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At least 37 records · Page 2Linked to original sources
PubMed1997

Analysis of the intracellular processing of proteins: application of fluorescence polarization and a novel fluorescent probe.

Previous studies indicated that fluorescein derivatized bovine serum albumin was an ideal probe to monitor the time-dependent kinetics of antigen processing in the murine macrophage cell line J774. Whereas previous work focused on fluorescence intensity measurements, the present study relied on fluorescence polarization to dissect the local environment of the fluorescent hapten-protein within the endocytic system of the cell. A steady increase in both fluorescence intensity and fluorescence polarization of the cell population was detected for the first 100 min. However, at 100 min, a plateau in both fluorescence intensity and polarization was observed and was followed by a decrease in fluorescence polarization and a corresponding increase in fluorescence intensity. Western blot analyses revealed that the decrease in fluorescence polarization was due to proteolytic degradation of the probe within the cell. Using a combination of in vitro experiments and an additional fluorescent probe, it was determined that the initial increase in fluorescence polarization was due to movement of the probe through a pH gradient within the cell, suggestive of transport through the endocytic system. By combining fluorescence polarization, flow cytometry, and a unique fluorescent enhancement substrate, these studies represented a novel approach for monitoring intracellular trafficking and processing of proteins within macrophages.

Animals↗
PubMed1961

Studies on fluorescent antibody staining. I. Non-specific fluorescence with fluorescein-coupled sheep anti-rabbit globulins.

1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules.

Animals↗
PubMed2000

The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band.

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.

Chlorophyll↗
PubMed2006

Structural basis of fluorescence fluctuation dynamics of green fluorescent proteins in acidic environments.

Green fluorescent proteins (GFPs) have become powerful markers for numerous biological studies due to their robust fluorescence properties, site-specific labeling, pH sensitivity, and mutations for multiple-site labeling. Fluorescence correlation spectroscopy (FCS) studies have indicated that fluorescence blinking of anionic GFP mutants takes place on a time scale of 45-300 ms, depending on pH, and have been attributed to external proton transfer. Here we present experimental evidence indicating that conformational change in the protein &beta-barrel is a determining step for the external protonation of GFP-S65T (at low pH) using time-resolved fluorescence and polarization anisotropy measurements. While the average anionic fluorescence lifetime of GFP-S65T is reduced by approximately 18% over a pH range of 3.6-10.0, the fluorescence polarization anisotropy decays mostly as a single exponential with a rotational time of phi = 17 +/- 1 ns, which indicates an intact beta-barrel with a hydrodynamic volume of 78 +/- 5 nm3. In contrast, the total fluorescence (525 +/- 50 nm) of the excited neutral state of S65T reveals a strong correlation between the fluorescence lifetime, structural conformation, and pH. The average fluorescence lifetime of the excited neutral state of S65T as a function of pH yields pKa approximately 5.9 in agreement with literature values using steady-state techniques. In contrast to the intact beta-barrel at high pH, the anisotropy of neutral S65T (at pH <or= pKa) decays as a biexponential (e.g., at pH 5.8, phi1 =1.86 ns, beta1 = 0.03, phi2 = 17.5 ns, and beta2 = 0.25), which suggests a segmental mobility of the chromophore associated with conformational changes of the protein. The segmental motion of the S65T chromophore becomes faster with an enhanced amplitude ratio as pH is reduced. For comparative purposes, we also provide complementary FCS results on fluorescence blinking of the excited neutral state of an EGFP mutant (F64L/S65T) on a much slower time scale. Our results indicate that conformational rearrangement of the beta-barrel and the amino acids surrounding the embedded chromophore is a rate-determining step for external proton transfer and possibly cis/trans isomerization as nonradiative pathways that underlie fluorescence blinking of GFP mutants in an acidic environment. In addition, the neutral-state transition is likely to be involved in the blinking process previously observed for the anionic-state transition in several GFP mutants.

Anions↗
PubMed1998

Enhanced detection sensitivity of "fluorescence reduction" by shifting the analyte absorbance spectrum and use of a fluorescent paper with higher signal/noise ratio.

Nonfluorescing protein bands can be detected by the fluorescence optics of the commercial gel electrophoresis apparatus with automated scanning of the migration path (HPGE-1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins ("fluorescence reduction"). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of "fluorescence reduction" equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by "fluorescence reduction" provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 microg/lane of the gel tray. A theory of "fluorescence reduction" is presented which accounts for the existence of a linear relationship between band area and load.

Animals↗
PubMed2005

Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay.

Different from organic fluorescence dyes, fluorescent lanthanide complexes have the fluorescence properties of long fluorescence lifetime, large Stokes shift and sharp emission profile, which makes them favorable be used as the fluorescent labeling reagents for microsecond time-resolved fluorescence bioassay. Lanthanide complex-based fluorescence labels have been successfully used for highly sensitive time-resolved fluorescence immunoassay, DNA hybridization assay, cell activity assay, and bio-imaging microscopy assay. Since the technique allows easy distinction of the specific fluorescence signal of the long-lived label from short-lived background noises associated with biological samples, scattering lights (Tyndall, Rayleigh and Raman scatterings) and the optical components (cuvettes, filters and lenses), the sensitivity of fluorescence bioassay has been remarkably improved. This paper summarized the recent developments of lanthanide complex-based fluorescence labels and their applications in time-resolved fluorescence bioassays mainly based on the authors' researches and relative publications.

Animals↗
PubMed2003

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease, and 1 phosphatase.

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.

Biological Assay↗
PubMed1982

Fluorescence titration and fluorescence stopped-flow studies on skeletal muscle troponin labeled with fluorescent reagent.

Incorporation of skeletal muscle troponin C (TN-C) subunit into skeletal muscle troponin (TN) induces a large increase in the apparent binding constant of Ca2+ to the low affinity Ca2+-binding sites of TN-C (from 1 X 10(5) M-1 to 5.6 X 10(6) M-1 in the presence of 2 mM MgCl2), and a large decrease in the rate constant of the Ca2+ removal reaction from the low affinity Ca2+-binding sites of TN-C (from 230 s-1 to 37 s-1 in the presence of 2 mM MgCl2). On the other hand, no significant modification in the molecular kinetic mechanism of the local conformational change due to the Ca2+ binding or removal reaction with the high affinity Ca2+-binding sites of TN-C is observed as TN-C is incorporated into TN.

Animals↗
PubMed2000

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres.

BACKGROUND: Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS: Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS: The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS: Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake. Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.

Animals↗
PubMed1992

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics.

A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (greater than 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650-800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters: a) the rise-time of the fluorescence to the maximum level fm, b) the changes in the shape of the fluorescence emission spectra during the induction kinetics, c) the induction kinetics in the fluorescence ratio F690/F735 as well as d) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.

Chlorophyll↗
PubMed1998

pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation.

The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and nuclear fluorescence was observed. Cytosolic fluorescence constituted over 80% of the total. Excitation scanning of transfected cells revealed two GFPmut1-specific regions that were pH-sensitive over the physiologic range, and each region exhibited a unique pH "bias" in fluorescence emission. Excitation at or near the expected maximum of 488 nm (region II) uniformly resulted in fluorescence that was preferentially altered at acidic pH. In contrast, a novel "wild-type" excitation peak at 400 nm (region I) resulted in alkaline-biased fluorescence similar to that described for the wild-type chromophore in vitro, suggesting that wild-type spectral features disrupted in vitro by mutagenesis may be recovered in intact cells. Calibration of intracellular pH (pHi) with in situ fluorescence following excitation in either region revealed a semilogarithmic relationship between fluorescence intensity and pH within the physiologic range. We therefore measured pHi changes attributable to altered Na/HCO3 cotransport (NBC) activity both in GFPmut1-expressing cells and in paired untransfected cells loaded with BCECF. Basal NBC activity was the same in each group, as was the stimulation of activity by 10% CO2, thus validating the utility of GFPmut1 as a fluorescent probe for pHi and establishing a novel, useful, and practical application for GFPmut1 in monitoring pHi in real time.

Animals↗
PubMed2003

Measurement of fluorophore concentrations and fluorescence quantum yield in tissue-simulating phantoms using three diffusion models of steady-state spatially resolved fluorescence.

Steady-state diffusion theory models of fluorescence in tissue have been investigated for recovering fluorophore concentrations and fluorescence quantum yield. Spatially resolved fluorescence, excitation and emission reflectance Carlo simulations, and measured using a multi-fibre probe on tissue-simulating phantoms containing either aluminium phthalocyanine tetrasulfonate (AlPcS4), Photofrin meso-tetra-(4-sulfonatophenyl)-porphine dihydrochloride The accuracy of the fluorophore concentration and fluorescence quantum yield recovered by three different models of spatially resolved fluorescence were compared. The models were based on: (a) weighted difference of the excitation and emission reflectance, (b) fluorescence due to a point excitation source or (c) fluorescence due to a pencil beam excitation source. When literature values for the fluorescence quantum yield were used for each of the fluorophores, the fluorophore absorption coefficient (and hence concentration) at the excitation wavelength (mu(a,x,f)) was recovered with a root-mean-square accuracy of 11.4% using the point source model of fluorescence and 8.0% using the more complicated pencil beam excitation model. The accuracy was calculated over a broad range of optical properties and fluorophore concentrations. The weighted difference of reflectance model performed poorly, with a root-mean-square error in concentration of about 50%. Monte Carlo simulations suggest that there are some situations where the weighted difference of reflectance is as accurate as the other two models, although this was not confirmed experimentally. Estimates of the fluorescence quantum yield in multiple scattering media were also made by determining mu(a,x,f) independently from the fitted absorption spectrum and applying the various diffusion theory models. The fluorescence quantum yields for AlPcS4 and TPPS4 were calculated to be 0.59 +/- 0.03 and 0.121 +/- 0.001 respectively using the point source model, and 0.63 +/- 0.03 and 0.129 +/- 0.002 using the pencil beam excitation model. These results are consistent with published values.

Algorithms↗
PubMed1997

Capillary electrophoresis with laser-induced fluorescence detection for the analysis of free and immune-complexed green fluorescent protein.

Naturally fluorescing green fluorescent protein (GFP) was separated by capillary electrophoresis (CE) and detected by laser-induced fluorescence (LIF). Exploiting recombinant technology and the natural fluorescence of GFP presents the capability of preempting the need for fluorescent derivatization. Such an approach would circumvent the obstacles typically associated with covalently labeling and purifying analytes that undergo fluorescent labeling. The unique property of GFP to fluoresce naturally was combined with CE-LIF to compare GFP isoforms prepared recombinantly or in vitro with wild-type GFP isoforms isolated from native jellyfish Aequorea victoria. Second, GFP antisera were reacted with wild-type GFP and the formation of the GFP antigen-antibody complex was monitored. A simple borate buffer, pH 8.5, was ample for resolving the two isoforms of the naturally fluorescent GFP in less than 5 min. The separation of GFP from GFP-Ab was complete in less than 7 min with the individual components detectable at the picogram level. A number of factors influence CE separation and/or LIF detection including sample buffer pH and incorporation of the additive 1,4-diamino butane. Remarkably, conditions that severely impair fluorescence detection of free GFP do not diminish fluorescence detection of the GFP antigen-antibody complex in a similar manner. Thus, the antibody appears to preserve the natural fluorophore of GFP. These data lend credence to the utility of coupling naturally fluorescent GFP to the speed, automation, and reduced sample size benefits of CE-LIF analysis for efficient separation and detection of an immunoreaction. In principle, a fusion protein of antibody with GFP as the label in CE-based immunoassays offers an advantageous alternative to the fluor-labeling process usually required in LIF detection.

Animals↗
PubMed1986

Fusion of fluorescently labeled Sendai virus envelopes with living cultured cells as monitored by fluorescence dequenching.

Fluorescently labeled (bearing N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE)) reconstituted Sendai virus envelopes (RSVE) were used to study fusion between the viral envelopes and cultured living cells such as lymphoma, Friend erythroleukemia cells (FELC) and L cells. Incubation of fusogenic viruses with the above cell lines resulted in a relatively high degree (40-45%) of fluorescence dequenching. On the other hand, incubation of unfusogenic (trypsin or phenylmethylsulfonylfluoride (PMSF)-treated) RSVE with these cells led to very little (6-9%) fluorescence dequenching. The degree of fluorescence dequenching was linearly correlated to the surface density of the virus-inserted N-NBD-PE molecules. Fluorescence photobleaching recovery experiments showed that fusion of fluorescent RSVE with FELC resulted in an infinite dilution of the fluorescent molecules in the recipient cell membranes. The fluorescent probe 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (N-NBD-Cl) was covalently attached to envelopes of intact Sendai virions without significantly impairing their biological activity. Incubation of fluorescently labeled, intact Sendai virions with cultured cells resulted in about 20% fluorescence dequenching. The present data clearly indicate that fluorescently labeled Sendai virions can be used for a quantitative estimation of the degree of virus-membrane fusion.

Animals↗
PubMed2000

Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506.

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5 degrees C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30 degrees C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30 degrees C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50 degrees C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost.

Colony Count, Microbial↗
PubMed1993

Studies on the constancy of the blue and green fluorescence yield during the chlorophyll fluorescence induction kinetics (Kautsky effect).

Blue (F450) and green (F530) leaf fluorescence were studied together with the red chlorophyll fluorescence (emission maxima F690 and F735) during light-induced chlorophyll fluorescence induction kinetics (Kautsky effect) in predarkened leaves of wheat (Triticum aestivum L.) and soybean (Glycine max L.). The intensity of the red chlorophyll fluorescence decreased from maximum fluorescence Fm to steady-state fluorescence Fs, and the fluorescence ratio F690/F735 decreased by about 10% from Fm to Fs. However, blue and green fluorescence intensities remained constant throughout the measuring time. Consequently, the ratio of blue to red fluorescence (F450/F690) increased during chlorophyll fluorescence induction kinetics, whereas the ratio of blue to green fluorescence (F450/F530) remained unchanged within the same period. The knowledge of these ratios will be a prerequisite for the interpretation of remote sensing data from terrestrial vegetation.

Chlorophyll↗
PubMed1982

Fluorescent nucleotides: application in a fluorescence immune assay.

A fluorescence immune assay designed to measure anti-nucleotide antibody activity is described based on the synthesis of a fluorescent nucleotide probe possessing a low fluorescence quantum yield when free in aqueous solution (neutral pH). The fluorophore, AmNS (1-naphthylamine-5-sulfonic acid), was covalently conjugated to various phosphate derivatives of nucleotides through carbodiimide activation to form the fluorescent probe. The quantum yield (phi) of the fluorescent nucleotide in solution (neutral pH) was approximately 0.025 based on an excitation maximum of 320 nm and an emission maximum of 460 nm. Anti-nucleotide antibodies elicited in rabbits and mice served as standard immunological reagents in development of the fluorescence assay. Upon binding of an AmNS-nucleotide conjugate with homologous anti-nucleotide antibodies, the fluorescence quantum yield of the conjugate was significantly enhanced (12-35 x). Fluorescence enhancement was not obtained upon incubation of the fluorescent probe with normal Ig, non-immune rabbit sera and murine ascites fluid, or bovine and rabbit serum albumin. Nucleotide inhibition reactions were quantitatively measured in the fluorescence assay. Nucleotide binding results obtained with the fluorescence assay were correlated with a modified radioimmune Farr assay.

Antibody Specificity↗