scholarly journals Solid-Phase PCR with Hybridization and Time-resolved Fluorometry for Detection of HLA-B27

2001 ◽  
Vol 47 (3) ◽  
pp. 498-504 ◽  
Author(s):  
Minna Sjöroos ◽  
Jorma Ilonen ◽  
Timo Lövgren
Keyword(s):  
Solid Phase ◽  
Detection System ◽  
Pcr Primers ◽  
Dried Blood Spots ◽  
Microtiter Plate ◽  
Covalent Immobilization ◽  
Hla B27 ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Consecutive Reactions

Abstract Background: Preactivated solid surfaces provide new possibilities for multiple consecutive reactions in a microtiter plate format. In this study, a combination of PCR and subsequent hybridization in the same microtiter well was applied for the detection of HLA-B27 alleles. Methods: A multiplex solid-phase PCR to amplify the HLA-B27 alleles together with β-actin as an amplification control gene was performed on the NucleoLinkTM (Nunc) surface. PCR was followed by hybridization and detection with time-resolved fluorescence. For the covalent capture of the PCR primers onto the solid support via a 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride-mediated reaction, different 5′-end modifications of oligonucleotides were tested [amination, phosphorylation, and a poly(dT)10 linker]. Results: For covalent immobilization of the primers, amination of the 5′ end combined with use of the poly(dT)10 linker was superior. At least 19.5% of the primer added per well was attached via a stable bond. When the standard time-resolved, fluorescence-based HLA-B27 detection system was compared with the newly developed method in a sample series of 82 genomic DNAs and the corresponding dried-blood spots, all results were in full agreement. Conclusions: The new solid-phase PCR approach can be applied for multiple-target DNA detection. PCR followed by hybridization can be accomplished in a few hours using precoated strips and dried-blood spot PCR templates.

Sensitive, rapid procedure for time-resolved immunofluorometry of lutropin.

1988 ◽  
Vol 34 (8) ◽  
pp. 1640-1644 ◽  
Author(s):  
M J Khosravi ◽  
R C Morton ◽  
E P Diamandis
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Detection System ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Practical Procedure ◽  
Daily Monitoring ◽  
Fluorescence Measurements ◽  
Capture Antibody ◽  
Europium Chelate

Abstract In this new immunofluorometric method for quantification of lutropin in serum, the "sandwich" principle is combined with time-resolved fluorescence measurements, with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) used as label. A monoclonal antibody to the alpha-subunit of lutropin is adsorbed onto the walls of white-opaque microtiter wells to form the solid-phase capture antibody, and a biotin-labeled soluble monoclonal antibody is used for antigen quantification. The detection system is completed with streptavidin, which has been linked to a protein bulking agent labeled with multiple BCPDA residues. In the presence of excess europium, the fluorescence of the final complex attached to captured lutropin molecules is measured on the dried solid phasse with an automated time-resolved fluorometer. The assay can be performed as a rapid (less than 60 min incubation) or regular (150 min incubation) procedure. The rapid assay is well-suited for routine daily monitoring of increasing or ovulatory lutropin concentrations; the regular assay, with its greater sensitivity (0.5 int. unit/L), is a practical procedure for lutropin measurements in hyposecretory states. The assay measures up to 240 int. units/L, and results compare well with those by a commercially available radioimmunoassay, an immunoradiometric assay, and another time-resolved immunofluorometric procedure.

scholarly journals Multispectral LIF-Based Standoff Detection System for the Classification of CBE Hazards by Spectral and Temporal Features

Sensors ◽  
2020 ◽  
Vol 20 (9) ◽  
pp. 2524 ◽  
Author(s):  
Lea Fellner ◽  
Marian Kraus ◽  
Florian Gebert ◽  
Arne Walter ◽  
Frank Duschek
Keyword(s):  
Spectral Data ◽  
Classification Accuracy ◽  
Detection System ◽  
Laser Source ◽  
Nanosecond Laser ◽  
Time Resolved Fluorescence ◽  
Data Set ◽  
Time Resolved ◽  
Standoff Detection

Laser-induced fluorescence (LIF) is a well-established technique for monitoring chemical processes and for the standoff detection of biological substances because of its simple technical implementation and high sensitivity. Frequently, standoff LIF spectra from large molecules and bio-agents are only slightly structured and a gain of deeper information, such as classification, let alone identification, might become challenging. Improving the LIF technology by recording spectral and additionally time-resolved fluorescence emission, a significant gain of information can be achieved. This work presents results from a LIF based detection system and an analysis of the influence of time-resolved data on the classification accuracy. A multi-wavelength sub-nanosecond laser source is used to acquire spectral and time-resolved data from a standoff distance of 3.5 m. The data set contains data from seven different bacterial species and six types of oil. Classification is performed with a decision tree algorithm separately for spectral data, time-resolved data and the combination of both. The first findings show a valuable contribution of time-resolved fluorescence data to the classification of the investigated chemical and biological agents to their species level. Temporal and spectral data have been proven as partly complementary. The classification accuracy is increased from 86% for spectral data only to more than 92%.

Laser-excited immunofluorometric assay of prolactin, with use of antibodies coupled to lanthanide-labeled diethylenetriaminepentaacetic acid.

1986 ◽  
Vol 32 (7) ◽  
pp. 1323-1327 ◽  
Author(s):  
H Déchaud ◽  
R Bador ◽  
F Claustrat ◽  
C Desuzinges
Keyword(s):  
Plasma Sample ◽  
Solid Phase ◽  
Nitrogen Laser ◽  
Diethylenetriaminepentaacetic Acid ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Step Procedure ◽  
Polystyrene Tube ◽  
Sandwich Type ◽  
One Step

Abstract We describe an immunofluorometric assay for prolactin based on lanthanide labeling of a monoclonal antibody and measuring time-resolved fluorescence. In this "sandwich"-type assay, the label (Eu3+) was bound to the second antibody by means of a simple, rapid method involving the anhydride of diethylenetriaminepentaacetic acid. To measure the photoluminescence of europium (or other lanthanides), we have developed a time-resolved fluorometer with a nitrogen laser as the pulsed excitation source. During the assay, the solid-phase antibody immobilized inside a polystyrene tube is incubated with the plasma sample and the second antibody in a one-step procedure. Results for 67 human plasmas correlated well (r = 0.98) with those by an immunoradiometric method.

Characterization of Plasma-Modified Fluoropolymer Surfaces Using Steady-State and Time-Resolved Fluorescence Spectroscopy

1994 ◽  
Vol 48 (5) ◽  
pp. 630-637 ◽  
Author(s):  
Ming Li ◽  
Michaeleen L. Pacholski ◽  
Frank V. Bright
Keyword(s):  
Steady State ◽  
Fluorescence Spectroscopy ◽  
Sulfonyl Chloride ◽  
Sodium Dodecylsulfate ◽  
Covalent Immobilization ◽  
Ambient Conditions ◽  
Time Resolved Fluorescence ◽  
Decay Kinetics ◽  
Time Resolved

Poly(hexafluoropropylene-co-tetrafluoroethylene) (FEP) has been widely used in biotechnology because of its unique surface properties and biocompatibility. Recent work from our group has shown that plasma discharge-modified FEP can be used as the substratum for development of a very stable immunosensor. This result has prompted us to study further this new surface under ambient conditions. In this paper, we report on the covalent immobilization of a pyrene residue (-Py) onto FEP-APS (FEP-aminopropyl silane) surfaces and the characterization of FEP-APS-Py using steady-state and time-resolved fluorescence spectroscopy. Among the immobilization schemes tested, we found that the covalent coupling of pyrene-sulfonyl chloride to FEP-APS is the easiest and yields the most photostable FEP-APS-Py derivative. Steady-state emission spectra of FEP-APS-Py in contact with H2O, β-cyclodextrin (β-CD), and sodium dodecylsulfate (SDS) aqueous solutions differ considerably from those of Py-SO3 in solution. Time-resolved fluorescence spectroscopy of FEP-APS-Py demonstrates that the decay kinetics are strongly affected by the presence of ionic quenchers and molecular oxygen, as well as β-CD and SDS. The results are consistent with the suggestion that the APS-Py moiety undergoes a slow time-dependent reconfiguration at the FEP/APS interface.

A Simple Time-Resolved Fluoroimmunoassay of Total Thyroxine in Serum

1989 ◽  
Vol 26 (3) ◽  
pp. 238-243 ◽  
Author(s):  
Anastasia Papanastasiou-Diamandis ◽  
Vipin Bhayana ◽  
Eleftherios P Diamandis
Keyword(s):  
Dicarboxylic Acid ◽  
Binding Proteins ◽  
Solid Phase ◽  
Time Resolved Fluorescence ◽  
Competitive Immunoassay ◽  
Time Resolved ◽  
Serum Thyroxine ◽  
Total Thyroxine

We describe a non-isotopic heterogeneous competitive immunoassay of total thyroxine in serum. Thyroxine, released from its binding proteins by merthiolate (thimerosal), competes with immobilised thyroxine (thyroxine-bovine globulin conjugate) for binding to a monoclonal biotinylated antibody. The amount of biotinylated antibody bound, which is inversely related to the amount of thyroxine in the sample, is then quantified by adding streptavidin labelled with the europium chelator 4,7-bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in the presence of excess Eu3+. The complex formed (bovine globulin-thyroxine-antibody-biotin-streptavidin-BCPDA-Eu3+) is measured on the solid-phase by time-resolved fluorescence. The assay is simple to perform and its characteristics are similar to those of other currently used immunoassay techniques.

Ultrasensitive Thyrotropin Immunoassay Based on Enzymatically Amplified Time-Resolved Fluorescence with a Terbium Chelate

1992 ◽  
Vol 38 (4) ◽  
pp. 545-548 ◽  
Author(s):  
A Papanastasiou-Diamandi ◽  
T K Christopoulos ◽  
E P Diamandis
Keyword(s):  
Alkaline Phosphatase ◽  
Detection Limit ◽  
Solid Phase ◽  
Thyroid Stimulating Hormone ◽  
Fluorescence Immunoassay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Highly Sensitive ◽  
Assay Time ◽  
Highly Sensitive Detection

Abstract We describe an ultrasensitive, enzymatically amplified time-resolved fluorescence immunoassay of thyrotropin (thyroid-stimulating hormone) in serum with use of a terbium chelate as the detectable moiety. In this assay, thyrotropin is first simultaneously reacted with a solid-phase (microtiter well) monoclonal antibody and a soluble biotinylated monoclonal detection antibody. After washing, a streptavidin-alkaline phosphatase conjugate is added, followed by another washing. Alkaline phosphatase acts on the substrate 5-fluorosalicyl phosphate (FSAP) to produce 5-fluorosalicylic acid (FSA). FSA, but not FSAP, can then form with Tb3+ and EDTA a highly fluorescent ternary complex of long fluorescence lifetime. This complex is quantified with time-resolved fluorometry. The thyrotropin assay is highly sensitive (detection limit approximately 0.003 milli-int. unit/L when a total assay time of 85 min is used), precise, and accurate. The thyrotropin assay can also be completed in less than 30 min (detection limit 0.013 milli-int. unit/L), thus making this procedure a candidate technology for high-throughput automated analyzers.

Testing and validating a homogeneous immunometric assay for interference

2004 ◽  
Vol 42 (2) ◽  
Author(s):  
Johan Bjerner ◽  
Kari Hauge Olsen ◽  
Ole P. Børmer ◽  
Kjell Nustad
Keyword(s):  
Monoclonal Antibody ◽  
Energy Transfer ◽  
Carcinoembryonic Antigen ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Microtiter Plate ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Assay Format

AbstractWe analyzed 95 sera, demonstrating interference in a previous study, with the Kryptor homogeneous time-resolved fluorescence resonance energy transfer carcinoembryonic antigen (CEA) immunoassay (Brahms AG, Berlin, Germany). Only one serum differed, i.e., 6.0 μg/l for Kryptor vs. 13.3 μg/l for a microtiter plate in-house immunofluorometric assay (IFMA), using both aggregated mouse immunoglobulins as blocker and capture monoclonal antibody (Mab) F(ab′)Kryptor-CEA assay results thus agreed with our inhouse CEA assay results, showing no interference. As the Kryptor-CEA assay antibodies were sensitive to interference and the Kryptor-CEA assay buffer did not reduce interference as efficiently as our in-house assay buffer, the Kryptor-CEA assay format was crucial for the absence of interference.

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