ABSTRACT
Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/H2O2/enhancer). The second immunoassay is based on the use of bovine serum albumin-daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu3+-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.
Subject(s)
Immunoenzyme Techniques , Isoflavones/analysis , Isoflavones/blood , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/blood , Cross Reactions , Dose-Response Relationship, Immunologic , Equilin/blood , Female , Fluorescent Dyes , Genistein/blood , Haptens/blood , Horseradish Peroxidase , Humans , Immunoglobulin Fc Fragments/blood , Lignans/blood , Luminescent Measurements , Molecular Structure , Phenanthrolines , Polystyrenes , Radioimmunoassay , Serum Albumin, Bovine , Steroids/bloodABSTRACT
We demonstrate direct three-dimensional (3-D) microfabrication inside a volume of silica glass. The whole fabrication process was carried out in two steps:(i) writing of the preprogrammed 3-D pattern inside silica glass by focused femtosecond (fs) laser pulses and (ii) etching of the written structure in a 5% aqueous solution of HF acid. This technique allows fabrication of 3-D channels as small as 10mum in diameter inside the volume with any angle of interconnection and a high aspect ratio (10mum -diameter channels in a 100mum -thick silica slab).
ABSTRACT
In the control of tomato transgenic cultivars it is important to determine the glycoalkaloid content, which requires the screening of many samples. We have developed a simple method for the extraction and determination of glycoalkaloids in the leaf and fruit extracts of tomatoes using a europium chelator entrapped in phosphatidylcholine-cholesterol containing liposomes. The concentration values were quantified from liposome lysis effected by glycoalkaloid action. The fluorescent signal is linear between 1 and 10 micrograms of tomatine, which makes the method useful for the analysis of tomato samples.
Subject(s)
Solanum lycopersicum/chemistry , Tomatine/analysis , Chelating Agents , Chromatography, High Pressure Liquid , Fluorometry , Liposomes , Tomatine/analogs & derivatives , Tomatine/chemistryABSTRACT
We show that, in the case of sum-frequency mixing, one can alleviate group-velocity mismatch between IR and UV pulses by choosing different pulse widths, thus extending the interaction length of ultrashort pulses within nonlinear crystals. By fifth-harmonic generation with a Nd:glass laser, we demonstrate efficient frequency upconversion of 195-fs 264-nm pulses under the envelope of 0.9-ps 1055-nm pulses in beta-barium borate crystal, yielding <270-fs pulses with energy of up to 110muJ at 211 nm.
ABSTRACT
We investigated the intensity-dependent loss properties of nonlinear crystals by using subpicosecond laser pulses at 264 and 211 nm. Two-photon absorption coefficients for potassium dihydrogen phosphate, beta-barium borate, and lithium triborate crystals were obtained from the intensity-dependent transmission measurements.
ABSTRACT
Theophylline (Th) has been selectively conjugated to the four amino groups of melittin (Mel) by solid phase peptide synthesis. The cytolytic activity of the resultant Th-Mel compounds was tested on liposomes trapping the bovine serum albumin (BSA) conjugate with 4,7-bis(chlorosulfophenyl)-1,10-phenanthrol ine-2,9-dicarboxylic acid (BCPDA). The loss of lytic activity was the highest for Th-K7-Mel. Th-G1-Mel retains almost the same lytic activity as Mel. A homogeneous liposome time-resolved fluoroimmunoassay (LITRFIA) of Th in serum has been carried out with Th-G1-Mel between 5 ng and 10 microg.
Subject(s)
Fluoroimmunoassay/methods , Liposomes/metabolism , Melitten/analogs & derivatives , Theophylline/analogs & derivatives , Theophylline/blood , Amino Acid Sequence , Antibodies/immunology , Calibration , Dose-Response Relationship, Drug , Europium , Fluorescent Dyes/metabolism , Haptens/immunology , Humans , Liposomes/chemistry , Melitten/metabolism , Melitten/pharmacology , Molecular Sequence Data , Permeability/drug effects , Phenanthrolines/metabolism , Sensitivity and Specificity , Serum Albumin/metabolism , Substrate Specificity , Theophylline/immunologyABSTRACT
In the cultivations of Cannabis, it is important to be able to distinguish fiber-type plants from drug-type plants by an easy observation of their phenotype. This study required the screening of many samples for their cannabinoid content. A simple and highly sensitive time-resolved fluoroimmunological method was developed for the determination of Delta(9)-tetrahydrocannabinol in the leaf extracts. The useful range of the calibration curve was between 10 pg and 25 ng of standard. Matrix effects were minimized by a high dilution of samples.
Subject(s)
Cannabis/classification , Dronabinol/analysis , Fluoroimmunoassay/methods , Animals , Chromatography, Gas , Mice , Mice, Inbred BALB CABSTRACT
Efficient generation of new spectral components owing to second-order cascading in a seeded broadband beta -barium borate typeI phase-matching parametric amplifier is demonstrated. One can vary the number and magnitude of these components by changing amplification bandwidth (wavelength) and phase-matching conditions. The phenomenon is treated theoretically by use of a formalism developed previously for the case of cascaded self-diffraction.
ABSTRACT
A time-resolved immunofluorometric assay of the IgG specific for Toxoplasma gondii has been compared with two commercial methods, one automated enzyme linked fluorescent assay (VIDAS) and one automated colorimetric enzyme immunoassay (BEIA). The coefficients of variation were 3.4% and 7.4% within-assay (n = 12), and 9.2% and 8.1% between-assay (n = 10) for two sera at low and high concentrations of specific IgG. The regression lines obtained with 96 samples were y = 1.04x + 2.1 and y = 0.98x - 1.1. We also compared a time-resolved capture fluoroimmunoassay for Toxoplasma-specific IgM with a commercial immunoenzymatic assay (BEIA TOXO-M). The sensitivity and specificity were 100%, calculated from assays of 78 samples.
Subject(s)
Immunoglobulin G/analysis , Immunoglobulin M/analysis , Reagent Kits, Diagnostic , Toxoplasma/immunology , Animals , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Humans , Immunoenzyme TechniquesABSTRACT
A time-resolved fluoroimmunoassay (TR-FIA) for the direct determination of clenbuterol residues in horse urine using a highly specific monoclonal antibody has been compared with an immunoenzymometric assay (IEMA). The sensitivity of both methods was 10 pg; the calibration curve was linear between 10 and 10(5) pg for the TR-FIA and between 10 and 10(4) pg for the IEMA.
Subject(s)
Clenbuterol/urine , Fluoroimmunoassay , Horses/urine , Immunoenzyme Techniques , Animals , Calibration , Fluoroimmunoassay/statistics & numerical data , Immunoenzyme Techniques/statistics & numerical data , Sensitivity and Specificity , Time FactorsABSTRACT
A time-resolved fluoroimmunoassay (TR-FIA) and an immunoenzymometric assay (IEMA) were applied for the measurement of aflatoxin B1 in soya seeds, dried figs and raisins. The extraction procedure was simple and no clean-up was found to be necessary. Limits of detection were 0.5 microgram kg-1 for TR-FIA (range of linearity of calibration graph 2.5-5 x 10(4) pg; inter-assay relative standard deviation Sr < 5%) and 0.2 microgram kg-1 for IEMA (linear range 1.0-5 x 10(3) pg; Sr < 11%).
Subject(s)
Aflatoxin B1/analysis , Fluoroimmunoassay , Fruit/chemistry , Glycine max/chemistry , Immunoenzyme Techniques , Calibration , Food Analysis , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
We compared two commercial methods with a time-resolved immunofluorimetric assay for alpha-foetoprotein in human serum using the europium complex of 4,7-bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label. The correlation coefficients were r1 = 0.94 and r2 = 0.96.
Subject(s)
Blood Proteins/metabolism , alpha-Fetoproteins/metabolism , Antibodies/immunology , Biomarkers, Tumor/blood , Down Syndrome/blood , Down Syndrome/diagnosis , Europium/chemistry , Fetal Diseases/blood , Fetal Diseases/diagnosis , Fluorescent Antibody Technique , Fluorometry , Humans , Linear Models , Prenatal Diagnosis , Reference Standards , alpha-Fetoproteins/immunologyABSTRACT
We compared two time-resolved fluoroimmunoassay systems for measuring free oestriol in human serum and progesterone in bovine milk. By reading the fluorescence of europium complex of 4,7-bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid in solution, the measuring range is increased for both oestriol (10-50,000 ng/l instead of 25-50,000 ng/l) and for progesterone (10-50,000 ng/l instead of 25-10,000 ng/l). In addition, the interassay coefficients of variation were lowered from 9.5 to 5.7% for oestriol and from 7.5 to 5.4% for progesterone, at the smaller hormone concentrations detectable by each method.
Subject(s)
Estriol/blood , Fluoroimmunoassay/methods , Milk/chemistry , Progesterone/analysis , Animals , Cattle , Fluorescent Dyes , Humans , Male , PhenanthrolinesABSTRACT
The interaction between biotin and avidin, used in a single-step enzyme-immunoassay for ferritin determination, has been studied. The antigen is simultaneously bound by an antibody coated to a polystyrene bead and by an antibody coupled to biotin which reacts with avidin conjugated to peroxidase. We have assessed the optimal ratio between avidin, conjugated to peroxidase, and biotin, coupled to antibodies, to give rise to the best signal for a quantitative enzyme-immunoassay. We have found that a careful balance between biotinylated antibody and conjugated avidin is necessary for our purpose and a biotinylated antibody excess should be avoided since it causes a signal decrease. This ratio is uninfluenced by both the presence and the absence of the antigen. Thus, an avidin-biotin single-step methodology, which has proved to be reliable for routine use, was developed.
Subject(s)
Avidin/chemistry , Biotin/chemistry , Enzyme-Linked Immunosorbent Assay , Ferritins/analysis , Antigen-Antibody Complex , Humans , Nephelometry and Turbidimetry , Polystyrenes/chemistry , Reproducibility of ResultsABSTRACT
Proteins A and G were each labelled with two different europium chelates (p-isothiocyanatophenyl-ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid). Their affinities for IgG from rabbit, goat, horse, sheep, mouse, pig, and rat were then measured using time-resolved fluorescence. Protein G labelled with the second chelate was found to be especially effective in binding to goat and horse IgG.
Subject(s)
Europium , Immunoglobulin G/analysis , Nerve Tissue Proteins/metabolism , Staphylococcal Protein A/metabolism , Affinity Labels , Animals , Binding, Competitive , Chelating Agents , Edetic Acid/analogs & derivatives , Fluoroimmunoassay , Goats , Horses , Immunoglobulin G/metabolism , Mice , Pentetic Acid , Rabbits , Rats , Sheep , Species Specificity , SwineABSTRACT
A rapid, direct, solid-phase, time-resolved fluoroimmunoassay for free estriol in serum, using Europium-chelate-protein A as a label, is described. The coefficient of correlation with the results of RIA was 0.983.
Subject(s)
Estriol/blood , Fluoroimmunoassay/methods , Animals , Europium , Rabbits , Staphylococcal Protein AABSTRACT
A direct, solid-phase, time-resolved fluoroimmunoassay for progesterone in cow's and goat's milk, using europium-chelate-protein A as a label, is described. The coefficients of correlation with the results by RIA were 0.987 and 0.989.
Subject(s)
Immunoassay , Milk/analysis , Progesterone/analysis , Animals , Cattle , Europium , Female , Goats , Immunoassay/statistics & numerical data , Insemination, Artificial/veterinary , Pregnancy , Pregnancy Tests/veterinary , Quality Control , Radioimmunoassay , Staphylococcal Protein AABSTRACT
Progesterone has been assayed in several serum samples by a time-resolved fluoroimmunoassay (TR-FIA). The solid phase was 11 alpha-hydroxyprogesterone hemisuccinate bound covalently to ovalbumin and adsorbed on wells of polystyrene. The assay was based on competitive reaction of solid phase-bound hormone and samples with specific antibody labelled in situ with protein-A prelabelled with europium. The bound Eu was dissociated from the solid phase by an enhancement solution and measured by time-resolved fluorometry. The coefficient of correlation between TR-FIA and RIA was 0.97.
Subject(s)
Progesterone/blood , Europium , Fluorescent Antibody Technique , Humans , Indicators and Reagents , Staphylococcal Protein A , Time FactorsABSTRACT
A fluoroimmunoassay for progesterone is described. It involves a labelled antigen synthesized by linking phenanthroimidazole-2-amine to progesterone-11 alpha-hemisuccinate and specific antiserum (rabbit anti-progesterone-11 alpha-hemisuccinate-bovine serum albumin) with a heterogeneous procedure.