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Results for “Fluorescence Polarization”

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PubMed1999

Preparation of a diagnostic antigen of human melanoma based on lymphocyte activation as measured by intracellular fluorescein fluorescence polarization.

The intracellular fluorescein fluorescence polarization (IFFP) test indicates that peripheral blood lymphocytes (PBL) of cancer patients display stimulatory sensitivity to a short incubation with specific tumor protein extracts. In this work, a human lymphocyte activation melanoma antigen (LAMA) was purified from supernatant of a human melanoma cell line (L1M1), which could specifically stimulate lymphocytes of melanoma patients. The results showed a significant stimulation of lymphocytes from healthy donors after incubation with phytohaemagglutinin (PHA), while no stimulation was observed after incubation with LAMA. On the other hand, lymphocytes from melanoma patients showed a significant stimulation with LAMA, while generally showing minor or no stimulation with PHA. Melanoma specificity of LAMA was demonstrated by no response in lymphocytes from patients of lung, colon, or breast cancer. The purified fraction is therefore considered to be a shared tissue-specific antigen which may be useful in immunodiagnosis and immunotherapy of melanoma.

Antigens, Neoplasm↗
PubMed1991

Clinical evaluation of teicoplanin fluorescence polarization immunoassay.

A teicoplanin fluorescence polarization immunoassay (FPIA) developed by International BioClinical (IBC) was evaluated by using serum samples from patients who had been receiving teicoplanin at Detroit Receiving Hospital (DRH) as part of a clinical investigation. Patient samples collected over a 1-year span were assayed at DRH and at IBC, and the results were compared with those of a standard microbiological assay performed at Merrell Dow Research Institute, Indianapolis, Ind. The FPIA has a rapid turnaround time (circa 20 min), utilizes small sample volumes (less than 100 microliters) and is sensitive and accurate in determining concentrations in the range of 5 to 100 micrograms/ml. The intra-assay and interassay coefficient of variation for controls (7, 35, and 75 micrograms/ml) was less than or equal to 13%. Concentrations greater than 100 micrograms/ml must be diluted prior to the assay, which may introduce additional error in determination. The FPIA compared well with the bioassay (r = 0.901) for 193 clinical samples. The results obtained utilizing the FPIA system were reproducible at two different sites, as illustrated by the high degree of correlation between the results at DRH and IBC (r = 0.92). There was less than 7% interference noted when teicoplanin was assayed in the presence of other antibiotics. Patient samples stored for up to 1 year retained their potency: the mean recovery rate in these samples was 107%. The FPIA should be useful for monitoring and adjusting teicoplanin dosage regimens in patients.

Anti-Bacterial Agents↗
PubMed1988

Analysis of early lymphocyte activation events by fluorescence polarization flow cytometry.

After short-term (up to 4 h) stimulation with mitogen or antigen, lymphocytes were incubated with fluorescein diacetate and the polarization of fluorescence from intracellular fluorescein was measured on a specially adapted FACS II. This flow cytofluorimetric method to assay early changes in activated lymphocytes gave a reproducible response to the mitogens phytohaemagglutinin (PHA), concanavalin-A and the monoclonal antibody OKT3, recognized at 1 h by decreased polarization. A response by immune spleen cells to the antigen dinitrophenyl-ovalbumin was revealed at 4 h. The calcium ionophore A23187 induced an increase in polarization after only 10 min. The PHA polarization response was shown to be dependent on PHA binding, PHA dose, T cells, calcium ions and an intact cytoskeleton. The cellular events monitored by the polarization change are presumably altered fluidity of the probe's microenvironment due to conformational change in macromolecules to which the probe has bound or to dissociation of the probe into the aqueous phase. The fluorescein fluorescence polarization assay is a reliable and sensitive monitor of early lymphocyte activation events and, coupled with the use of a flow cytometer, permits study of particular subpopulations of responding cells.

Acetylgalactosamine↗
PubMed1999

Capillary electrophoresis coupled with laser-induced fluorescence polarization as a hybrid approach to ultrasensitive immunoassays.

Immunoassays using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a powerful approach to the determination of trace amounts of analytes in a complex biological matrix. However, its applicability is limited by the requirement that the free and bound tracer (fluorescently labeled compound) be resolved for their identification and quantitation. Here we show that replacing LIF with laser-induced fluorescence polarization (LIFP) permits ultrasensitive immunoassays to be performed with or without the separation of the free and bound tracer. A binding system involving cyclosporin A (CyA) and monoclonal antibody to CyA was chosen to demonstrate both homogeneous and heterogeneous immunoassay approaches. In the homogeneous scheme where the free and bound tracer were not separated, the fluorescence polarization of the mixture was a quantitative measure of the antibody-bound tracer. The concentration and mass detection limits for CyA using the homogeneous competitive assay were found to be 1 nM and 1 amol (10(-18) mol), respectively. The heterogeneous assay involved a nearly baseline separation of the free and bound tracer using CE with a phosphate running buffer of pH 7.0. The complex of the tracer with the antibody had a fluorescence polarization of approximately 0.24 whereas the free tracer had negligible polarization. The fluorescence polarization was independent of analyte concentration, and the fluorescence intensity of either the free or bound tracer was used for quantitation. Results from both assays suggest that the CE-LIFP approaches may have a wider application than the immunoassays based on either CE-LIF or fluorescence polarization alone.

Antibodies, Monoclonal↗
PubMed2004

The effect of common clinical contaminants on amniotic fluid fluorescence polarization results.

OBJECTIVE: To determine the effect of blood, meconium, and vaginal secretions on amniotic fluid (AF) fluorescence polarization results. METHODS: Amniotic fluid was collected by transabdominal amniocentesis from women at 20-41 weeks of gestation and contaminated with blood, meconium, and vaginal secretions to concentrations of 0.5, 1, 2, 5, and 10%. An additional 20% concentration was performed with meconium and vaginal secretions. Fluorescence polarization was determined by a TDx Analyzer with the NBD-PC fluorescent probe. Results were compared for each contaminant by concentration level using repeated-measures analysis of variance. RESULTS: Forty-eight samples from women at a mean gestational age of 35 weeks (range 20-41.5 weeks) were evaluated. Before contamination, 16 (33%) samples had fluorescence polarization values greater than 290 mPol (immature), 10 (21%) were 260- 289 mPol (transitional), and 22 (46%) were less than 260 mPol (mature). Contamination with blood significantly altered fluorescence polarization values in AF samples with baseline values in the immature and mature categories such that values trended toward the transitional range. Contamination of baseline immature samples with vaginal secretions at 20% contamination level resulted in more mature fluorescence polarization values. Contamination with meconium more than 2% in the baseline immature category or more than 20% in the baseline transitional category also resulted in significantly more mature fluorescence polarization values. CONCLUSION: Amniotic fluid contamination with blood can result in more transitional range fluorescence polarization values, whereas contamination with meconium and vaginal secretions can result in more mature fluorescence polarization values.

Amniocentesis↗
PubMed1987

[Structural changes of the chromatin DNA in situ in partial deproteinization of DNP by a 0.6 M NaCl solution. A polarization-fluorescence microscopic study].

By means of polarization fluorescence microscopy changes in polarization degree of fluorescence of DNA-bound of Acridine orange in situ were observed. A marked decrease in polarization degree of chromatin fluorescence was found after the treatment of cell nuclei with 0.6 M NaCl. It shows that the removal of histone H1 from the chromatin results in destabilization of its structure. The data obtained show that the measurements of polarization degree of fluorescence allow to obtain relevant information about structural changes in DNA chromatin.

Acridine Orange↗
PubMed1979

Fluorescence polarization immunoassay for cortisol.

A fluorescence polarization immunoassay for serum cortisol was established using cortisol 21-amine which was readily coupled with fluorescein isothiocyanate. The proposed method is sufficiently sensitive, reliable, specific and simple for routine determination of serum cortisol. The assay is rapid, without separation of antibody-bound and free ligands. The minimal amount of cortisol detected was 1.5 ng/tube and the measurable range was from 1.5 to 100 micrograms/dl. There was a good correlation between values obtained from radioimmunoassay and the proposed method.

Cross Reactions↗
PubMed2002

Fluorescence polarization detection for affinity capillary electrophoresis.

Affinity capillary electrophoresis (ACE) with laser-induced fluorescence polarization (LIFP) detection is described, with examples of affinity interaction studies. Because fluorescence polarization is sensitive to changes in the rotational motion arising from molecular association or dissociation, ACE-LIFP is capable of providing information on the formation of affinity complexes prior to or during CE separation. Unbound, small fluorescent probes generally have little fluorescence polarization because of rapid rotation of the molecule in solution. When the small fluorescent probe is bound to a larger affinity agent, such as an antibody, the fluorescence polarization (and anisotropy) increases due to slower motion of the much larger complex molecule in the solution. Fluorescence polarization results are obtained by simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes. Applications of CE-LIFP to both strong and weak binding systems are discussed with antibody-antigen and DNA-protein binding as examples. For strong affinity binding, such as between cyclosporine and its antibody, complexes are formed prior to CE-LIFP analysis. For weaker binding, such as between single-stranded DNA and its binding protein, the single-stranded DNA binding protein is added to the CE separation buffer to enhance dynamic formation of affinity complexes. Both fluorescence polarization (and anisotropy) and mobility shift results are complementary and are useful for immunoassays and binding studies.

Antibodies↗
PubMed1990

Measurement of cyclosporine in plasma of cardiac allograft recipients by fluorescence polarization immunoassay.

The Abbott TDx fluorescence polarization immunoassay (FPIA) procedure for measuring cyclosporine A (CsA) was evaluated and compared with the Sandoz polyclonal radioimmunoassay (CsA RIA kit) method. This drug assay was evaluated for precision, calibration, stability, and accuracy. Within-run precision studies utilizing 25 replicate analyses of the three control preparations (containing CsA in the 60-800 ng/ml range) resulted in coefficients of variation (CV) ranging from 1.0 to 9.1%. The CVs of between-run precision determined by assaying the same control drug levels for five consecutive working days ranged from 3.9 to 4.6%. Calibration curve stability was assessed by examining the drift in control values over a 2-week period. Maximum plasma ranged from 82.6 to 108.2%. Four hundred plasma samples were obtained from 30 heart-transplant patients during the first 6 months of CsA therapy and each sample was analyzed simultaneously by TDx and RIA. Linear regression analysis of the results obtained for each patient (x = RIA, y = FPIA) revealed the following mean values: r = 0.87, (CV = 13.7%), slope = 1.47 (CV = 39.2%). Moreover, the concentration of CsA was determined in 35 patient samples both by TDx and high-performance liquid chromatography (HPLC). FPIA results up to 12 times higher than HPLC results have been noted.

Chromatography, High Pressure Liquid↗
PubMed2005

Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy.

To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <<10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments.

Actins↗
PubMed1981

Cellular blood fluorescence polarization: a possible prognostic tool in human acute lymphatic leukemia.

We measured the fluorescence polarization of 1-6 diphenyl hexatriene in mononucleated cells from the peripheral blood of 49 acute lymphoid leukemic patients in complete hematological remission with an Elscint MV-1 microviscosimeter. The measurements of fluorescence polarization were made at various times after the remission was achieved. Our results represent two years of this prospective study. We found that lymphoid leukemic patients in remission could be separated into two groups according to the lowest value of fluorescence polarization of their blood cells. 1) The first group of 26 patients showed values superior to 3.44 poises, in all samples, corresponding to the lowest value of normal controls. 2) The second group showed at least one value of blood fluorescence polarization lower than 3.44 poises. This second group had a significantly lower median duration of remission and survival compared to the first. We have subdivided these two groups according to other established prognostic parameters such as cytological subtype and initial volume of the disease: we found that cellular fluorescence polarization could give additional prognostic information. These data suggest that fluorescence polarization measurements have a prognostic value to follow acute lymphoid leukemia patients in complete remission: when a low value is found there is a significant risk of relapse.

Adolescent↗
PubMed1985

Fluorescence polarization applied to cellular microrheology.

The fluorescence polarization of probe molecules gives information on the "fluidity" of probe environment. Although the data cannot be related with absolute values of microviscosity, the method is largely used for probing the "fluidity" of lipid regions of biological membranes. Therefore, fluorescence polarization is of interest in clinical research, for membrane alterations are associated with either pathological processes of red cells, platelets, leukocytes or important cell functions.

Blood Platelets↗
PubMed1985

Change in the physical state of platelet plasma membranes upon ionophore A23187 activation. A fluorescence polarization study.

Human platelets were isolated and fluorescence-labelled by 1,6-diphenylhexatriene. Diphenylhexatriene was essentially localized in the plasma membrane, as indicated by trinitrobenzenesulfonate-quenching experiments. A decrease of the fluorescence polarization of diphenylhexatriene was observed upon ionophore A23187 addition in the absence of aggregation. 0.3 microM ionophore allowed to reach the maximum rate of the decrease of fluorescence polarization; it also maximally stimulated the light transmission change, the serotonin release and the thromboxane B2 synthesis. The amplitude of the fluorescence polarization decrease was maximum at platelet concentrations between 4 X 10(7) and 7 X 10(7)/ml. The presence of Ca2+ in the medium increased the rate constant of the polarization change. Chlorpromazine (60 microM) completely inhibited this transition, but at 30 microM its inhibitory effect was reversed by Ca2+. The membrane events implied in platelet activation very likely lead to fluidization of the plasma membrane, perhaps by its fusion with the membranes of internal granules which are relatively depleted of cholesterol. Ca2+ plays a central role in the triggering of the observed effects at the membrane level.

Blood Platelets↗
PubMed1985

Real-time fluorescence polarization measurements: interaction of phospholipase A2 with a fluorescent lecithin derivative.

We have modified an SLM 4800 fluorometer to allow continuous measurement of fluorescence polarization. The modifications included construction of simple mechanical and electronic accessories which allowed polarizer position to be program-controlled by an HP 9815 calculator. With these modifications we were able to follow the interaction of the fluorescent lipid analog 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminododecanoyl phosphatidylcholine (I) with Naja-naja venom phospholipase A2 (PLA2) (E.C.3.1.1.4). Upon addition of aliquots of PLA2 containing up to 85 ng protein to a cuvette containing 1-2 microM I, the total measured polarization decreased linearly with time for at least 1000 s. Concomitant analyses of equivalent incubation mixtures and analyses of the contents of the cuvette after incubation and collection of fluorescence data revealed time-dependent formation of N-4-nitrobenzo-2-oxa-1,3-diazole-aminododecanoic acid (II). The decrease in total measured polarization was accelerated by Ca2+ and inhibited by EDTA. These data suggest that PLA2 activity in Naja-naja venom can be measured rapidly at low concentrations of both enzyme (0.01 microgram protein) and substrate (1 microM). Since this technique can be used to collect polarization data over time intervals as short as 4 s, it should be possible to measure the early changes in polarization during the interaction of fluorescent probes with proteins or membranes.

Animals↗
PubMed1987

Pitfalls in measuring fluorescence polarization in mitogen-stimulated T-lymphocytes.

Fluorescence polarization measurements with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to detect changes in the fluidity of plasma membranes from T-lymphocytes stimulated with mitogens. When the cells were incubated with succinyl-concanavalin A an increase in fluorescence polarization was observed. This, however, could be shown to be due to the interaction of the mitogen with the label DPH and did not reflect changes in the plasma membrane. In purified plasma membranes a decrease rather than an increase of fluorescence polarization was observed.

Animals↗
PubMed1996

A homogeneous fluorescence polarization assay for detection of antibody to Brucella abortus.

A homogeneous fluorescence polarization (FP) assay (FPA) was developed for detection of antibody in bovine sera to Brucella abortus. The assay used O-polysaccharide prepared from B. abortus lipopolysaccharide in the molecular weight range of 20-30 kDa which was conjugated with fluorescein isothiocyanate and used as a tracer. Fluorescence polarization was measured with a FPM-1 fluorescence polarization analyzer. Sample (20 microliters) was added to 2.0 ml of diluent buffer at ambient temperature. A serum blank reading was taken and tracer (10 microliters) to yield approx. 1.5 nM fluorescein equivalents was added. The FP of the tracer was determined after a period of greater than 2 min. A positive reaction was indicated by a significant elevation of the FP reading over the negative control. In a blind study, 9480 bovine sera were tested in addition to sets of four controls which were included with each lot of 100 samples tested. The controls were a strong positive, a weak positive, a negative and a serum derived from a B. abortus strain 19 vaccinated cow. Test sera included 8669 sera from Canadian cattle which were negative by routine serological tests, 561 sera from cows from which B. abortus had been isolated either from tissues or milk and 250 sera from cattle previously vaccinated with B. abortus strain 19 at various times. One lot of O-polysaccharide tracer was used for all tests. The initial cut-off for negative samples in the fluorescence polarization assay was set at 107.2 mP. This resulted in a sensitivity estimate of 98.1 +/- 1.1% and the specificity was 99.8 +/- 0.09%. After decoding the samples and retesting false positive and negative reactions, the sensitivity estimate was 98.5 +/- 1.0% and the specificity was 100%. It became evident that the initial cut-off value was set too high and, using ROC analysis, a cut-off of 90 mP increased the sensitivity to 99.02% while the specificity decreased to 99.96%. Of the 250 sera from vaccinated cattle, 248 were negative giving a point specificity value of 99.2%.

Animals↗