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T Lövgren

Publications and source records attributed to T Lövgren.

At least 73 records · Page 4Linked to original sources
PubMed1994

Expression of prostate specific antigen on the surface of a filamentous phage.

We have constructed two phagemid vectors containing the gene coding for human Prostate Specific Antigen (PSA) translationally fused to a pelB signal sequence and minor coat protein III of phage fd leading to phage particles that carry PSA on their tips. Phages were characterized by Western blotting and by panning, using biotinylated monoclonal anti-PSA antibodies immobilized onto streptavidin-coated microtitration wells. Binding of PSA-phages was 10(2)- to 5 x 10(3)-fold more effective than that of wild-type phages. Trypsin treatment of the phage abolished the specific binding. Competitive panning with different concentrations of added purified PSA demonstrated that binding was PSA specific. Finally, Western-blot analysis confirmed that full-length and shorter degradation products of the fusion between PSA and protein III were visible as probed with anti-PSA antibody.

Antibodies, Monoclonal↗
PubMed1994

Detection of a point mutation using short oligonucleotide probes in allele-specific hybridization.

Two nonradioactive and simple procedures were developed to detect the A985G point mutation that causes medium-chain acyl-CoA deficiency. In both of these assays, short oligonucleotide probes were used in allele-specific hybridization combined with DNA amplification. The lower limit for a useful probe was found to be between 9 and 12 base pairs. Time-resolved fluorometry was utilized as the label technology and microtitration plates as the solid support. In one of the assay formats, probes labeled with europium and samarium chelates were used to simultaneously detect the mutant and normal alleles from the same hybridization reaction. In addition, the discrimination efficiency of different probes was characterized by cross-reactivity determinations and by measuring affinities of the probes towards fully complementary as well as towards mismatch-forming target oligonucleotides. All of the 80 coded patient samples analyzed were correctly typed in both of the assay formats used.

Alleles↗
PubMed1993

Double-label time-resolved immunofluorometric assay of prostate-specific antigen and of its complex with alpha 1-antichymotrypsin.

We have developed a procedure for simultaneous immunofluorometric assay (IFMA) of prostate-specific antigen (PSA) and its complex with alpha 1-antichymotrypsin (ACT). A PSA-specific monoclonal antibody, which captures both free PSA and the PSA-ACT complex, was used as solid-phase antibody. Total PSA immunoreactivity was measured with a Eu(3+)-labeled PSA antibody that reacted with both free PSA and PSA-ACT. PSA-ACT was assayed simultaneously with a Sm(3+)-labeled polyclonal ACT antibody as a tracer. As standard we used pooled serum in which most of the PSA occurred as the PSA-ACT complex. The assay range was 0.03-500 micrograms/L for total PSA and 0.16-450 micrograms/L for PSA-ACT. In comparison with the assay for total PSA, assay of the PSA-ACT/PSA ratio improved the clinical specificity for cancer by reducing the number of false-positive results in prostatic hyperplasia.

Chromatography, Gel↗
PubMed1993

Europium-labeled oligonucleotides to detect point mutations: application to PIZ alpha 1-antitrypsin deficiency.

We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive time-resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-amplified nucleic acid fragment. The hybrids formed were then collected onto streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy underlying the design of the Eu-labeled ASO probes for the solution hybridization assay. The method was applied to the detection of the Z-mutation in the alpha 1-antitrypsin gene. Evaluation of whole-blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.

Base Sequence↗
PubMed1992

Simultaneous quadruple-label fluorometric immunoassay of thyroid-stimulating hormone, 17 alpha-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM isoenzyme in dried blood spots.

We describe a quadruple-label fluorometric immunoassay for simultaneously measuring four analytes: thyroid-stimulating hormone (TSH), 17 alpha-hydroxyprogesterone (17 alpha-OHP), immunoreactive trypsin (IRT), and creatine kinase MM (CK-MM). The assay is based on immunoreagents labeled with four different lanthanide ions (Eu3+, Tb3+, Sm3+, and Dy3+), on dissociative fluorescence enhancement applying the principle of co-fluorescence, and on time-resolved fluorometry. The monoclonal anti-alpha-TSH and anti-IRT antibodies and the polyclonal anti-CK-MM antibody were labeled with Eu3+, Sm3+, and Dy3+, respectively; 17 alpha-OHP was labeled with Tb3+. The assay was performed in microtitration strip wells coated with a mixture of monoclonal antibodies against beta-TSH, IRT, and CK-MM and a polyclonal goat anti-rabbit IgG for capture of the rabbit anti-17 alpha-OHP antibodies. After completion of the immunoreactions, the bound fractions of the lanthanides were dissociated into the co-fluorescence enhancement solution, creating highly fluorescent chelates. The four lanthanide-specific signals were subsequently measured in a time-resolved fluorometer. The detection limits of the assay were 0.1 mIU/L for TSH, 2 nmol/L for 17 alpha-OHP, 2 micrograms/L for IRT, and 4 U/L for CK-MM.

17-alpha-Hydroxyprogesterone↗
PubMed1991

Preparation of europium-labelled DNA probes and their properties.

A chemical method for labelling DNA with a europium chelate is presented. First, primary aliphatic amino groups are introduced onto DNA in a transamination reaction. The transamination reaction is altered by adjusting temperature and duration of the reaction. Subsequently, the modified DNA is reacted with an isothiocyanate derivative of a Eu chelate. The optimum amount of Eu chelates on a DNA probe is 4-8% of total nucleotides. There is a decrease of 0.7 degrees C in the melting temperature of DNA for each incorporated Eu chelate on 100 bases. Hybridization efficiency is lowered by the introduction of Eu chelates but this effect can be partly overcome by using high DNA probe concentrations. The detection limit of the Eu-labelled probe is 0.15 attomoles of target DNA in a mixed-phase hybridization assay on microtitration wells. In addition to high sensitivity the Eu-labelled probes offer convenience in use and results which are quantitative and easy to interpret.

Affinity Labels↗
PubMed1991

Co-fluorescence of europium and samarium in time-resolved fluorimetric immunoassays.

In the presence of an excess of Y3+, the fluorescence intensities of Eu3+ and Sm3+, chelated with benzoyltrifluoroacetone (BTA) or thenoyltrifluoroacetone (TTA) in an aqueous solution containing 1,10-phenanthroline, were increased by factors ranging from 209- to 811-fold. This co-fluorescence phenomenon was used in a highly sensitive time-resolved fluorimetric detection of the lanthanides, Eu3+ and Sm3+. The detection limits of Eu3+ in the BTA- and TTA-based solutions were 4 and 15 fmol dm-3, respectively. The detection limits of Sm3+ were 0.11 and 0.12 pmol dm-3, respectively. The co-fluorescence enhancement systems were also applied in the double-label time-resolved fluorimetric immunoassay of luteinizing hormone and follicle stimulating hormone using specific antibodies labelled either with Eu3+ or Sm3+. The co-fluorescence enhancement solution was superior as compared with the commercial 'direct' fluorescence enhancement solution based on the acidic solution of beta-naphthoyltrifluoroacetone, trioctylphosphine oxide and Triton X-100, in respect to the signal level obtained and the sensitivity. It is suited to time-resolved fluorimetric immunoassays in which particularly high detection sensitivities are required, and it can also be used in double-label assays employing Eu3+ and Sm3+ chelate labels.

Europium↗
PubMed1991

Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin.

Immunologic measurements of the serum concentration of prostate-specific antigen (PSA), an abundant prostatic-secreted serine proteinase, are frequently used to monitor patients with prostate cancer, though it has not been ascertained whether this immunoreactivity represents a PSA zymogen, the active proteinase, or PSA complexed to extracellular proteinase inhibitors. To characterize the PSA immunoreactivity in serum, we used monoclonal antibodies produced against PSA and a polyclonal rabbit IgG against alpha 1-antichymotrypsin in the design of three noncompetitive PSA assays: assay T, which detected PSA both when present as the active proteinase and when complexed to alpha 1-antichymotrypsin; assay F, which recognized the active proteinase but most poorly detected PSA complexed to alpha 1-antichymotrypsin; and assay C, which was specific for PSA complexed to alpha 1-antichymotrypsin. We used the three assays to measure PSA immunoreactivity in 64 patients' sera and in the effluent after gel chromatography of sera from four patients. This identified an 80- to 90-kDa complex between PSA and alpha 1-antichymotrypsin as the predominant fraction of the PSA immunoreactivity in blood plasma; an immunoreactive 25- to 40-kDa compound was the minor fraction.

Animals↗
PubMed1989

Measurement of estrone-3-glucuronide in urine by rapid, homogeneous time-resolved fluoroimmunoassay.

We describe a liquid-phase nonseparation time-resolved fluorescence immunoassay for measuring estrone-3-glucuronide in undiluted urine. The sensitivity, specificity, and accuracy are similar to those for a conventional separation fluoroimmunoassay or radioimmunoassay, but the speed, convenience, precision, reliability, and clinical utility of the new method are more advantageous. The labeled antigen, a fluorescent europium chelate covalently linked to estrone-3-glucuronide, is incubated for 10 min with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-bovine serum albumin and 10 microL of standard or sample (undiluted urine) in microtiter wells. The fluorescence emanating from the antibody-free label, which is proportional to the concentration of estrone-3-glucuronide in the standard or sample, is then measured in a time-resolved fluorometer. The method is useful for monitoring ovarian function in women.

Antibodies, Monoclonal↗
PubMed1988

Determination of cytotoxic T lymphocyte activity by time-resolved fluorometry using europium-labelled concanavalin A-stimulated cells as targets.

Time-resolved fluorometry was used to detect the cytolysis of concanavalin A-stimulated blast cells by employing cells stimulated in a mixed lymphocyte culture as effectors. The target cells were labelled with europium diethylenetriaminopentaacetate (EuDTPA) chelates. The results obtained showed that this method, which has been successfully applied to the measurement of natural killer cell activity, was also applicable to the more specific cytotoxic T lymphocyte reaction. The specific release was higher with EuDTPA-labelled target cells than with 51chromium-labelled target cells. As with 51chromium, EuDTPA can be used to distinguish between the cytolysis of specific and non-specific target cells.

Chromium Radioisotopes↗
PubMed1988

Homogeneous time-resolved fluoroimmunoassay of thyroxin in serum.

We describe a rapid, simple nonseparation fluoroimmunoassay for determination of thyroxin in serum. The assay is based on the labeling of thyroxin directly with a fluorescent europium chelate, the fluorescence of which is quenched on binding to an antithyroxin antibody. With the assay buffer we used, maximum quenching is 90%. The rapid achievement of equilibrium in the assay solution, regardless of the sequence of reagent additions, allows fast measurement of thyroxin. Precision was good (CV less than 5%) within the clinical range for total thyroxin (50-300 nmol/L), and results correlated well with those by a commercial radioimmunoassay.

Antibodies, Monoclonal↗
PubMed1987

Sensitive detection of genes by sandwich hybridization and time-resolved fluorometry.

Europium has been used as a non-radioactive marker in immunoassays as this metal can be detected with high sensitivity by time-resolved fluorometry. In this work streptavidin labeled with europium was used to detect biotinylated probes in a sandwich nucleic-acid hybridization assay with microtitration strips as the solid phase. pBR 322 plasmids were detected with a sensitivity of 4 x 10(5) molecules. As the sample is added in solution in sandwich hybridization, fast and simple sample pre-treatment can be used without encountering background problems. The method was applied to test bacterial samples of uropathogenic Escherichia coli strains for the presence of the beta-lactamase gene.

Animals↗
PubMed1987

Time-resolved immunofluorometric assay for the ovarian carcinoma-associated antigenic determinant CA 125 in serum.

A time-resolved immunofluorometric assay (IFMA) is described for quantifying the ovarian carcinoma-associated antigenic determinant CA 125 in human serum. Monoclonal antibody to CA 125 is immobilized onto a microtiter well, and the same antibody labeled with a europium chelate is used as a tracer. After the immunoreaction the bound portion of the labeled antibody is quantified by dissociating the Eu3+ in a fluorescence-enhancement solution and measuring its fluorescence with a time-resolved fluorometer. The detection limit of the IFMA is 1.5 arb. units/mL, being about the same as that of a commercially available immunoradiometric assay (IRMA) for CA 125 (1.4 arb. units/mL). The analytical range of the IFMA extends to 2000 arb. units/mL, whereas the range of the IRMA is 500 arb. units/mL. For 29 serum samples from ovarian-cancer patients measured simultaneously in the IFMA and IRMA, orthogonal regression analysis gave the equation CA 125 (IFMA) = 0.9937 CA 125 (IRMA) - 1.211 arb. units/mL (Syx = 6.8681, r = 0.9932). Apparently, the IFMA for CA 125 is a convenient alternative to the IRMA for CA 125 because of short counting times, the use of nonradioactive, stable reagents, and the much-extended measuring range. Additionally, the microtiter format should lend itself to more fully automated procedures in laboratories doing many such analyses.

Antigens, Neoplasm↗
PubMed1987

Double-label time-resolved immunofluorometry of lutropin and follitropin in serum.

We describe a procedure for the simultaneous immunofluorometric assay of lutropin and follitropin in human serum, based on the use of monoclonal antibodies and of the fluorescent lanthanides Eu3+ and Tb3+. The alpha-chain-specific antibody was used as a common capture antibody on the surfaces of microtitration strips. The anti-beta-follitropin antibody was labeled with Tb3+, the anti-beta-lutropin antibody with Eu3+. After the immunoreactions had taken place, the bound fractions of the labels were dissociated in a fluorescence enhancement solution of pivaloyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100 surfactant. In this solution both lanthanides can be measured successively with a time-resolved fluorometer. The detection limit of the assay is 0.1 int. unit/L for lutropin and 1 int. unit/L for follitropin. Results correlated well with those by commercial immunofluorometric assays and radioimmunoassays.

Antibodies, Monoclonal↗
PubMed1986

Europium-labelled target cells in an assay of natural killer cell activity. II. A novel non-radioactive method based on time-resolved fluorescence. significance and specificity of the method.

The significance, specificity and high sensitivity of a new method to determine the natural killer cell cytolysis of europium diethylenetriaminepentaacetate (EuDTPA)-labelled target cells has been confirmed. The targets used in this release assay were the NK sensitive cell line K-562 and the resistant cell line Raji. The released EuDTPA was detected by a method based on time-resolved fluorometry. The specific EuDTPA release was higher than specific 51chromium (51Cr) release. Competitive assays, where half of the target cells were labelled with EuDTPA and the other half with 51Cr and the use of double labelled target cells showed that the results were identical with those of single labelled cells. The reliability of the EuDTPA release assay was further confirmed by performing experiments using NK cells from a patient whose complete lack of NK activity had earlier been demonstrated with the 51Cr release assay. Furthermore, our studies show that the amount of incorporated EuDTPA was directly proportional to the concentration of marker used. Due to the proportional incorporation of EuDTPA the labelling conditions can be chosen to obtain a sensitivity which allows even single cells to be detected.

Cells, Cultured↗