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PubMed1996

Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization.

The principle of fluorescence polarization described by Perrin (F. Perrin, J. Phys. Radium 7:390-401, 1926) was applied to the development of a novel assay that used fluorescein-labeled Mycobacterium bovis secretory protein MPB70 for rapid detection of anti-MPB70 antibodies in selected sera from three M. bovis-infected species (elk, Ilama, and bison). Labeling of purified MPB70 with fluorescein isothiocyanate resulted in the incorporation of 0.96 +/- 0.08 (mean +/- standard deviation; n = 3) fluorescein group per MPB70 molecule. The labeled protein fluoresced strongly with an emission maximum at 518 nm when excited with light of a wavelength near 493 nm, and its immunoreactivity with anti-MPB70 monoclonal antibody 4C3/17 was not altered by modification with fluorescein. The fluorescence polarization assay protocol was optimized for analysis of serum samples by incorporating into the assay buffer 0.05% lithium dodecyl sulfate, which prevents the occurrence of some nonspecific interactions. Sera from M. bovis-infected animals, selected on the basis of exhibiting the presence of anti-MPB70 antibodies, as detected by enzyme-linked immunosorbent assay (ELISA), reacted with fluorescein-labeled MPB70, resulting in an increase in polarization of up to 330 milli-polarization units, in contrast to the values for noninfected sera (167 to 178 mP), which were close to that obtained in the absence of specific antibodies (164.7 +/- 3.3 mP; n = 6). These results demonstrated the feasibility of using fluorescein-labeled MPB70 to detect anti-MPB70 antibodies by fluorescence polarization and suggested that the assay described here can be an alternative to ELISA or other antibody assay systems. The advantages of this original methodology and its general applicability to the diagnosis of infectious diseases are discussed.

Animals↗

PubMed2003

Fluorescence polarization: past, present and future.

Fluorescence polarization was first observed in 1920 and during the next few decades the theoretical foundations of the phenomenon were clearly established. In the last two decades of the 20(th) century, fluorescence polarization became one of the most prevalent methods used in clinical and biomedical sciences. In this article we review the history of fluorescence polarization, its theoretical foundations and some of the more important practical developments, which helped to popularize the method. We also discuss important, but often misunderstood, practical considerations including the wavelength dependence of the limiting polarization and the effect of energy transfer on polarization. The present state of fluorescence polarization, both in pure research as well as in the applied biosciences is also reviewed. Finally, we speculate on possible future developments in the field, such as the use of multi-photon techniques.

Energy Transfer↗

PubMed1981

Effect of centrifugation on fluorescence polarization of amniotic fluid.

Fluorescence polarization was measured on amniotic fluids of different gestational ages before and after centrifugation at relative centrifugal forces of 34 to 4955 x g. Centrifugation significantly increased the fluorescence polarization values over those of uncentrifuged amniotic fluids. The greatest increase was observed in the most "mature" fluids centrifuged at 34 to 230 x g. Almost without exception, use of centrifugal forces greater than 1239 x g did not further increase fluorescence polarization values.

Amniotic Fluid↗

PubMed1983

Evaluation of an automated fluorescence polarization immunoassay for vancomycin.

An automated fluorescence polarization immunoassay for the determination of vancomycin levels in serum was evaluated. The vancomycin assay is a homogeneous competitive inhibition immunoassay based on changes in fluorescence polarization that occur with antibody binding. This assay was compared with a liquid chromatographic assay and an agar well diffusion bioassay method by using clinical serum specimens and controls. Linear regression analysis of the data obtained on clinical specimens by the three methods resulted in correlation coefficients of 0.97 for the fluorescence polarization immunoassay versus the liquid chromatographic assay (n = 60), 0.90 for the fluorescence polarization immunoassay versus the bioassay (n = 57), and 0.90 for the liquid chromatographic assay versus the bioassay (n = 57). Repetitive analysis of control sera containing 7, 35, and 75 micrograms of vancomycin per ml by the fluorescence polarization immunoassay yielded coefficients of variation of 3.0, 1.7, and 2.3, respectively. No interference was demonstrated in spiked hemolytic, lipemic, or icteric sera, and the assay was free of matrix effects. The automated fluorescence polarization immunoassay system offers a rapid, efficient, and accurate method for monitoring vancomycin serum levels for both toxicity and efficacy.

Biological Assay↗

PubMed2005

An automated homogeneous method for quantifying polysorbate using fluorescence polarization.

An automated fluorescence polarization (FP) assay has been developed for the quantitation of polysorbate in bioprocess samples. Using the lipophilic probe 5-dodecanoylaminofluorescein (DAF), polysorbate concentrations above the critical micelle concentration can be quantified by the FP increase that results when DAF inserts into the detergent micelles. The specificity, accuracy, and precision of this assay were defined for samples obtained from vaccine purification processes. Spike recoveries were 98-106% for purified products and 110-120% for crude process intermediates. The coefficients of variation for intra- and interassay precision were less than 9 and 14%, respectively. Because of the operational simplicity of the assay, all of the assay steps from sample preparation to data reduction were automated on a Tecan liquid-handling workstation. The combination of a rapid assay and an automated format makes this method well suited to the routine analysis of samples from trial purification processes which are carried out during the development of a vaccine or therapeutic protein. This method should be adaptable for the quantitation of other detergents into which DAF will insert.

Automation↗

PubMed1995

Fluorescence polarization study of the rigor complexes formed at different degrees of saturation of actin filaments with myosin subfragment-1.

A serine residue located in the active site of myosin head (S1) was labelled by 9-anthroylnitrile, an amino group located in the central domain of S1 was labelled by 7-diethylamino-3-(4'-isothio-cyanato-phenyl)-4-methylcoumari n, a cysteine residue located near the C-terminus of S1 was labelled by 5-[2-((iodoacetyl)-amino)ethyl]-amino-naphthalene-1-sulfonic acid (1,5-IAEDANS) and a cysteine residue located near the C-terminus of the alkali light chain 1 was labelled with iodoacetamido-tetramethyl-rhodamine. Polarization of fluorescence of S1 was measured in solution (where it indicated the mobility of actin-bound S1) and in myofibrils (where it indicated orientation of probes) to check whether the anisotropy of S1 labelled at different positions depended on the molar ratio S1:actin. In solution, when increasing amounts of actin were added to a fixed amount of labelled S1 (i.e. when myosin heads were initially in excess over actin), anisotropy saturated at 1 mol of S1 per 1 mol of actin. When increasing amounts of S1 were added to a fixed amount of F-actin (i.e. when actin was initially in excess over S1), the anisotropy saturated at 1 mol of S1 per 2 mols of actin. In myofibrils, orientation of S1 was different when S1 was added at nanomolar concentration (intrinsic actin was in excess over extrinsic S1) then when it was added at micromolar concentration (excess of S1 over actin). The fact that the anisotropy of S1 labelled at different positions depended on the molar ratio excluded the possibility that changes were confined to one part of the cross-bridge and supports our earlier proposal that the two rigor complexes which S1 can form with F-actin differ globally in conformation.

Actins↗

PubMed1988

The influence of abnormal pregnancies on fluorescence polarization of amniotic fluid lipids.

The fluorescence polarization of amniotic fluid, a measure of fetal lung maturity, was determined in 518 amniotic fluid samples obtained by amniocentesis. The subjects were divided into seven clinical groups: premature contractions, premature rupture of the membranes, pregnancy-induced hypertension, diabetes, intrauterine growth retardation, vaginal bleeding, and "other" for gestational-age groups of 30 weeks or less, 31-36 weeks, and 37 or more weeks. The proportion of mature values (fluorescence polarization 0.285 or lower) increased progressively from 12.5% at 27-28 weeks to 100% at 39-40 weeks. In the 31-36-week gestation group, the proportion of mature values in subjects with premature rupture of the membranes (84.6%) was significantly higher than in those with premature contractions (60%), severe pregnancy-induced hypertension (50%), mild pregnancy-induced hypertension (55.2%), diabetes class A (50%), insulin-dependent diabetes (60%), and other (63.5%). The mean +/- SD fluorescence polarization value was significantly lower in premature rupture of the membranes (0.256 +/- 0.030) than in premature contractions (0.274 +/- 0.032), mild and severe pregnancy-induced hypertension (0.280 +/- 0.027 and 0.280 +/- 0.035, respectively), and class A and insulin-dependent diabetes (0.285 +/- 0.018 and 0.277 +/- 0.030, respectively). The severity of pregnancy-induced hypertension and diabetes did not appear to influence either the fluorescence polarization value or the proportion of mature results. With the exception of a marked influence of premature rupture of the membranes, abnormal pregnancy conditions did not appear to have a significant effect on fluorescence polarization of amniotic fluid.

Amniotic Fluid↗

PubMed1983

Automated fluorescence polarization immunoassay for monitoring streptomycin.

The fluorescence polarization immunoassay technique has been used previously for the assay of vancomycin and the aminoglycoside antibiotics (gentamicin, tobramycin, and amikacin). We extended this technique to assay streptomycin. Fluorescein-labeled streptomycin was used as the tracer, and antiserum specific for streptomycin was raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum were combined, and the polarization of tracer fluorescence was determined in a specially designed fluorometer (Abbott TDx). Because of the instrument design, the possibility of fluorescent interferences was eliminated. The coefficient of variation within run was 4% (n = 5), and between run it was 5% (n = 5). We compared the fluorescence polarization immunoassay technique (TDx streptomycin) with a conventional bioassay, using Bacillus subtilis for clinical specimens (n = 39). A least-squares linear regression analysis gave a slope of 1.16, an intercept of 2.41 mg/liter, and a correlation coefficient of 0.885. Repeat analyses by both techniques showed that the largest discrepancies could be explained by the imprecision of the bioassay.

Antibody Specificity↗

PubMed1996

Binding of biologically important molecules to DNA, probed using electro-fluorescence polarization spectroscopy.

When small fluorescent tag molecules are bound with a single geometry to larger carrier molecules the fluorescence excited using polarized light may itself be partially polarized. If this is the case then alignment of the carrier molecules changes the fluorescence polarization. Electro-fluorescence polarization spectrometry (EFPS) makes use of the fluorescence polarization changes which occur when the carrier molecules are aligned by an electric field. EFPS allows the determination of the binding geometry of the small tag molecules to the larger carrier molecules. In this study, EFPS has been used to probe the interactions between DNA and a series of chromosomal stains and anti-neoplastic (anti-cancer) agents. Results are presented for calf thymus DNA, treated with the dyes acridine orange, ethidium bromide, proflavine, Hoechst 33258, Hoechst 33342, 4',6-diamidino-2-phenylindole and the anti-tumour agents, doxorubicin, daunomycin, actinomycin C and actinomycin V.

Animals↗

PubMed1975

Degree of dissociation of apohemoglobin studied by nano-second fluorescence-polarization technique.

A fluorescent dye 1-anilino-8-naphthalene sulfonate was complexed with human apohemoglobin and sperm whale apomyoglobin. Nanosecond fluorescence-polarization kinetics were measured for each of these complexes in KC1 solutions to obtain their fluorescence lifetimes and rotational correlation times. The rotational correlation time of apohemoglobin-dye complex was found to be 21 ns, which was about twice that of apomyoglobin-dye complex, 11 ns. These values were constant over an ionic strength range from 0 to 1.7. Circular dichroism spectra (215-300 nm) and fluorescence lifetimes of the complexes were also found to be independent of the ionic strength, indicating that no gross conformational change occurs with the change in the salt concentration, These results suggest that apohemoglobin remains dimeric over the ionic-strength range examined.

Anilino Naphthalenesulfonates↗

PubMed2001

Universal SNP genotyping assay with fluorescence polarization detection.

The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.

Alkaline Phosphatase↗

PubMed2003

Development and application of fluorescence polarization assays in drug discovery.

Fluorescence polarization technology has been used in basic research and commercial diagnostic assays for many decades, but has begun to be widely used in drug discovery only in the past six years. Originally, FP assays for drug discovery were developed for single-tube analytical instruments, but the technology was rapidly converted to high-throughput screening assays when commercial plate readers with equivalent sensitivity became available. This review will discuss fluorescence polarization assays in current use in drug discovery research as well as those in development that will likely be used in the near future. These assays include targets such as kinases, phosphatases, proteases, G-protein coupled receptors, and nuclear receptors.

Drug Evaluation, Preclinical↗

PubMed1974

The site of force generation in muscle contraction as deduced from fluorescence polarization studies.

The fluorescent dye, N(-iodoacetylamino)-l-naphthylanine-5-sulfonic acid, labeled exclusively the myosin cross-bridges in rabbit glycerinated psoas muscle fibers, without impairing their function. Fluorescence polarization was used to study cross-bridge orientation in rigor, relaxation, and contraction, as a function of sarcomere length. At a length where no overlap between thick and thin filaments occurs, rigor-inducing, relaxation-inducing, and contraction-inducing solutions all induced the relaxation attitude. At lengths where overlap does exist, the slowly-hydrolyzing ATP analog, "alpha,beta-methylene ATP," induced the relaxation attitude. The data were consistent with the A. F. Huxley-Simmons model of force generation. Combined with our earlier results, the data indicated that torque was generated at the actin-myosin interface.

Animals↗

PubMed1993

Characterization of liposomes and other lipid assemblies by multiprobe fluorescence polarization.

This minireview describes the use of fluorescence polarization to characterize the dynamics and organization of biological membranes, lipoproteins, liposomes and other lipid-based assemblies. The advantages, disadvantages and limitations of this method are discussed. There is special emphasis on the multiprobe approach, which can monitor various parts of the lipid membrane or assembly. The fluorescence polarization, anisotropy and the total fluorescence intensity are defined and their graphic presentation described. A user guide for the actual measurements is given. It describes how to apply fluorescence polarization, especially how to select the proper extrinsic probe and to optimize the measurement regarding theoretical and practical issues, the determination of a fluorescent probe location in the assembly, how to introduce the probe to the lipid assembly, what is the ideal ratio of the probe to the lipid, dealing with the spectrofluorimeter problems and how to overcome disturbances introduced by light scattering. It is shown here, that even though there are disadvantages in using fluorescence polarization, this approach provides a highly sensitive and simple means for assessing the subtle and more pronounced changes in the structure of lipid assemblies.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Fluorescence polarization for monitoring ribozyme reactions in real time.

Fluorescence polarization has been used recently to monitor diverse macromolecular interactions. In this report, the application of fluorescence polarization has been extended to monitor ribozyme reactions in real time. With fluorescently labeled substrate RNAs, group I ribozyme ligation and hammerhead ribozyme cleavage reactions were studied by fluorescence polarization in substrate excess (multiple turnover) conditions. These results also show that fluorescently labeled RNAs remain active substrates for ribozymes. Furthermore, a direct comparison of fluorescence polarization with fluorescence resonance energy transfer showed that both techniques were comparable for monitoring ribozyme reactions.

Automation↗

PubMed1985

Fluorescence polarization assay by flow cytometry.

Fluorescence polarization measurement on cell suspensions provides a highly sensitive means for detecting subtle changes in the cells, such as occur early after lymphocyte activation or on malignant transformation. We review here the principles of fluorescence polarization, its measurement by a commercially available flow cytometer and application of such assays especially in cellular immunology.

Animals↗

PubMed1985

Influence of gestational age on prediction of fetal lung maturity by fluorescence polarization of amniotic fluid.

Compared are the amniotic fluid fluorescence polarization values and the neonatal outcomes of 201 pregnant women who delivered from 28 through 37 weeks of gestation within 48 hours of the fluorescence polarization determinations. Thirty-five neonates developed hyaline membrane disease. The corresponding fluorescence polarization values ranged from 0.275 to 0.391. Eight of those 35 tests results were less than 0.325. The predictiveness of the method was studied using different threshold fluorescence polarization values. At the authors' own threshold of less than or equal to 0.325, the overall predictive value was as follows: false mature predictions: 6.2%, false immature predictions: 62.5%, sensitivity: 77.1%, and specificity: 72.8%. However, the false mature prediction rate was 21 to 40% from week 28 through week 33 versus 3.4 to 5.8% from week 34 through week 37, depending on the selected cutoff fluorescence polarization value. The sensitivity and specificity before, at, or after week 34 were significantly different at all tested fluorescence polarization values (P less than .05 to P less than .01) with the exception of the sensitivity at 0.310 and at 0.316 (P = .057). Caution is advised against relying on the fluorescence polarization method to predict fetal lung maturity at least before 34 weeks of gestation.

Amniotic Fluid↗

PubMed1997

A homogeneous, fluorescence polarization assay for src-family tyrosine kinases.

A nonradioactive, simple, sensitive fluorescence polarization assay was developed to assay protein tyrosine kinase activity. This assay involves incubation of a fluorescenylated peptide substrate with the kinase, ATP, and anti-phosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the anti-phosphotyrosine antibody resulting in an increase in the polarization signal as measured in a fluorescence polarization analyzer. Among several anti-phosphotyrosine antibodies examined, monoclonal antibody PY54 was found to give the best polarization signal with the test peptide. For validation of the fluorescence polarization assay, Lck activity was compared with a 32PO4 transfer assay. In both the fluorescence polarization and 32PO4 transfer assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentrations. Both assays gave similar inhibition constants with a known tyrosine kinase inhibitor staurosporine and the Lck inhibitor, 4-amino-5-(methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. These results show that the fluorescence polarization assay can detect inhibitors and is comparable to the 32PO4 transfer assay. The fluorescence polarization method is advantageous compared to the 32PO4 transfer assay or ELISA or DELFIA because it is a one-step assay that does not involve several washings, liquid transfer, and sample preparation steps. It has the added advantage of using nonisotopic substrates. The fluorescence polarization assay thus is environmentally safe and minimizes handling problems. The homogeneous nature of the assay makes it readily adaptable to high-throughput screening for small-molecule drug discovery.

Adenosine Triphosphate↗