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PubMed1987

Probe studies of the MgADP state of muscle cross-bridges: microscopic and wavelength-dependent fluorescence polarization from 1,5-IAEDANS-labeled myosin subfragment 1 decorating muscle fibers.

The microscopic and wavelength-dependent fluorescence polarization signals from the 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (1,5-IAEDANS) labeled subfragment 1 of myosin (S-1) decorating muscle fibers in rigor and in the presence of MgADP are measured. Using microscopic fluorescence polarization, we select a small uniform volume (approximately 0.1 micron 3) from the muscle fiber and detect a high degree of angular order. From these data we show that the probe angular distribution from fibers in rigor is quantitatively different from that present when MgADP is bound to S-1. Using wavelength-dependent fluorescence polarization, we vary the wavelength of the excitation light and thereby change the direction that the probe absorption dipole makes with a reference frame fixed in S-1. From these data we show that the binding of MgADP to S-1 causes an angular reorientation of S-1 relative to the actin filament. The difference between the angular distribution of probes for the rigor vs. MgADP states cannot be accounted for by the addition of random probes. The microscopic fluorescence polarization experiments suggest that the earlier attempts to distinguish a rigor from a MgADP probe angular distribution by using the 1,5-IAEDANS probe failed due to the lower resolution of the optical technique employed. The wavelength-dependent fluorescence polarization experiments indicate that the probe dipole orientation on S-1, at the typically used excitation and emission wavelengths, is not ideal for detecting the orientation change in the rigor to MgADP angular transition.

Adenosine Diphosphate↗
PubMed2003

The use of fluorescence polarization assays for the detection of infectious diseases.

Fluorescence Polarization Assays (FPAs) have been shown to have great utility in the detection of infectious diseases. Examples are presented of the use of O-polysaccharides (OPSs) for the detection of antibodies in serum, whole milk and whole blood to gram negative organisms (Brucella spp., Salmonella spp.). The use of proteins and peptides are also described for the detection of Mycobacterium bovis and Equine Infectious Anemia Virus. Fluorescence Polarization Inhibition Assays (FPIAs) are discussed for the specific and sensitive detection and quantitation of Salmonella spp. cells from culture. An example of the detection of enterohemorrhagic E. coli (EHECS) by Strand Displacement Amplification (SDA), coupled with FP, down to the single cell level, within thirty minutes, is described.

Animals↗
PubMed1986

The use of disulfonatonaphthalimide fluorescent dyes for the fluorescence polarization immunoassay of steroids.

A series of fluorescent disulfonatonaphthalimide derivatives of testosterone and estriol have been synthesized and their fluorescent properties investigated. The fluorescence lifetimes of these derivatives were higher than that of the unreacted fluorescent dye while the quantum yields were of the same order. The compounds were therefore compared in terms of their utilizability in steroid fluorescence polarization immunoassays. The assay sensitivity and precision with each compound is discussed in terms of the position, type, and length of the chemical "bridge" linking the steroid to the fluorescent dye. It is proposed that these fluorescent labels are highly appropriate to this type of immunoassay.

Estriol↗
PubMed1999

Capillary electrophoretic immunoassays for digoxin and gentamicin with laser-induced fluorescence polarization detection.

New immunoassays for therapeutic drugs digoxin and gentamicin have been described, which involved the separation of free and antibody-bound drug by capillary electrophoresis (CE) and the detection by laser-induced fluorescence polarization (LIFP). While the fluorescein-labeled digoxin and gentamicin (tracers) displayed negligible fluorescence polarization in solution, the complex formation between these small molecules and their antibodies resulted in substantial increases in fluorescence polarization due to the increase in molecular size. The LIFP detection, capable of measuring vertically and horizontally polarized fluorescence components simultaneously, provides enhanced capability for the identification of complex in capillary electrophoretic immunoassays. Proper adjustments of the running buffer pH and the ratio of antibody to tracer are essential for optimization of the performance of these assays. The digoxin-antibody complex remained stable during CE separation with running buffer pH ranging from 9.3 to 12. Calibration curves covering a concentration range of 0.05 to 0.5 ng/ml were obtained with a running buffer of pH 12. The concentration and mass detection limits were 0.02 ng/ml and 26 zmol, respectively. For gentamicin assay, the running buffer pH 10 was used to reduce the adsorption of the tracer while minimizing the dissociation of the antibody-tracer complex during the separation. The calibration curves covered a concentration range 0.05-1.0 microg/ml, with a concentration detection limit of 25 ng/ml and a mass detection limit of 52 amol of gentamicin.

Anti-Arrhythmia Agents↗
PubMed1978

Determination of fetal lung maturity by fluorescence polarization of amniotic fluid.

The degree of fluorescence polarization (P value) of 1,6-diphenyl-1,3,5-hexatriene has been shown to be an excellent indicator of the lecithin/sphingomyelin (L/S) ratio in laboratory prepared liposomal dispersions. In order to test the validity of this technique in amniotic fluid, the L/S ratio and P value of 161 amniotic fluid specimens were determined. In 127 the P value was less than or equal to 0.336 and the L/S ratio was greater than or equal to 2. In 26 the P value was greater than 0.336 and the L/S ratio was less than 2 (P less than 0.05). The amniotic fluid P value is affected by blood or meconium but not by surgical lubricants or bilirubin. We concluded that the degree of fluorescence polarization of amniotic fluid indicates fetal lung maturity as reliably as the L/S ratio. However, this method has important technical and practical advantages that make it more applicable for a clinical laboratory.

Amniotic Fluid↗
PubMed2003

High-throughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogeneous assay for protein kinases.

Protein kinases are one of the most important target classes in high-throughput screening today. The use of generic assay technologies facilitates assay development for new targets and decreases the time needed for implementation of assays in robotic screening. For tyrosine kinases, several generic assay technology platforms are available. These technologies make use of high-affinity antibodies that discriminate between phosphorylated tyrosines and non-phosphorylated tyrosines. Similar generic antibodies specific for phosphoserine or phosphothreonine are lacking. Recently, a non-antibody-based fluorescence polarization assay for protein kinases has become available, called IMAP (Molecular Devices, Sunnyvale, CA). In this assay, a fluorescently labeled peptide substrate that is phosphorylated by kinase is captured on metal-derivatized nanoparticles. We have evaluated IMAP in high-throughput screening, and compared this technology with a competition fluorescence polarization immunoassay based on an antibody specific for a phosphorylated peptide substrate. A random collection of >250000 compounds was screened with the two assays. Fluorescent library compounds were identified by calculation of fluorescence intensity values from the screening data, and by assaying in the absence of fluorescent reagents. Fluorescence polarization artifacts were filtered out further by testing in an ELISA-based kinase assay. Our data show that IMAP is a robust technology for high-throughput screening of kinase targets, and suggest that it is less susceptible to fluorescence polarization artifacts than the competition fluorescence polarization immunoassay.

Artifacts↗
PubMed1983

Automated fluorescence polarization immunoassay for monitoring vancomycin.

We have extended fluorescence polarization immunoassay (FPIA) technology for the measurement of drugs to include the complex amphoteric glycopeptide antibiotic vancomycin (molecular weight, 1,449). Fluorescein-labeled vancomycin was employed as a tracer, and antisera specific for vancomycin were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum were combined, and the polarization of tracer fluorescence is determined in a specially designed fluorometer (Abbott TDx). Because of instrument design, the possibility of fluorescent interferences is minimized. The assay can measure as little as 0.6 mg/L of vancomycin and is free of interferences from hemolysis, lipemia, bilirubin, and changes in protein concentration. The coefficient of variation within assay was 3% (n = 5) and between assays was 5% (n = 5). The FPIA assay (TDx Vancomycin) was compared to a liquid chromatographic (LC) assay for vancomycin and to a commercially available radioimmunoassay (RIA) for 98 clinical specimens. A linear least-squares regression analysis gave a correlation coefficient for LC of 0.980 from the equation FPIA = 1.09 LC + 3.04, and a correlation coefficient for RIA of 0.957 from the equation FPIA = 1.036 RIA + 1.66.

Antibody Specificity↗
PubMed1984

Fluorescence polarization in gastric aspirate and in amniotic fluid and its relationship to the respiratory distress syndrome.

Fluorescence polarization of diphenylhexatriene (DPH), added to amniotic fluid can be used to estimate fetal lung maturity. In this study we estimated the fluorescence polarization (FP) value of gastric aspirates of newborns and of amniotic fluids and studied the relationship between the FP value and neonatal lung function. Gastric aspirates of 51 newborns and amniotic fluids of 13 cases were mixed with a DPH-phosphate buffered saline solution and incubated for 30 minutes at 37 degrees C. Fluorescence polarization was measured at 22 degrees C. The FP values of amniotic fluid are in accordance with results previously found. A highly significant negative correlation was present between the FP value in amniotic fluid and gestational age. The highest FP values were found in amniotic fluids of infants in whom respiratory distress developed. The FP values of gastric aspirates decreased also with gestational age. A rather poor negative correlation, however, was found between these FP values and gestational age. We also noticed that, before the 36th week of gestation, the FP values in gastric aspirates tend to be lower than those in amniotic fluids. Both high and low FP values were estimated in gastric aspirates of infants with RDS and of infants without respiratory problems. These results show that the measurement of the FP value in gastric aspirates of newborns is not an useful method to determine whether the infant will develop RDS or not.

Amniotic Fluid↗
PubMed1981

Role of cytoplasmic lipids in altering diphenylhexatriene fluorescence polarization in malignant cells.

Prior studies of fluorescence anisotropy (polarization) with diphenylhexatriene in normal and malignant cell populations have shown differences which have been attributed to an altered membrane lipid composition in cancer. We studied fresh tumor cells from patients with diverse lymphoid neoplasms and found a discrete range of whole-cell fluorescence polarization values (P values) for each type of neoplasm. Following cell fractionation, however, the P values of isolated plasma membranes from malignant cells did not differ significantly from the values obtained with normal donor lymphocytes. Therefore, the altered whole-cell fluorescence polarization measurements in malignant cells are not likely to be due to gross lipid changes in the plasma membrane. Histochemical staining and cell fractionation revealed the presence of cytoplasmic lipid accumulations, and these had extremely low P values, which could account for the low P values of malignant cells. Complementary studies of lymphoid cell lines showed whole-cell fluorescence polarization measurements to be extremely sensitive to exogenous lipid supplements, but membrane values remained stable. We conclude that alterations in membrane lipid fluidity, as measured by the diphenylhexatriene probe, are not consistently found in lymphoid neoplasms and hence cannot presently be invoked to account for the malignant behavior of these cells. However, intracellular neutral lipid accumulation appears to be a common feature of the lymphoid neoplasm. The lipid alterations described could be characteristic of cell immaturity or proliferation rather than malignancy; nevertheless, they may convey unappreciated biological consequences.

Animals↗
PubMedDate not supplied

Analysis of digoxin concentrations in serum by fluorescence polarization immunoassay: an evaluation.

The Abbott TDx fluorescence polarization immunoassay was evaluated for the determination of serum digoxin concentrations. Within-assay precision was less than 4% coefficient of variation (CV) for concentrations ranging from 0.64 to 3.75 ng/mL. Between-assay precision was 14.5% CV at 0.75 ng/mL, 5.7% CV at 1.50 ng/mL, and 4.9% CV at 3.48 ng/mL. Sensitivity to 0.2 ng/mL digoxin was confirmed. Correlation of 86 patient specimens assayed by radioimmunoassay (RIA) with the TDx showed the following: correlation coefficient r = 0.94, slope = 0.93, intercept = 0.11, and Sy/x = 0.19. Recovery from serum at concentrations of 0.97 ng/mL and 4.50 ng/mL averaged 98%. No significant interference from lipemia, icteria, or hemolysis was observed. Spironolactone showed no cross-reactivity with the antibody, while digitoxin exhibited significant cross-reactivity. Compared to the RIA procedure, the TDx assay was more rapid, reliable and, in this clinical situation, more cost effective.

Digoxin↗
PubMed1989

Fluorescence polarization immunoassay for zidovudine.

We report a fluorescence polarization immunoassay (FPIA) for zidovudine (azidothymidine; Retrovir). This assay is accurate and specific over the clinically relevant range of zidovudine concentrations in serum (from 1 to 1,250 ng/ml; from 0.004 to 4.8 microM) and is unaffected by potentially interfering compounds in the sera of patients with renal or hepatic failure. Cross-reactivity with structural analogs of zidovudine (including zidovudine glucuronide) is less than 0.05%, except for cross-reactivities of 0.2, 0.3, and 0.4% with 3-methylthymidine, 3',5'-dideoxythymidine, and A22U (the optical isomer of zidovudine), respectively. The FPIA for zidovudine is more sensitive and more specific than high-performance liquid chromatography (HPLC); it requires 50 to 60 or 200 versus 500 microliters of serum and is faster to perform (45 specimens per h with the FPIA versus 3 specimens per h with HPLC). The zidovudine FPIA compares well with the radioimmunoassay. A correlation coefficient of 0.992 was observed with 31 serum specimens examined by both methods. All three assays (FPIA, radioimmunoassay, and HPLC) are unaffected by the heat treatment used to inactivate human immunodeficiency virus. The zidovudine FPIA should be particularly useful for analyzing specimens from large numbers of human immunodeficiency virus-infected patients receiving zidovudine in current clinical trials.

Antibody Specificity↗
PubMed2004

Fluorescence polarization imaging for delineating nonmelanoma skin cancers.

We present a method for detecting nonmelanoma skin cancers using exogenous fluorescence polarization. We built an automated system that permits exogenous fluorescence polarization imaging. It includes a tunable linearly polarized monochromatic light source and a CCD camera equipped with a rotating linear polarizer and a filter to reject excitation light. Two fluorophores that are retained in tumors, toluidine blue and methylene blue, are employed. We demonstrate that fluorescence polarization imaging can be used for accurate delineation of nonmelanoma cancers. The results suggest that this optical technique may be suitable for real-time noninvasive demarcation of epithelial cancers.

Fluorescence Polarization↗
PubMed1987

Prospective clinical evaluation of an improved fluorescence polarization assay for predicting fetal lung maturity.

We performed a prospective clinical evaluation of our newly developed fluorescence polarization procedure to predict fetal lung maturity (Clin Chem 1986;32:248-54). Net fluorescence polarization was measured at 34 degrees C after a 6.5-min incubation of amniotic fluid with fluorophore. For the 26 cases of neonatal respiratory distress syndrome encountered in 196 deliveries, the net polarization exceeded 0.287 for 22 (85%) of these, and exceeded 0.260 for all 26. The specificity of the polarization assay equaled or exceeded the specificity of the lecithin/sphingomyelin ratio for all sensitivities greater than 70%. Neither assay was a good predictor of the clinical severity of respiratory distress. For a separate group of 21 amniotic fluid specimens clinically contaminated with blood or meconium, the discriminatory power of the polarization assay was decreased, but six of seven respiratory-distress cases still had polarization values greater than 0.260. We conclude that this fluorescence polarization assay is a better overall predictor of fetal lung maturity than is the lecithin/sphingomyelin ratio, and that polarization values less than 0.260 are associated with little risk of respiratory distress.

Amniotic Fluid↗
PubMed2004

Fluorescence polarization studies of B-phycoerythrin oriented in polymer film.

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment-protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 --> State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 --> State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.

Fluorescence Polarization↗
PubMed1980

Amniotic fluid fluorescence polarization value as a predictor of respiratory distress syndrome.

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in amniotic fluid lipid aggregates was measured to determine the ability of this technique to prospectively predict respiratory distress syndrome. Prior retrospective studies have shown that an FP value of less than or equal to 0.336 correlated best with a lecithin to sphingomyelin ratio of greater than 2.0. Fluorescence polarization values of amniotic fluid samples obtained within 48 hours of birth from 161 pregnancies were correlated with neonatal outcome. Samples from 149 pregnancies had FP values of less than or equal to 0.336 and samples from 12 pregnancies had FP values of greater than or equal to 0.336. No infants delivered from the former group developed RDS and eight of 12 infants from the latter group developed RDS (P less than 0.001). In the entire population the test had a sensitivity of 100% and a specificity of 97%. When 37 infants with birth weights less than 2,500 gm were studied, the FP value remained a highly reliable predictor of the infant at risk for developing RDS (P less than 0.001). In this subset the test had a sensitivity of 100% and a specificity of 90%. L-S ratios were performed on 96 samples and the results correlated well with FP values (P less than 0.001). We conclude that the amniotic fluid FP value is a reliable index of fetal lung maturity and the risk for developing RDS. The FP value also has specific technical and diagnostic advantages over the L-S ratio.

Amniotic Fluid↗
PubMed1975

Fluorescence polarization and viscosities of membrane lipids of 3T3 cells.

Studies of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in membranes of normal, transformed, and revertant 3T3 cells allowed estimation of microviscosities (eta) of the lipid bilayers of these cells. Fluorescence polarization values observed with normal cells were significantly lower than those observed with cells transformed either by polyoma virus or by simian virus 40. The values of fluorescence polarization obtained with revertant Py6R1 cells were lower than those obtained with the normal cells. The calculated microviscosities (eta) show a 50% increase upon transformation. Possible correlations between the bilayer viscosity and the mobility of the receptors in the membrane are discussed.

Cell Line↗