes and they rapidly and specifically hybridized with their target RNA sequences. NASBA could be sufficiently used for the combination and DNA/RNA hybridization could be detected in the fluorescence polarization.

Base Sequence↗
PubMed1990

POLAR: a menu driven computerized analysis of fluorescence polarization data.

While fluorescence polarization is a powerful tool for the measurement of ligand-receptor (e.g. antigen-antibody) interactions, its application in routine clinical chemistry and immunochemistry has been limited because collection of data was error prone, and because analysis of data previously required tedious and imprecise manual graphical methods. We present the series of linear and curvilinear regressions required to calculate parameters (p', Q', pf, Qf, Pb, Qb, Fb max, a and Ko) and their corresponding standard errors. The values of a and Ko are comparable to those obtained by the method of finding the minimal the sum of squares. A menu operated computer program that uses these regressions, simplifies the collection, analysis and presentation of fluorescence polarization data. A simple spread sheet system permits the insertion, deletion or modification of data. The results are presented in graphical and tabular form, either on a monitor, a printer or an X-Y plotter. The system has its own graphical routines.

Algorithms↗
PubMed1990

Adaptation of fluorescence polarization immunoassay to the assay of macromolecules.

This paper describes an original methodology for determining macromolecular antigen levels by polarization of fluorescence. it involves the use of fluorescent derivatives of Fab fragments of a monoclonal antibody (Mr 50,000), whose fluorescence polarization rises significantly when it combines with a macromolecular antigen. An experimental system (Fab anti-aldosterone and aldosterone--bovine serum albumin (BSA)) is studied to test this methodology, which was then used to develop an immunoassay for human immunoglobulin M (IgM), using anti-mu chain Fabs. In the two assays, the binding stoichiometry of Fab/antigen was 10/1 and 8/1 for aldosterone--BSA and IgM, respectively. The lower limit of detection of the IgM assay was 0.8 microgram/ml and thus it was applicable to clinical detection of IgM concentrations.

Aldosterone↗
PubMed2006

Study of binding stoichiometries of the human immunodeficiency virus type 1 reverse transcriptase by capillary electrophoresis and laser-induced fluorescence polarization using aptamers as probes.

Binding stoichiometries between four DNA aptamers (RT12, RT26, RTlt49, and ODN93) and the reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1) were studied using affinity CE (ACE) coupled with LIF polarization and fluorescence polarization (FP). The ACE/LIF study showed evidence of two binding stoichiometries between the HIV-1 RT protein and aptamers RT12, RT26, and ODN93, suggesting that these aptamers can bind to both the p66 and p51 subunits of the HIV-1 RT. Only one binding stoichiometry for aptamer RTlt49 was found. The affinity complexes were easily separated from the unbound aptamers; however, the different stoichiometries were not well resolved. A complementary technique, FP, was able to provide additional information about the binding and supporting evidence for the ACE/LIF results. The ACE/LIFP study also revealed that the FP values of the 1:1 complexes of the HIV-1 RT protein with aptamers RT12, RT26, and ODN93 were always much greater than those of the 1:2 complexes. This was initially surprising because the larger molecular size of the 1:2 complexes was expected to result in higher FP values than the corresponding 1:1 complexes. This phenomenon was probably a result of fluorescence resonance energy transfer between the two fluorescent molecules bound to the HIV-1 RT protein.

Aptamers, Nucleotide↗
PubMed1979

Direct fluorescence polarization immunoassay.

Direct fluorescence polarization immunoassay of serum cortisol was established. Non-specific binding of fluorescent-labelled cortisol to serum proteins was successively eliminated by sodium dodecyl sulfate. The minimal amount of cortisol detected was 0.1 ng/tube and serum concentration of 1.0 microgram/dl to 100 microgram/dl of cortisol could be measured. This fluorescence polarization immunoassay satisfied the standard criteria of accuracy and precision. The values correlated well with those obtained by radioimmunoassay.

Fluorescent Antibody Technique↗
PubMed1990

Slow rotational mobilities of antibodies and lipids associated with substrate-supported phospholipid monolayers as measured by polarized fluorescence photobleaching recovery.

Polarized fluorescence photobleaching recovery has been used to monitor slow rotational motions of a fluorescently-labeled anti-dinitrophenyl mouse IgGl monoclonal antibody (ANO2) specifically bound to substrate-supported monolayers composed of a mixture of distearoylphosphatidylcholine (DSPC) and dinitrophenyldioleoylphosphatidylethanolamine (DNP-DOPE). ANO2 antibodies were labeled with a new bifunctional carbocyanine fluorophore that has two amino-reactive groups; steady-state fluorescence anisotropy data confirmed the expected result that the ANO2-conjugated bifunctional probe had less independent flexibility than ANO2-conjugated unifunctional fluorescence labels. Rotational mobilities were also measured for the fluorescent lipid 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine (dil) in DSPC and in mixed DSPC/DNP-DOPE monolayers in the presence and absence of unlabeled ANO2 antibodies. The apparent rotational correlation time and fractional mobility of ANO2 on supported monolayers were approximately 70 and approximately 0.3 s, respectively. These measured parameters of rotational mobility did not depend on the ANO2 surface density or on kinetic factors, but addition of unlabeled polyclonal anti-(mouse IgG) antibodies significantly decreased the apparent mobile fraction. The measured fluorescence recovery curves for dil were consistent with two fluorophore populations with rotational correlation times of approximately 4 and approximately 100 s and a population of immobile fluorescent lipid. No difference in fluorescence recovery and decay curves was measured for dil in DSPC monolayers, DSPC/DNP-DOPE monolayers, and DSPC/DNP-DOPE monolayers treated with unlabeled ANO2 antibodies.

Animals↗
PubMed1988

[Intravital research on hydrophobic interactions in protein fibrils using a fluorescence polarization method].

Fluorescent probe of acridine orange (AO) is known to be bound exclusively by materials of thick protofibril muscle fibres. The dependence of fluorescence anisotropy of AO in frog muscle fibres on the temperature has been studied. It has been found that the fluorophore orientation decreases with temperature, i.e. here the adsorbent cold denaturation takes place. This phenomenon was demonstrated for living, skinned and glycerinated muscle fibres. It shows that hydrophobic interactions contribute much to stabilization of the myosin structure. After contraction of glycerinated muscle fibres at the action of ATP, the AO fluorescence anisotropy grows independently of the temperature which is indicative of structural rearrangements in materials of thick protofibrils upon contraction.

Acridine Orange↗
PubMed2005

Energy transfer efficiency on lymphocytes of leukemia patients measured by fluorescence polarization.

Measurements of fluorescence resonance energy transfer efficiency (E), via fluorescence polarization, have been applied to distinguish between chronic lymphocytic leukemia (CLL) patients and healthy persons. Capping of Concanavalin-A receptors is more pronounced in normal lymphocytes than in those of CLL patients. Membrane capping decreases the distance between donor and acceptor molecules embedded in the membrane (r), and thus increases the monitored energy transfer efficiency (E approximately 1/r6). Blood samples of 10 healthy subjects and 16 CLL patients were examined. In the healthy subjects, the mean E value for capped lymphocytes was 19 +/- 3, whereas in the CLL patients it was significantly lower (8 +/- 5) (P < 0.01).

Concanavalin A↗
PubMed1989

Influence of auranofin on lymphocyte membrane fluidity in rheumatoid arthritis. A fluorescence polarization study.

Using the fluorescence polarization of 1.6-diphenyl-1, 3, 5-hexatriene, the membrane fluidity of lymphocytes obtained from rheumatoid arthritis patients was measured during 12 months of auranofin treatment. Significant differences in fluorescence polarization values were observed following auranofin treatment. The correlations shown between fluorescence polarization values and the indices of rheumatoid arthritis activity, such as erythrocyte sedimentation rate, alpha 2-globulins, plasma fibrinogen levels and platelet count suggest that changes in lymphocyte membrane fluidity could be used to monitor the effects of auranofin therapy.

Adult↗
PubMed2006

A simple theoretical model for fluorescence polarization binding assay development.

Fluorescence polarization is a screening technology that is radioactivity free, homogeneous, and ratiometric. The signal measured with this technology is a weighted value of free and bound ligand. As a consequence, saturation curves are accessible only after calculation of the corresponding concentrations of free and bound ligand. To make this technology more accessible to assay development, the authors propose a simple mathematical model that predicts fluorescence polarization values from ligand and receptor total concentrations, depending on the corresponding dissociation constant. This model was validated using data of Bodipy-NDP-alphaMSH binding to MC(5), obtained after either ligand saturation of a receptor preparation or, conversely, receptor saturation of a ligand solution. These experimental data were also used to calculate the actual concentration of free and bound ligand and receptor and to obtain pharmacological constants by Scatchard analysis. A general method is proposed, which facilitates the design of fluorescence polarization binding assays by relying on the representation of theoretical polarization values. This approach is illustrated by the application to 2 systems of very different affinities.

Binding Sites↗