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Biomedical subjects

T K Christopoulos

Publications and source records attributed to T K Christopoulos.

At least 37 records · Page 2Linked to original sources
PubMed1995

Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragments.

A novel immunoassay is reported which uses an enzyme-coding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with O2 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50,000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/translation and the substrate turnover.

Antigens↗
PubMed1995

Time-resolved fluorometric hybridization assays with RNA probes synthesized from polymerase chain reaction-generated DNA templates.

DNA templates suitable for direct synthesis of RNA probes are produced by the polymerase chain reaction. The nucleic acid sequence of interest is amplified using a downstream primer carrying the T7 RNA polymerase promoter sequence. The modified primer is incorporated into the amplified DNA, which is subsequently used for RNA probe synthesis in the presence of T7 RNA polymerase and a hapten-labeled ribonucleotide (digoxigenin-UTP). As a model, we prepared RNA probes specific for the BCR-ABL mRNA characteristic of chronic myelogenous leukemia. The probes are used in time-resolved fluorometric hybridization assays. Mixtures of BCR-ABL positive and negative cells, as well as whole blood samples, are analyzed. The sample mRNA is amplified using a biotinylated upstream primer. The amplified product (target DNA) is captured onto streptavidin-coated wells and hybridized to the RNA probe. The hybrids are detected with an alkaline phosphatase (ALP)-labeled antibody. ALP hydrolyzes the phosphate ester of fluorosalicylic acid, and the fluorosalicylate produced forms highly fluorescent ternary complexes with Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. As low as 0.4 fmol of target DNA can be detected. Also, a single leukemic cell may be detected in the presence of 0.5 million "normal" cells.

Base Sequence↗
PubMed1995

Detection of prostate-specific antigen mRNA by reverse transcription polymerase chain reaction and time-resolved fluorometry.

We have developed a time-resolved fluorometric hybridization assay for detecting prostate-specific antigen (PSA) mRNA amplified by reverse transcription polymerase chain reaction. During PCR, digoxigenin-11-dUTP is incorporated into the amplified product. An oligonucleotide internal to the primers is used as a specific probe, being biotinylated and captured on streptavidin-coated microtiter wells. Denatured PCR product hybridizes with the probe, and the hybrids are detected with an alkaline phosphatase-labeled antidigoxigenin antibody. We used the phosphate ester of fluorosalicylic acid as the substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which we can measure by time-resolved fluorometry. A signal-to-background ratio of 10 was obtained when 160 PSA cDNA molecules were present in the preamplification sample. Also, mRNA corresponding to one LNCaP cell in the presence of 10(6) PSA-negative cells can be detected (signal-to-background ratio of 3.1). Samples containing 100, 1000, and 50,000 LNCaP cells gave CVs of 12.4%, 4.9%, and 6.8%, respectively (n = 10).

Fluorometry↗
PubMed1995

Detection of BCR-ABL transcripts from the Philadelphia translocation by hybridization in microtiter wells and time-resolved immunofluorometry.

Two hybridization assays have been developed to detect BCR-ABL mRNA transcripts arising from the Philadelphia translocation. Both assays use time-resolved immunofluorometric detection of polymerase chain reaction-amplified BCR-ABL mRNA sequences hybridized to specific probes. In configuration I, biotinylated amplified target is immobilized onto streptavidin-coated wells and hybridized to a probe labeled with the hapten digoxigenin. Hybrids are detected via an alkaline phosphatase-labeled antibody and fluorosalicylylphosphate as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb(3+)-EDTA. In configuration II, biotinylated probe is immobilized onto streptavidin-coated wells. PCR, performed in the presence of hapten-labeled deoxyribonucleotide, generates labeled product, which is hybridized to immobilized probe and quantified as above. BCR-ABL transcripts from one leukemic cell amidst mRNA from 500,000 normal granulocytes are detectable with signal/background ratios as high as 36.4 and 24.6 for configurations I and II, respectively. The respective CVs for the assays were 6.6-9.0% and 5.1-12.5%.

Base Sequence↗
PubMed1994

Time-resolved immunofluorometric determination of specific mRNA sequences amplified by the polymerase chain reaction.

We report a highly sensitive time-resolved immunofluorometric method for quantification of polymerase chain reaction (PCR)-amplified mRNA sequences. The PCR primers are labeled at their 5' ends, one with biotin and the other with a hapten. The modified primers are incorporated, during PCR, in the amplified product. The PCR product is captured, through its biotin moiety, to a streptavidin-coated solid phase and subsequently is detected with an alkaline phosphatase-labeled antibody. The phosphate ester of fluorosalicylic acid is used as a substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which is measured by time-resolved fluorometry. We chose the determination of PCR-amplified chronic myelogenous leukemia-specific mRNA as a model system. mRNA molecules corresponding to 0.1 leukemic cell in the presence of 0.5 million normal cells may be detected (signal-to-background ratio of 1.5). The method provides a sensitive and rapid nonisotopic alternative to Southern blot and hybridization with radioactive probes.

Alkaline Phosphatase↗
PubMed1992

Quantitative western blot analysis and spot immunodetection using time-resolved fluorometry.

We describe a new method of staining and quantification of proteins blotted or spotted on nitrocellulose. Blotted or spotted proteins are first reacted with specific antibodies followed by reaction with biotinylated secondary antibodies. The immunocomplex is then reacted with a streptavidin-based macromolecular complex labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl) 1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). The fluorescent spots or bands can then be assessed by visual inspection under UV illumination, by instant photography or quantified by scanning with a time-resolved fluorometer. The method does not involve enzyme detection, is simple, sensitive and gives sharp bands which remain fluorescent for long periods of time (months to years).

Animals↗
PubMed1992

Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates.

We report an ultrasensitive, enzymatically amplified, time-resolved fluorescence immunoassay with a terbium chelate as the detectable moiety. In this immunoassay, the primary label is the enzyme alkaline phosphatase (ALP). ALP cleaves phosphate out of a fluorogenic substrate, 5-fluorosalicyl phosphate, to produce 5-fluorosalicylic acid (FSA). 5-Fluorosalicylic acid can then form a highly fluorescent ternary complex of the form FSA-Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. In this assay, exceptional sensitivity is achieved because of the enzymatic amplification introduced by ALP and the quantification by laser-induced microsecond time-resolved fluorometry. Time-resolved fluorometry is applicable because of the long fluorescence lifetime of the Tb3+ complexes. It is shown that in a model AFP assay 10(6) or 1.5 x 10(5) molecules can be detected (final assay volume, 100 microL) by using monoclonal or polyclonal detection antibodies, respectively. The assay demonstrates excellent precision (approximately 4%), and it seems to be highly suited for automated, sensitive, and rapid immunoassays.

Alkaline Phosphatase↗
PubMed1992

Multianalyte immunoassay based on spatially distinct fluorescent areas quantified by laser-excited solid-phase time-resolved fluorometry.

We describe a new multianalyte immunoassay principle and apply it to the simultaneous immunoassay of lutropin, follitropin, choriogonadotropin, and prolactin in serum. The method is based on the coating of distinct areas of polystyrene with analyte-specific antibodies. These antibodies react with the analyte and immobilize it in a specific area while another biotinylated antibody also reacts with the analyte to form a sandwich. After addition of streptavidin labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid, fluorescent areas are formed, the intensity of which is related to the amount of each analyte present in the sample. The fluorescent areas are quantified on the dry solid phase with laser-excited time-resolved fluorometric measurements. The assays developed are highly sensitive, precise, and accurate. We believe that this system shows potential for multianalyte immunoassay of diverse groups of compounds in disciplines such as endocrinology, infectious disease, hematology, and oncology.

Chorionic Gonadotropin↗
PubMed1992

Ultrasensitive thyrotropin immunoassay based on enzymatically amplified time-resolved fluorescence with a terbium chelate.

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.

Alkaline Phosphatase↗
PubMed1991

Quantification of nucleic acids on nitrocellulose membranes with time-resolved fluorometry.

We use a streptavidin-based macromolecular complex (SBMC) labelled with the europium chelate of 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as a staining reagent for biotinylated DNA present on nitrocellulose filters. The fluorescent spots or bands obtained can either be observed under UV illumination, photographed by instant camera photography or quantified by using a specially designed instrument working as a high resolution time-resolved fluorometric scanner. The detection limit is approximately 10 pg of target DNA. Various experiments with use of biotinylated DNA probes hybridized to Southern transferred targets have shown that the new procedure is a useful versatile non-isotopic methodology for staining DNA on solid supports.

Bacterial Proteins↗
PubMed1991

Time-resolved immunofluorometric detection of antigens separated by high-performance liquid chromatography and coated to polystyrene.

We use high-performance liquid chromatography with fraction collection to separate an antigen of interest. The antigen is then immobilized on polystyrene microtiter wells and detected with a specific antibody, followed by a second biotinylated antibody and streptavidin labeled with a fluorescent europium chelate. Fluorescence can be quantified with the use of time-resolved fluorescence. Antigen detectability down to 2-3 x 10(-17) mol was achieved. This method could be used as an alternative to Western blot in certain applications.

Antigens↗
PubMed1991

The biotin-(strept)avidin system: principles and applications in biotechnology.

The biotin-(strept)avidin system has been used for many years in a variety of different applications. Here we present a general overview of the system, describe its components and advantages, and show how the system is used in various applications, with emphasis on immunological and nucleic acid hybridization assays. This system is now considered a versatile independent technology with broad applications in many branches of biotechnology. Clearly, its use will continue to grow in the years to come.

Animals↗
PubMed1990

Binding studies using ion-selective electrodes. Examination of the picrate-albumin interaction as a model system.

We are studying the binding of ligands to macromolecules by using ligand ion selective electrodes as transducers. The picrate-bovine albumin interaction is examined in detail as a model system. A picrate ion selective electrode is used to monitor the free picrate concentration directly in the presence of albumin and bound ligand. The binding parameters are estimated and the effect of protein concentration, ionic strength, pH, and temperature is studied. The experimental data are interpreted with a specially designed computer program that performs nonlinear least-squares fitting of the generalized Scatchard model with an infinite number of classes of binding sites directly to the raw potentiometric data. The binding parameters (binding constant and maximum number of ligands that can be bound), the nonspecific binding as well as their standard deviations are estimated by this program. The principles described can be used for the potentiometric study of any ligand-binder interaction.

Electrodes↗
PubMed1990

Ultrasensitive time-resolved fluorescence method for alpha-fetoprotein.

We have examined the maximum sensitivity of a newly developed and optimized time-resolved fluorescence immunoassay system. The system, originally described elsewhere (Clin Biochem 1988:21:139-50), has undergone significant improvements in sensitivity through improvements of the labeled reagent used. We have chosen an alpha-fetoprotein (AFP) assay as a model and used monoclonal "capture" antibodies and monoclonal or polyclonal biotinylated antibodies in "sandwich-type" assay configurations. Streptavidin labeled with the europium chelator 4.7-bis(chlorosulfophenyl)-1,10-phenanthroline-2.9-dicarboxylic acid was used for detection. We can measure as few as 3 x 10(5) molecules of AFP with the optimized system. We have applied this assay to measure AFP in the serum of normal individuals after a 10-fold sample dilution. We conclude that this system is extremely sensitive and can be used in immunoassay or other applications where biotinylated reagents can be applied.

Adult↗
PubMed1990

Assay of creatine kinase isoenzyme MB in serum with time-resolved immunofluorometry.

We describe the first time-resolved immunofluorometric assay for creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB) in serum. The assay is based on the formation of the complex: solid-phase anti-CK-MB-CK-MB-biotinylated anti-CK-BB-streptavidin-BCPDA-Eu3+, where anti-CK-MB and anti-CK-BB are monoclonal antibodies against the CK isoenzymes MB and BB, respectively, and BCPDA is the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid. The solid-phase complex is fluorescent and is measured on the dry solid-phase (microtiter well) in a specially designed time-resolved fluorometer that uses laser excitation. The assay requires 25 microL of serum and is not affected by the presence of either CK-MM (up to 5000 micrograms/L) or CK-BB (up to 1000 micrograms/L) in the sample. Precision and accuracy indices for the assay were satisfactory.

Antibodies, Monoclonal↗
PubMed1987

Dissolution studies of drug formulations using ion-selective electrodes as sensors in an air-segmented continuous flow analyzer.

The application of drug ion-selective electrodes as sensors for the direct determination of the released drug in a continuous-flow analyzer for automated dissolution studies is described. Flow-through electrodes, selective to chlorpromazine, amitriptyline, propantheline, cimetidine, and ranitidine, have been constructed and used for the dissolution studies of 18 dosage forms using the rotating basket apparatus. The dissolution profiles are obtained in the form of potential peaks versus time.

Amitriptyline↗
PubMed1986

A general method for the assay of binders. Application to the potentiometric determination of albumin in human serum, plasma and whole blood.

From the generalized theory of binding (stepwise equilibrium model, Scatchard model) it can be derived that in a solution containing the binder and a specific ligand, the bound ligand concentration is linearly related to the total binder concentration only if the free ligand concentration is constant. We designed a general method which is based on that principle and we have shown its validity for the albumin assay in serum, plasma and whole blood. We have tested the ligands bromcresol purple and picrate and used ligand-ion selective electrodes to monitor free ligand concentration in a homogeneous solution. The method has excellent linearity. We chose picrate (PIC) as ligand, because of better specificity. Comparison with the bromcresol purple (BCP) and bromcresol green (BCG) spectrophotometric procedures for 93 samples gave the regression equations: y(PIC) = 0.98x(BCP) + 0.76 (g/L, r = 0.979) and y(PIC) = 0.95x(BCG) - 1.3 (r = 0.947), respectively. Within-day and day-to-day precision was up to 1.5% and 2.3%, respectively. Results for albumin in plasma, when transformed to expected whole blood values, were in close agreement with those obtained by direct analysis of whole blood (in the presence of erythrocytes).

Bromcresol Green↗