Fluorometric Detection System Research Articles

Dual enzymes-mimic activity of nanolayered manganese-calcium oxide for fluorometric determination of metformin

There are different analytical methods available for the determination of metformin, as an oral hypoglycemic and antidiabetic drug, in biological samples. However, most of these methods suffer from some drawbacks, including high-priced materials and equipment, damaging chemical reagents, time-consuming nature, and tedious operation procedures. So, in this work a new, sensitive and simple method was reported for the detection of metformine. In this regard, nanolayered manganese-calcium oxide (NL-MnCaO2) were synthesized and characterized using scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy, and X-ray powder diffraction (XRD) techniques. Also, we studied the enzyme-like activity of synthesized particles and reported a bifunctional nanozyme, which performs the dual roles for peroxidase and catalase-mimicking. The results demonstrated the hindering effect of metformin on the peroxidase-mimic activity of NL-MnCaO2 and this effect was increased by raising metformin concentration. So, a sensitive fluorometric detection system was designed for the analytical assay of metformin, based on the terephthalic acid (TA)-H2O2 reaction with NL-MnCaO2. An acceptable linearity was observed between the metformin concentration and fluorescence quenching of the system in the range of 0.07–0.77 mM. Limit of detection (LOD) and limit of quantification (LOQ) were 0.17 μM and 0.96 μM, respectively. The proposed system was applied for the estimation of metformin concentration in serum samples by recoveries of 86.68–106%. So, the proposed fluorometric method provides some main advantages such as wide linear range, low detection limit, rapid detections, high sensitivity, and good practicability for the determination of metformin in biological samples.

A convenient fluorometric test method for skin sensitization using glutathione in chemico

ABSTRACT A convenient fluorometrical test method to identify skin sensitizers in chemico was developed using reactivity with glutathione (GSH), a low molecular weight endogenous substance. Following incubation of test chemicals with GSH, the remaining GSH was quantitated fluorometrically by using monobromobimane (mBBr), a thiol-detecting agent, for determining % depletion of this endogenous substance by test chemicals. The experimental conditions optimized were: (1) reactivity of thiol compounds including GSH with mBBr, (2) effects of vehicles on reactivity, (3) molar ratios of GSH to test chemicals, and (4) reactivity of endogenous substance with test substances under different incubation times. When an optimized condition with DMSO as a vehicle for test chemicals and in 1:60 ratio for 24 hr at 4°C was applied to classify 48 well-known skin sensitizers and non-sensitizers, the predictive capacity was as follows: 88.2% sensitivity, 78.6% specificity, and 85.4% accuracy with 95.8% consistency of three trials when 10.3% depletion of GSH was used as a cutoff value. Because the present method employed relatively simple GSH as an acceptor for sensitizers and/or a relatively convenient fluorometric detection system in 96-well plates for a high throughput test, it would be a useful test tool for screening skin sensitization potential of test chemicals.

Study on bisphenol F, a bisphenol A analogue, at a dairy company: Health hazard and risk assessment

The occurrence of analogues of bisphenol A (BPA), including bisphenol F(BPF) in milk is still not well known. BPF may enter the milk chain at the farm and during milk processing at the dairy company. This study identified the main BPF contamination pathways using a monitoring model based on the identification of the hazard at three stages along the dairy chain: raw milk from the storage tank, pasteurized milk from the storage tank, and cardboard packaged milk. Quantitative analysis was performed by high-performance liquid chromatography with fluorometric detection (HPLC/FD) system. BPF was detected in all analysed stages (from <LOQ to 2.686μg/L). The structural and toxicological similarity between BPF and BPA suggested considering both bisphenols for a more comprehensive risk evaluation. The daily intake of BPF and of the sum of BPF and BPA, and the worst-case scenario through the consumption of packaged milk were calculated. Exposure levels below the temporary daily intake, fixed for only BPA, were detected in all consumer age classes. Nevertheless, the use of BPA substitutes represents a risk to human health because of their potential synergic effects. The application of a monitoring program at each stage of milk processing at the dairy company may represent a useful strategy to ensure food safety in the milk chain.

Assessment of skin sensitizing potential of metals with β-galactosidase-expressing E. coli culture system

ABSTRACTIt has been a challenge to develop in vitro alternative test methods for accurate prediction of metallic products which may exert skin sensitization, as several test methods adopted by OECD were relatively ineffective in assessing the capacity for metallic compounds to exert sensitizing reactions, compared with organic test substances. Based upon these findings, a system that incorporates β-galactosidase producing E. coli cultures was tested for its predictive capacity to well-known metallic sensitizers. In this system, E. coli cells were incubated with metal salts at various concentrations and β-galactosidase suppression by each test metal was determined. Fourteen local lymph node assay (LLNA) categorized metal salts were examined. Although color interference from metal salts was minimal, a fluorometric detection system was also employed using 4-methylumbelliferyl galactopyranoside as a substrate for β-galactosidase to avoid the color interference, concomitantly with the original UV-spectrometric method. Data demonstrated that two detection methods were comparable and complementary. In addition, most of the metallic sensitizers were correctly identified at 0.6 and 0.8 mM concentrations. Despite the lower specificity obtained in the current study and small number of substances tested, the developed method appears to be a relatively simple and effective in vitro method for detecting metallic sensitizers. When 61 chemicals tested in the β-galactosidase producing E. coli cultures including the present study were collectively analyzed, the prediction capacity was as high as other OECD-adopted tests: 95.6% of sensitivity, 66.7% of specificity, and 88.5% of accuracy. It is important to emphasize that animals or mammalian cell cultures were not required in the current method, which are in accordance with the EU guidelines on restricted or banned animal testing.

Application of surface molecular imprinted magnetic graphene oxide and high performance mimetic behavior of bi-metal ZnCo MOF for determination of atropine in human serum

Herein, high performance peroxidase-like activity of zinc and cobalt bi-metal metal-organic framework (ZnCo MOF) is reported and applied for the sensitive measurement of atropine. ZnCo MOF was synthesized by the reaction of 2-methylimidazole with Zn and Co (II) cations in aqueous media. The colorimetric and fluorometric experiments were applied to investigate the catalytic activity of obtained MOF, using o-phenylenediamine (OPD) and terephthalic acid (TA) peroxidase substrates, respectively. The results demonstrated the more efficient mimetic behavior of ZnCo MOF compared with common Zn or Co MOFs. Besides, it was found that atropine hindered the catalytic action of ZnCo MOF and this effect was intensified by increasing the atropine concentration. So, it was considered to design a sensitive analytical assay for atropine detection. To assure a high specific recognition, molecularly imprinted polymer (MIP)-based extraction using magnetic graphene oxide supports was applied to extract atropine before its determination. The combination between the high specific extraction and great catalytic activity of ZnCo MOF led to the ultrasensitive and reliable determination of atropine. The best linear range of calibration graph was achieved using fluorometric detection system for 0.1–45 ng mL−1 atropine concentrations, and detection limit (3Sb/m) was 27 pg mL−1. The relative standard deviations (RSD %) for the determination of 1, and 10 ng mL−1 atropine (n = 5) were 2.13% and 3.08%, respectively. The explained fluorometric assay was examined for the measurement of atropine in biological fluids (Recoveries were in the range of 95.90–103.57%), and the results were validated by an official method.

Sensitive biosensing of organophosphate pesticides using enzyme mimics of magnetic ZIF-8.

Development of a sensitive detection method for the reliable screening of widely used organophosphorus (OP) toxins is a crucial request to control their side-effects. Herein, a novel fluorometric assay based on the acetylcholinesterase (AChE) inhibited enzymatic activity and the new peroxidase-like Fe3O4 nanoparticles@ZIF-8 composite (Fe3O4 NPs@ZIF-8) was developed for the determination of OPs. Magnetic Fe3O4 NPs were encapsulated into ZIF-8 and the high mimetic activity of produced composite was assessed on the oxidation of substrates. This observation was applied to the rapid detection of diazinon as a model OP compound. The sensing tool contains AChE and choline oxidase (CHO) enzymes, peroxidase colorimetric or fluorometric substrate, and Fe3O4 NPs@ZIF-8 as the catalyst. In the presence of mimic Fe3O4 NPs@ZIF-8, the generated H2O2 from the enzymatic reactions of acetylcholine is decomposed to hydroxyl radicals. The radicals oxidize the peroxidase substrates to generate a detectable signal. However, due to the inhibition effect of OPs on the enzymatic activity of AChE, lower H2O2 amounts are produced in the presence of diazinon. Using the fluorometric detection system, the generated signal is decreased proportionally by increasing diazinon concentration in the range of 0.5-500 nM. The limit of detection was obtained 0.2 nM. Consequently, the usage of high performance peroxidase-mimic Fe3O4 NPs@ZIF-8 provided a sensitive bio-assay with a potential to be applied as screening tool for toxic OP compounds. The developed assay was successfully applied for the determination of diazinon in water and fruit juices.

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid-liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30-1000ng for nicotine and 0.06-1000ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8-15ng/mL and 156-372ng/mL, respectively.

Impact of Sampling Parameters on the Radical Scavenging Potential of Olive (Olea europaea L.) Leaves

The impact of sampling parameters, that is, cultivar, leaf age, and sampling date, on the radical scavenging potential of olive leaf extracts was examined via the DPPH(*) and other assays. Total phenol content was estimated colorimetrically and by fluorometry, whereas phenol composition was assessed by RP-HPLC coupled with diode array, fluorometric, and MS detection systems. Oleuropein was not always the major leaf constituent. Considerable differences noted in individual phenol levels (hydroxytyrosol, oleuropein and other secoiridoids, verbascoside, and flavonoids) among samples were not reflected either in the total phenol content or in the radical scavenging potential of the extracts. It can be suggested that olive leaf is a robust source of radical scavengers throughout the year and that differentiation in the levels of individual components depends rather on sampling period than on cultivar or age. The latter does not present predictable regularity. Exploitation of all types of leaves expected in an olive tree shoot for the extraction of bioactive compounds is feasible.

Fluorometric Identification of 5-Methylcytosine Modification in DNA: Combination of Photosensitized Oxidation and Invasive Cleavage

An efficient fluorometric detection system of DNA methylation has been developed by a combination of a photooxidative DNA cleavage reaction with 2-methyl-1,4-naphthoquinone (NQ) chromophore and an invasive cleavage reaction with human Flap endonuclease-1. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at 5-methylcytosine mC) and hairpin-like probe oligomer possessing a fluorophore (F) and a quencher (D) resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing cytosine but not mC at the target sequence was extremely weak. In addition, by monitoring the fluorescence change, this system allows for the detection of mC in DNA at subfemtomole amounts. This system would provide a highly sensitive protocol for determining the methylation status in DNA by fluorescence emission.

Regulation of Neuronal Nitric-oxide Synthase Activity by Somatostatin Analogs following SST5 Somatostatin Receptor Activation

Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.

A new time-resolved fluorometric microarray detection system using core–shell-type fluorescent nanosphere and its application to allergen microarray

We have developed a new time-resolved fluorometric (TRF) microarray detection system consisting of fluorescent NH2 nanosphere, TRF microarray detector and gamma-irradiated polystyrene chip. Using the TRF microarray detector, we detected 500 particles of the fluorescent nanosphere in one channel. Cross-talk fluorescence from the adjacent channels was little observed in the TRF microarray detector (<0.0004 %). The TRF microarray detection system was further applied for serum allergen-specific immunoglobulin E (IgE) multi-analyses. As a labeled tag antibody, an anti-human IgE Fab' fragment-conjugated fluorescent nanosphere (Fab' nanosphere) was prepared as described previously. As a chip surface appropriate for allergen immobilization, the polystyrene chip surface was modified by gamma irradiation. The immunoassay reactivity using the gamma-irradiated polystyrene chip was approximately 2.5-times improved compared with that of the non-treated polystyrene chip. Non-specific adsorption of the Fab' nanosphere onto the gamma-irradiated polystyrene chip surface was very low level (<0.0009 %). In only 20 mul of serum, six allergen-specific IgEs could be simultaneously determined in one reaction well in fewer than 90 min. Good correlation curves were obtained between the microarray immunoassay and the CAP RAST fluoro-enzyme immunoassay (CAP/RAST FEIA) method (r > 0.961). Reproducibility (CVs) of the microarray immunoassay was 8.6 % to 19.0 % (n = 5).

25] Fluorescent imaging of mitochondrial nitric oxide in living cells

Publisher Summary The chapter presents a study related to fluorescent imaging of mitochondrial nitric oxide in living cells. Nitric oxide (NO) is an important modulator of mitochondrial respiration and membrane potential. The presence of a mitochondrial NO synthase (mtNOS) has been described in the inner membrane of isolated rat liver mitochondria. However, because NO is a highly diffusible, short-lived (0.5–5 sec), highly reactive radical, its determination poses considerable technical problems. In fact, until a while ago, the production of NO by mtNOS has only been indirectly characterized in broken mitochondria using techniques, such as spectroscopy or the citrulline assay. Two novel fluorometric detection systems have been developed allowing the demonstration of the presence of NO within mitochondria via microscopy. 4,5-Diaminofluorescein diacetate (DAF-2/DA) permits the direct detection of NO production, and the MitoTracker allows the detection of mitochondrial potential changes. Thus, whereas DAF-2/DA can be used as a semiquantitative method of evaluation of NO production in living cells, the combination of these two systems provides an ideal tool for the spatial and direct visualization of NO within mitochondria. A variety of products from Molecular Probes allow the detection of mitochondrial activity.

Development of a Time-Resolved Fluorometric Detection System Using Diffusion-Enhanced Energy Transfer

A novel detection system using both emission energy transfer and time-resolved fluorometry (TRF) was developed, with a europium chelate as the energy donor and a novel fluorophore SNR1, excitable with long-wavelength light corresponding to europium emission, as the energy acceptor. When the donor and acceptor molecules were mixed in solution, energy transfer was observed without direct attachment of the donor and the acceptor, via a diffusion-enhanced energy-transfer mechanism. Thus, the acceptor emission can be detected as a long-lifetime fluorescence in TRF. When the fluorescence properties of the acceptor molecule are changed by interaction with an enzyme or other bioactive molecule, the change can be detected as a long-lived sensitized emission. If we develop or select suitable acceptor molecules, this simple and convenient system should be applicable to a wide variety of bioactive molecules. Since it is based on TRF, it can be used for high-resolution assay.

Direct Evidence of Nitric Oxide Presence within Mitochondria

Nitric oxide (NO) has been implicated in the modulation of mitochondrial respiration, membrane potential, and subsequently in apoptosis. Although the presence of a mitochondrial NO synthase (mtNOS) has been described, there is no direct evidence in vivo of the presence of NO within mitochondria. It was the aim of this study to demonstrate the in vivo production of NO within mitochondria. Using the novel fluorometric NO detection system, 4,5-diaminofluorescein diacetate (DAF-2/DA), we observed the presence of NO production in PC12 and COS-1 cells by conventional and confocal fluorescence microscopy. Part of the overall NO signal was colocalized within a subpopulation of mitochondria, labeled with the potential-dependent probe MitoTracker red. These findings demonstrate for the first time that the subcellular distribution of NO production is consistent with the presence of a mitochondrial NOS. Our results provide a new tool to directly study the modulatory role of NO in mitochondrial respiration and membrane potential, in vivo.

Detection of Irradiation of Meats by HPLC Determination for o-Tyrosine Using Novel LASER Fluorometric Detection with Automatic Pre-column Reaction.

An o-Tyrosine method for detection of irradiation of foods was studied by HPLC using a novel light amplification by stimulated emission of radiation (LASER) fluorometric detection system with pre-column reaction. Sample was prepared and purified by eliminating fat and sugars using a mixture of acetone and chloroform, and then the purified protein was hydrolyzed using hydrochloric acid at 110°C for 24h in a vacuum. The sample was reacted with 4-fluoro-7-nitrobenzofurazan (NBD-F) reagent by an automatic pipetting system and was introduced into the HPLC system. Irradiated chicken, pork, beef, and tuna were examined by irradiating at 0, 1, 5, 10 kGy. Irradiation of chicken and pork irradiated at or over 10 kGy was successfully detected, but that of beef and tuna were more difficult to detect. After 3 months storage at-20°C, the irradiation was still detectable in chicken irradiated at 10 kGy. Thus this detection procedure can be used to detect irradiation in some chilled meats irradiated at 10 kGy. Non-irradiated o-tyrosine formation and reduction of o-tyrosine by hydroxylation are also discussed.

Temporal and anatomical distribution of nitric oxide synthase mRNA expression and nitric oxide production during central nervous system inflammation

Nitric oxide (NO) has important roles in inflammatory processes. It was the aim of this study to ascertain whether changes in nitric oxide synthase (NOS) mRNA expression lead to similar temporal and anatomical changes in NO production in an experimental model of CNS inflammation. NOS-II (inducible NOS) mRNA expression was analyzed 2, 4, 6 and 24 h after intracerebroventricular (i.c.v.) injection of interleukin-1β (IL-1β) or vehicle. Increased expression of NOS-II mRNA was observed surrounding the microinjection site and meninges. The changes were significantly higher than controls at 4 and 6 h, returning to baseline at 24 h. Using the novel fluorometric NO detection system, 4,5-Diaminofluorescein diacetate (DAF-2/DA), for the direct detection of NO production, we observed a significant increase in NO production after 4 and 6 h. NO production was observed in areas surrounding the injection site, meninges surrounding the brain and perivascular cells and neuron-like cells throughout the cortex. However, increases in NO production in these areas remained significantly higher than controls at 24 h. These findings demonstrate for the first time that, in fresh frozen tissue, that the anatomical distribution of NOS-II mRNA is consistent with the distribution of NO production. We conclude that increases in NOS-II mRNA following i.c.v. administration of IL-1β lead to increases in NO production. While the mRNA is degraded by 24 h post treatment, the enzyme remains active. We propose that the DAF-2/DA method can be used as a potential marker in the diagnosis of CNS inflammation.

Early detection of bacteremia in the neonatal intensive care unit using the new BACTEC system.

With the newer techniques of culture analysis such as the BACTEC 9240 fluorometric detection system, detecting bacteremia in the neonate may be possible in a significantly shorter time. We hypothesized that neonatal bacteremia can be detected in less than 48 hours by this method. Our study included a retrospective review of 613 blood cultures obtained during the period August 1, 1995 to March 18, 1996 taken from 325 infants who had cultures drawn with a sepsis work-up and/or repeat cultures who had initial positive cultures in our Neonatal Intensive Care Unit. Results of blood cultures were studied in conjunction with the variables of body weight, gestational age, organism grown, complete blood count (CBC), and timing of positive cultures. Statistical analyses were performed using Fisher's and two-tailed Student's t tests. The results showed that of 325 infant blood cultures 49 (15%) were positive. Of these, 64% were coagulase-negative staphylococci, 14% viridans streptococci, 8% Escherichia coli, 4% Enterococcus sp., 4% Pseudomonas sp., 2% Enterobacter sp., 2% Klebsiella sp., and 2% Candida albicans. Of the positive blood cultures taken from infants not on antibiotics at the time of culture, 54% were detected positive at 18 hours, 71% at 24 hours, and 100% by 30 hours. Detection time by organism type was as follows: coagulase-negative staphylococci, 21.7 hours; viridans streptococci, 15.6 hours; E. coli, 7.5 hours; Enterococcus sp., 12 hours; Enterobacter sp., 5 hours; Klebsiella sp., 10 hours; and Pseudomonas sp., 12 hours. Our results indicate that the BACTEC 9240 fluorometric detection system helps in early identification of neonatal bacteremia (in 24 to 30 hours), with Gram-negative organisms being detected earlier than Gram-positive organisms (p < 0.05) and having significantly higher immature neutrophils in a CBC (I:T ratio of > or = 0.2 (p < 0.001). Early detection of neonatal bacteremia using this method will allow earlier diagnosis and appropriate treatment of the potentially bacteremic and bacteremic infant.

Carcinogenic Polynuclear Aromatic Hydrocarbons in the Atmosphere in Chiang Mai, Thailand.

Ambient concentrations of 7 mutagenic and/or carcinogenic polycyclic aromatic hydrocarbons (PAHs) were measured in Chiang Mai, Thailand, and compared to those in Tokyo. In Chiang Mai, airborne particulate samples were collected at two sites, i.e., commercial and residential sites (two stations in each site) from July through December in 1989. In Tokyo, sampling was carried out at a commercial/residential site in November 1989. PAHs were extracted by sonication, and analyzed by a multicolumn HPLC/computerized fluorometric detection system. Major observations are as follows ; 1) In both cities, ambient concentrations of PAHs and airborne particulates showed log normal distribution. Geometric mean/standard deviation is appropriate to express the situation of pollution. 2) In Chiang Mai, there was not a large difference in PAH concentrations between commercial and residential sites. This suggested wide-spreaded pollution over the city. On the other hand, concentrations of airborne particulates showed large differences among the sites. PAH concentrations in 1989 were higher than those in 1986. 3) Ambient concentrations of PAHs and airborne particulates concentrations were both low in the rainy season and high in the dry season in Chiang Mai. 4) Ambient concentrations of PAHs (except for rather volatile 4 ring PAHs) and airborne particulates in Chiang Mai were suggested to be higher than those in Tokyo. 5) In Tokyo, a fairly good correlation among concentrations of 7 PAHs and airborne particulates has been observed. In Chiang Mai, there has been good correlation among PAHs concentrations, but a poor correlation between PAHs and airborne particulate concentrations. This suggested that large content of rough particulates in Chiang Mai air may be higher than that in Tokyo. These results suggest that air pollution by PAHs and airborne particulates may be more serious in Chiang Mai than in Tokyo. Size distribution of airborne particulates seems to be different between these two cities.

Analytical application of the co-fluorescence effect in detection of europium, terbium, samarium and dysprosium with time-resolved fluorimetry

Application of the co-fluorescence effect has been examined for the simultaneous detection of the lanthanide ions Eu 3+, Tb 3+, Sm 3+ and Dy 3+. In the presence of Y 3+ and 2,2′-bipyridine (BP), the fluorescence intensities of the pivaloyltrifluoroacetone chelates of these lanthanides were greatly enhanced by an inter-chelate energy transfer process. Under optimized conditions, the following detection limits were obtained; 0.019 p M for Eu 3+, 0.27 p M for Tb 3+, 3.8 p M for Sm 3+ and 20 p M for Dy 3+, when a sensitive time-resolved fluorometer was used for the measurement. A co-fluorescence based fluorescence enhancement solution was also tested in a dual-label time-resolved immunofluorometric assay of luteinizing and follicle stimulating hormones (LH and FSH) based on the use of Eu 3+ and Tb 3+ as the label ions. The present fluorometric detection system is particularly well suited for multilabel time-resolved fluorometric immunoassays utilizing two, three or four ion labels simultaneously.

Fiber type-specific distribution of parvalbumin in rabbit skeletal muscle

A highly sensitive sandwich ELISA for parvalbumin (PA), based on a fluorometric detection system, was developed. This assay detected PA concentrations as low as 20 pg/ml (2 pg per assay) and was used for measuring PA contents in fragments of single muscle fibers isolated from freeze-dried 100-150 microns thick cross sections. The fibers were typed according to their histochemically assessed mATPase in parallel cross sections. Type I fibers from rabbit tibialis anterior (TA) and vastus lateralis (VL) muscles contained extremely low PA concentrations (2-5 micrograms/g w.wt.). Type IIA fibers displayed slightly higher values with mean values of 17 and 29 micrograms/g w.wt. (range 5-65) in TA and VL, respectively. Much higher PA concentrations were found in type IIB fibers with wide ranges from 75-1150 micrograms/g w.wt. in TA and 440-1370 micrograms/g w.wt. in VL. Whereas the IIB fibers of the TA displayed a continuum, two subgroups were distinguishable according to their PA contents (means of 590 and 1230 micrograms/g w.wt.) in VL. Possibly, the population with the lower PA content which was histochemically defined as type IIB in the present study, corresponds to fiber type IID. The finding that PA is predominantly present in type IIB fibers was also confirmed by the parallel decay of PA and type IIB fibers during chronic low-frequency stimulation. The use of freeze substitution, or alternatively, of freeze-drying, made it possible to demonstrate PA immunohistochemically without artifacts and to evaluate the staining intensity by microphotometry.(ABSTRACT TRUNCATED AT 250 WORDS)