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Research Interests: Microbiology, Chemistry, Medical Microbiology, Filtration, Vibration, and 14 moreMedicine, Saccharomyces cerevisiae, Alpha-Glucosidase, Pubmed, Ultrasonics, Yeast, Gel Permeation Chromatography, Chemical Precipitation, Yeasts, Quaternary Ammonium Compounds, Culture Media, Molasses, Sulfates, and Antonie van Leeuwenhoek
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Abstract Two different ATP extraction methods, one using perchloirc acid and the other boiling Tris bufer, for ATP determinations in myocardial samples werer investigated and compared. A bioluminescent assay was used to quantify the... more
Abstract Two different ATP extraction methods, one using perchloirc acid and the other boiling Tris bufer, for ATP determinations in myocardial samples werer investigated and compared. A bioluminescent assay was used to quantify the extracted ATP. In spite of some drawbacks, eprchloric acid extraction is zix times more efficient thatn boiling Tris buffer extraction for releasing ATP from myocardial tissue. The perchloric acid extraction was used to determine ATP depletion in the endocardium of isolated ischemic rat hearts.
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Strains of fresh clinical isolates of Trichomonas vaginalis and Candida albicans have been tested in vitro for their sensitivity to eight drugs used in the therapy of monilial and trichomonal vaginitis. Three of the chemotherapeutic... more
Strains of fresh clinical isolates of Trichomonas vaginalis and Candida albicans have been tested in vitro for their sensitivity to eight drugs used in the therapy of monilial and trichomonal vaginitis. Three of the chemotherapeutic agents, chlorchinaldol, clotrimazole and broxyquinoline, were effective against both organisms. Tinidazole and metronidazole were active against T. vaginalis. The strains of C. albicans were also sensitive to trichomycin, natamycin and nystatin. Tinidazole was the most effective trichomonacide, clotrimazole and chlorchinaldol were most effective against C. albicans, while chlorchinaldol had the best in vitro effect against both organisms. The ranges of the MICs are compared to values previously reported.
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Research Interests: Chemistry, Immunology, Molecular Biology, Kinetics, Medicine, and 15 morePregnancy, Humans, Female, Monoclonal Antibodies, Time Resolved, Coefficient of Variation, Biotin, Streptavidin, Optometry and Ophthalmology, Reproducibility of Results, Monoclonal Antibody, Biotinylation, Human Chorionic Gonadotrophin, Reference standards, and plasma proteins
BACKGROUND Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. METHODS The method... more
BACKGROUND Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. METHODS The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. RESULTS The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 10(6) copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 +/- 200 to 44 100 +/- 4900 (mean +/- SD) in the samples, corresponding to approximately 1-100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 +/- 100 and 2000 +/- 900 (mean +/- SD) PSA mRNA copies in 5 mL of blood for the 2 males]. CONCLUSIONS The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.
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Research Interests: Prostate Cancer, Medicine, Humans, Male, The, and 15 moreProstate, Differential Diagnosis, Hyperplasia, Clinical Sciences, Aged, Middle Aged, Adult, Benign Prostatic Hyperplasia, Age Factors, Reference Values, Normal Distribution, Prostate Specific Antigen, Benign Prostate Hyperplasia, Antigen, and rectal examination
We describe an immunofluorometric assay for human pancreatic phospholipase A2 based on time-resolved fluorescence. The labeled antibody technique in combination with the time-resolved 1-s fluorometric detection of the europium label,... more
We describe an immunofluorometric assay for human pancreatic phospholipase A2 based on time-resolved fluorescence. The labeled antibody technique in combination with the time-resolved 1-s fluorometric detection of the europium label, which essentially eliminates all background fluorescence, resulted in a high sensitivity (20 ng/L) and a wide (5000-fold) linear range. Nonspecific binding was minimized by treating the solid-phase antibody with NaSCN before coating, to remove endogenous antigen, and by immunosorbent purification of the antibody before labeling with europium. This is a one-incubation, multi-site, solid-phase assay on polystyrene microtiter strips, even though a polyclonal antibody was used. As measured by this assay, activity of immunoreactive phospholipase A2 was found to be above normal in sera of patients suffering from acute pancreatitis.
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We describe a method for the detection of two type 1 (insulin-dependent) diabetes susceptibility (*0201, *0302) alleles and two protective (*0301, *0602/0603) alleles of the HLA-DQB1 gene on the human major histocompatibility complex... more
We describe a method for the detection of two type 1 (insulin-dependent) diabetes susceptibility (*0201, *0302) alleles and two protective (*0301, *0602/0603) alleles of the HLA-DQB1 gene on the human major histocompatibility complex (MHC). The test is based on DNA amplification with PCR followed by simultaneous, allele-specific triple-label hybridization performed in microtitration wells. In the hybridization, very short allele-specific oligonucleotides labeled with europium (Eu), terbium (Tb) or samarium (Sm) are used. The labeled probes could be detected using time-resolved fluorometry with sensitivities of 1 x 10(7), 3 x 10(8) and 3 x 10(8) molecules, respectively. Cross-reactions were not found among samples containing 14 common DQB1 alleles. To test the utility of the developed assay, 100 DNA and 14 dried blood spot samples with known DQB1 alleles were analyzed. A 100% agreement with the reference method was reached. Thus, this triple-label hybridization assay proved to be suitable even for detection of a large number of samples.
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Abstract A simple procedure is described for the use of amido black 10 B-stained BSA as an internal standard protein for estimation of sedimentation coefficients by density gradient centrifugation. The main advantage of the method is that... more
Abstract A simple procedure is described for the use of amido black 10 B-stained BSA as an internal standard protein for estimation of sedimentation coefficients by density gradient centrifugation. The main advantage of the method is that the standard protein does not interfere with the detection of isotopically labeled fractions.
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. High affinity cytoplasmic estrogen receptors in the endometrium, myometrium and ovary of 15 climacteric women were studied. In addition, the concurrent serum estradiol and progesterone level of each woman was estimated and the... more
. High affinity cytoplasmic estrogen receptors in the endometrium, myometrium and ovary of 15 climacteric women were studied. In addition, the concurrent serum estradiol and progesterone level of each woman was estimated and the endometrium examined histologically. The cytoplasmic estrogen receptor level of the endometrium and myometrium had remained extremely high in some cases several years after the menopause and in the presence of a completely atrophied endometrium. The lowest endometrial and myometrial estrogen receptor levels in premenopausal women were measured towards the end of the menstrual cycle. The endometrial estrogen receptor level was roughly 2‐3 times the comparable myometrial level. Estrogen receptors were also encountered in all cases in the cervical myometrium. The estrogen receptor levels of the ovary were low in all cases.
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Abstract The human myometrial estrogen receptor in cytosol from pre-menopausal uterine samples has been characterized. At 0° estradiol (KD 0.38 × 10−10M) has the highest affinity to the receptor followed by estrone (KD 0.76 × 10−10M) and... more
Abstract The human myometrial estrogen receptor in cytosol from pre-menopausal uterine samples has been characterized. At 0° estradiol (KD 0.38 × 10−10M) has the highest affinity to the receptor followed by estrone (KD 0.76 × 10−10M) and estriol ((KD 1.33 × 10−10M). The association rate constant is 2.8 × 105M−1s−1 for estradiol, 2.1 × 105M−1s−1 for estrone and 0.79 × 105M−1s−1 for estriol. The dissociation constants and the association rate constants increase with temperature. The calculated thermodynamic parameters indicate a positive change in entropy for the formation of the estrogen receptor complex. The cytoplasmic estrogen receptor has a sedimentation coefficient of 4 s in low salt sucrose gradients. In buffer containing diisopropylfluorophosphate (DFP) to inhibit proteolytic activity the estrogen receptor complex sediments solely as an 8 s peak if [3H]-estradiol is added to the buffer prior to homogenization and the tissue sample is used immediately after hysterectomy. Estrogen receptor complexes that sediment at 4 s and 8 s are found if [3H]-estradiol is omitted from the homogenization buffer and instead added after the cytosol preparation. Most likely a protease is involved the activity of which is not completely inhibited by DFP. Addition of low concentrations of Cu2+ (10 μM) to the cytosol increases the dissociation constant and decreases the estrogen-binding capacity of the receptor. The rate of association is reduced in the presence of 20 μM Cu2+. The estrogen receptor complexes do not show any change in their sedimentation profiles in the presence of Cu2+.
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Research Interests: Cognitive Science, Chemistry, Medicine, Adsorption, Clinical Chemistry, and 15 moreIn Vitro, Humans, Medical Biotechnology, Mice, Animals, Epitope mapping, Male, Clinical Sciences, Antibody, Monoclonal Antibody, Drug Stability, Prostate Specific Antigen, Antigen, epitope, and Medical biochemistry and metabolomics
We developed a simple one-step dual-label immunoassay for simultaneous measurement of the free, noncomplexed form of prostate-specific antigen (PSA) and total PSA. The assay is based on time-resolved fluorescence and includes a stable... more
We developed a simple one-step dual-label immunoassay for simultaneous measurement of the free, noncomplexed form of prostate-specific antigen (PSA) and total PSA. The assay is based on time-resolved fluorescence and includes a stable fluorescent chelate of Eu to label a monoclonal antibody (mAb) that detects only free PSA, whereas a second mAb labeled with a fluorescent chelate of Tb provides equimolar detection of both free PSA and PSA complexed to alpha 1-antichymotrypsin. A third mAb on a solid phase captures the free and complexed forms of PSA in an equimolar fashion. The simultaneous measurement of the free-to-total PSA ratio (F/T) with the one-step dual assay is not sensitive to variations in the sample volume. The discrimination between benign prostatic hyperplasia and prostate cancer patients, i.e., the area under the receiver-operating characteristic curve, increased from 0.64 (total PSA assay) to 0.78 and 0.81 when the F/T ratio was measured with single and dual assays, respectively.
Research Interests: Chemistry, Chromatography, Kinetics, Medicine, Clinical Chemistry, and 15 moreHumans, Medical Biotechnology, Female, Clinical, Male, Monoclonal Antibodies, Clinical Sciences, Fluorescent Antibody Technique, Benign Prostatic Hyperplasia, Immunoassay, Biochemistry and molecular biology, Monoclonal Antibody, Antigen, Europium, and Medical biochemistry and metabolomics
Prostate-specific antigen (PSA) was detected in microtitre wells coated with a PSA-specific antibody using biotinylated antibody and streptavidin-coated, highly fluorescent 107 nm nanoparticles, which contained more than 30000 europium... more
Prostate-specific antigen (PSA) was detected in microtitre wells coated with a PSA-specific antibody using biotinylated antibody and streptavidin-coated, highly fluorescent 107 nm nanoparticles, which contained more than 30000 europium ions entrapped by beta-diketones. PSA was monitored directly on the surface of a well without any additional enhancement step. The sensitivity of the assay was 1.6 ng/L, corresponding to 50 fmol/L or 250 zeptomoles (250 x 10(-21) mol/L) of PSA. The high specific activity and low non-specific binding of the streptavidin-coated nanoparticles improved the sensitivity of the PSA assay 100-fold compared to the conventional europium-labelled streptavidin tracer in the same assay format. Additionally, the streptavidin-coated nanoparticle label made very rapid assays possible, due to the high affinity of the streptavidin-biotin complex and a high number of binding sites available for tracing the biotinylated antibody on the surface. Due to the inherent problems of tracing analyte with a complex of biotinylated antibody and streptavidin-coated nanoparticles, the streptavidin-coated nanoparticles reacting with the surface-captured analyte and biotinylated antibody was favoured and factors influencing this are discussed. This universal labelling technology can be applied to detect any biotinylated molecule, either in solution or on a solid phase, in order to improve detection sensitivities in many areas of biochemical analysis, such as cyto- and histochemistry, multianalyte DNA-chip assays and single-particle assays.
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ABSTRACT
Research Interests: Biochemistry, Chemistry, Molecular Biology, Mass Spectrometry, Hydroxyapatite, and 13 moreBiology, Bone Metabolism, Escherichia coli, Glutathione, Time Resolved, Osteocalcin, Recombinant DNA, Protein expression and purification, Western blot, Amino Acid Profile, Amino Acid Sequence, Fusion Protein, and Biochemistry and cell biology
A dissociation-enhanced lanthanide fluoroimmunoassay of serum cortisol based on time-resolved fluorescence is described. The assay is a direct assay, where cortisol immobilized on the wall of a microtiter-strip well competes with cortisol... more
A dissociation-enhanced lanthanide fluoroimmunoassay of serum cortisol based on time-resolved fluorescence is described. The assay is a direct assay, where cortisol immobilized on the wall of a microtiter-strip well competes with cortisol in the sample for the europium-labeled polyclonal antibody. The amount of bound europium-labeled antibody is inversely proportional to the amount of cortisol in the sample. Separation is accomplished by washing the strip well. The assay is carried out in 2 h, at room temperature; it is easy to perform and gives accurate and reliable results. A chaotropic agent, trichloracetic acid, was very effective in releasing cortisol from binding proteins. This finding will have practical importance in the immunoassay field.
Research Interests: Chemistry, Chromatography, Kinetics, Fluorescence, Medicine, and 15 moreClinical Chemistry, Humans, Medical Biotechnology, Sheep, Animals, Lanthanum, Clinical Sciences, Fluorescent Antibody Technique, Time Factors, Polyclonal Antibodies, Immunoassay, Reference Values, Europium, hydrocortisone, and Medical biochemistry and metabolomics
Research Interests: Biochemistry, Chemistry, Chromatography, Kinetics, Biology, and 15 moreBiological Chemistry, Biological Sciences, Magnesium, Escherichia coli, Enzyme, CHEMICAL SCIENCES, Adenylate Kinase, Acylation, Biochemistry and cell biology, Adenosine monophosphate, binding sites, Isoleucine, Medical and Health Sciences, Medical biochemistry and metabolomics, and Aminoacylation
Research Interests: Biochemistry, Chemistry, Chromatography, Kinetics, Biology, and 15 moreBiological Chemistry, Medicine, Biological Sciences, Magnesium, Escherichia coli, CHEMICAL SCIENCES, Acylation, Protein Binding, Biochemistry and cell biology, Adenosine monophosphate, binding sites, Isoleucine, Medical and Health Sciences, Medical biochemistry and metabolomics, and Aminoacylation
Immunoreactive phospholipase A2 (EC 3.1.1.4) was measured by a new sensitive time-resolved fluoroimmunoassay in the serum of 58 healthy subjects and 103 patients with acute pancreatitis. Patients with acute pancreatitis were grouped... more
Immunoreactive phospholipase A2 (EC 3.1.1.4) was measured by a new sensitive time-resolved fluoroimmunoassay in the serum of 58 healthy subjects and 103 patients with acute pancreatitis. Patients with acute pancreatitis were grouped according to the etiology and clinical severity of the disease. The mean phospholipase A2 concentration in the reference (healthy) group was 5.5 (SD 1.9) micrograms/L. In acute pancreatitis the mean phospholipase A2 concentration was increased on the first day after hospital admission in all groups, and returned to normal somewhat more slowly than did serum amylase, especially in the patients with severe alcoholic pancreatitis. In this latter group the mean concentration of serum phospholipase A2 on the first day was 42.6 (SD 29.5) micrograms/L. In patients with pancreatic cancer, serum phospholipase A2 was 29.2 (SD 21.3) micrograms/L. The phospholipase A2 and amylase values were closely associated in all groups. The clinical sensitivities were 90.9% for severe alcoholic pancreatitis and 87.5% for pancreatic cancer. Immunochemical determination of phospholipase A2 in serum provides fast and specific detection of injury to pancreatic acinar cells. In addition to the early diagnosis of acute pancreatitis, follow-up determinations of phospholipase A2 seem to be useful in differentiating between mild and severe forms of pancreatitis.
Research Interests: Chemistry, Gastroenterology, Adolescent, Medicine, Clinical Chemistry, and 15 moreHumans, Internal Medicine, Medical Biotechnology, Female, Male, Acute Pancreatitis, Clinical Sciences, Aged, Middle Aged, Adult, Amylase, Acute Disease, Medical biochemistry and metabolomics, Biliary Tract Diseases, and amylases
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To design protocols for specific and quantitative immunohistochemical detection of human kallikrein 2 (hK2) using lanthanide chelate-labeled monoclonal antibodies (Mabs) and time-resolved fluorescence imaging. Anti-prostate-specific... more
To design protocols for specific and quantitative immunohistochemical detection of human kallikrein 2 (hK2) using lanthanide chelate-labeled monoclonal antibodies (Mabs) and time-resolved fluorescence imaging. Anti-prostate-specific antigen (PSA) Mabs were tested in microtiterplate assays for their ability to prevent PSA from cross-reacting with the anti-hK2 Mab 6H10. Europium-labeled 6H10 and terbium-labeled anti-PSA Mab 2E9, selected as the best blocker antibody, were used for dual-label immunodetection in routinely fixed benign (n = 7) and malignant (n = 5) prostate specimens. The amounts of IgG bound in tissue were calculated from drops containing known Mab concentrations. The use of anti-PSA Mab 2E9 for blocking diminished the cross-reaction from 5% to 0.3%. In the analyzed tissues, there was considerable variation in staining intensity for both proteins; PSA signals varied from 0.1 to 36.6 times that of hK2, with on average 10-fold more bound anti-PSA Mab than anti-hK2 Mab. In malignant tissue, the amounts of bound IgGs were lower and more variable than in benign tissue using both the anti-PSA Mab and the anti-hK2 Mab. The variation in signal intensities for PSA and hK2 correlated significantly in benign tissue (P >0.05), but not in benign hyperplastic and malignant specimens (P <0.05). Quantification of two lanthanide chelate-labeled antibodies bound in the same tissue section enabled comparison of PSA and hK2 content in individual cells. The average cellular content of hK2 relative to that of PSA was consistent with previous mRNA studies. The time-resolved fluorescence imaging-based quantification method has universal applicability in fixed tissue specimens.
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We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a... more
We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.
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Research Interests: Chemistry, Chromatography, Fluorescence, Medicine, Clinical Chemistry, and 13 moreHumans, Medical Biotechnology, Pubmed, Male, Monoclonal Antibodies, Terbium, Clinical Sciences, Green Reagent, Reproducibility of Results, Monoclonal Antibody, Prostate Specific Antigen, Europium, and Medical biochemistry and metabolomics
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... more
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.
Research Interests: Chemistry, Chromatography, Medicine, Clinical Chemistry, Humans, and 13 moreMedical Biotechnology, Monoclonal Antibodies, Terbium, Clinical Sciences, Antibody, Immunoassay, Monoclonal Antibody, Luteinizing Hormone, Fluorometry, Follicle stimulating hormone, Radioimmunoassay, Europium, and Medical biochemistry and metabolomics
Time-resolved fluorometry was used for the detection of DNA probes labeled directly with europium (Eu3+) chelate. The sensitivity of this nonisotopic hybridization assay was 100 pg of homologous DNA. Equivalent results with reference... more
Time-resolved fluorometry was used for the detection of DNA probes labeled directly with europium (Eu3+) chelate. The sensitivity of this nonisotopic hybridization assay was 100 pg of homologous DNA. Equivalent results with reference tests were obtained in the detection of adenoviruses in clinical specimens.
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Prostate-specific antigen (PSA) and human prostatic glandular kallikrein (hK2) have 79% identity with the primary structure. When we used recombinant hK2 protein, only 7 of 23 monoclonal anti-PSA IgGs (monoclonal antibodies, MAbs)... more
Prostate-specific antigen (PSA) and human prostatic glandular kallikrein (hK2) have 79% identity with the primary structure. When we used recombinant hK2 protein, only 7 of 23 monoclonal anti-PSA IgGs (monoclonal antibodies, MAbs) cross-reacted with hK2, which enabled us to design a novel immunofluorometric MAb-MAb assay for the specific detection of hK2. In the first incubation, an excess of MAb 2H11, which does not cross-react with hK2, is added to prevent both free and complexed PSA from reacting in subsequent immunoreactions. In the second incubation, biotinylated MAb H50, which cross-reacts with hK2 by an epitope overlapping with MAb 2H11, served to bind only hK2 to the microtitration wells coated with streptavidin. In the third step, Eu-labeled MAb H117, which cross-reacts with hK2, detected the immobilized hK2. The hK2 assay was calibrated with recombinant hK2. The detection limit of the assay was 0.1 microgram/L, and the cross-reactivity with recombinant PSA was < or = 0....
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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... more
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-fluores...
Research Interests: Endocrinology, Chemistry, Chromatography, Cystic Fibrosis, Medicine, and 15 moreClinical Chemistry, Humans, Internal Medicine, Medical Biotechnology, Muscular Dystrophies, Creatine Kinase, Hypothyroidism, Clinical Sciences, Isoenzymes, Immunoassay, Monoclonal Antibody, Dysprosium, Europium, Medical biochemistry and metabolomics, and Neonatal screening
Spectrophotometry of CK-B subunit activity based on immunoinhibition is rapid and convenient. However, the low sensitivity limits clinical applications to serum samples with CK-B activities considerably above normal. A sensitive, but not... more
Spectrophotometry of CK-B subunit activity based on immunoinhibition is rapid and convenient. However, the low sensitivity limits clinical applications to serum samples with CK-B activities considerably above normal. A sensitive, but not optimized, bioluminescent assay of CK-B, also based on immunoinhibition, has been described. We improved this method by the use of an ADP concentration saturating the CK reaction and by the combined use of AMP and diadenosine pentaphosphate for inhibition of adenylate kinase. The optimized bioluminescent method is similar to the corresponding spectrophotometric method (reaction conditions, analytical procedure, numerical results, etc.) and results by the two methods correlate well. However, the measuring time is shorter and, more important, the sensitivity of the bioluminescent method allows accurate determinations of CK-B, even in serum from healthy individuals (CV approximately 3%). Kits and instrumentation (manual and automatic) for determination...
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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... more
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.