Timo Lövgren

Europium as a label in time-resolved immunofluorometric assays

A nonisotopic immunoassay has been developed based on a sensitive detection of europium (III) in water solution using time-resolved fluorometry. The europium label is bound to the antibody with EDTA derivatives, either diazophenyl-EDTA-Eu or isothiocyanatophenyl-EDTA-Eu. After the immunometric assay has been completed the europium is preferably dissociated from the antibody at low pH and measured by time-resolved fluorescence in a micellar solution containing Triton X-100, beta-diketone, and a Lewis base. The detergent solubilizes the chelating compounds in the solution and excludes water from the fluorescent ligand-europium complex. Europium concentrations as low as 5 X 10(-14)M were measured using a 1-s counting time. The sensitivity of the immunoassay of rabbit IgG used as a model system was 25 pg/ml (6 pg/assay).

Strong Prediction of Fractures Among Older Adults by the Ratio of Carboxylated to Total Serum Osteocalcin

We examined serum total osteocalcin (TOC), carboxylated osteocalcin (COC), and their ratio (COC/TOC) by one‐step two‐site immunofluorescent assays in 87% (n = 792) of all home‐dwelling persons of 70 years or older living in a defined area in northern Finland. Other baseline subject‐related risk factors of fractures were assessed by postal questionnaires, interviews, clinical examinations, and tests. During a 5‐year follow‐up period, all falls and fractures (n = 106) were recorded by regular phone calls and by examining all the medical records yearly. Serum TOC and COC concentrations increased with advancing age and were higher in women than in men, but corresponding differences were not found in the case of COC/TOC. The adjusted relative risk of fracture was elevated in association with low (≤−1 SD from the mean) COC; hazard ratio (HR, 95% CI) 2.00 (1.20‐3.36) and low COC/TOC; HR 5.32 (3.26‐8.68), the relative risk being highest in the population older than 80 years; and HR 7.02 (2.42‐20.39). The predictive value of low COC/TOC lasted 3 years. The multivariable‐adjusted relative risk of hip fracture (n = 26) in regard to low COC/TOC ratio was 3.49 (1.12‐10.86), as compared with the persons who did not suffer hip fractures. Our results suggest that serum COC concentrations and, more strongly, COC/TOC, predict the occurrence of fractures in older community‐dwelling adults. The risk of fracture associated with low COC/TOC equals the hip fracture risk previously verified for concomitant high serum undercarboxylated OC concentrations and low bone mineral density.

In vitro stability of free prostate-specific antigen (PSA) and prostate-specific antigen (PSA) complexed to α1-antichymotrypsin in blood samples

OBJECTIVES To study the in vitro stability of free and complexed forms of prostate specific antigen (PSA) in blood samples in order to establish guidelines for specimen handling, in particular for the clinical utility of the analysis of percentage free PSA. METHODS Blood samples were collected and processed to generate serum, heparin plasma, and EDTA plasma. Three different two-site immunoassays were used to measure the concentrations of total PSA (PSA-T), free form of PSA (PSA-F), and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) in order to determine the effect of repeated freezing and thawing, delayed separation of serum from blood cells, and stability during storage at 4 degrees C and 30 degrees C. RESULTS Five cycles of freezing and thawing introduced no statistically significant changes in the measured concentrations of PSA-T, PSA-F, or PSA-ACT. The effect of storing blood samples at room temperature for 1-6 h before separation of serum revealed a statistically significant decrease only for PSA-F after 5.5 h of storage (mean decrease 3.5%). PSA-T and PSA-ACT showed good stability in both serum and plasma samples, whereas PSA-F, after 1 week of storage at 4 degrees C, decreased on average by 28.8%, 7.8%, and 5.6%, respectively, in serum, heparin plasma, and EDTA plasma. The decreases of PSA-F at 4 degrees C were statistically significant (P < 0.05) relative to the controls (samples stored at -20 degrees C) after storage for 23 h in serum, 86 h in heparin plasma, and 71 h in EDTA plasma. When the same samples were stored at 30 degrees C for 24 h, only the mean decrease of PSA-F (4.8%) in serum was statistically significant. CONCLUSIONS PSA-F in blood samples is less stable than PSA-ACT. It is not advisable to store samples on the clot, especially if time and temperature cannot be controlled. Serum samples should be stored frozen if not analyzed during the same day. After thawing, samples can be stored up to 23 h at 4 degrees C prior to analysis. The use of plasma samples improves the stability of free PSA.

Europium-labelled target cells in an assay of natural killer cell activity. I: A novel non-radioactive method based on time-resolved fluorescence

The use of a new marker for labelling cells used as targets for natural killer cells is described. The human erythroleukaemic cell line K-562 was used as target. The cells were labelled with europium diethylenetriaminopentaacetate (EuDTPA) chelates. The detection of the released marker is based on time-resolved fluorometry. The results obtained show that the method is sensitive, specific and rapid. The high specific activity of the marker and the sensitivity of the detection apparatus result in numeric values (counts per second) which are 10-20 times higher than the values (counts per minute) obtained with 51chromium. In comparison with 51chromium release assay the labelling of target cells is less time consuming, the marker release more rapid and the detection time of released marker only 1 second per tube. The use of this non-radioactive marker is an alternative way of measuring natural killer cell mediated cytolysis of target cells.

Determination of hormones by time-resolved fluoroimmunoassay

Immunoassays based on europium labels and time-resolved fluorescence as the detection method, have been developed. The specific activity of the label is several orders of magnitude higher than that of radioactive labels. Consequently, the technique provides great potential, especially in the determination of analytes which require high sensitivity. Both competitive and immunometric assays which use labelled antibodies have been worked out. In competitive assays the antigen is immobilized on a solid phase with a protein carrier. The antigen in the standard or sample then competes with the labelled antibody in solution. Separation is done simply by washing the wells in the microtitre strip where the assays are performed. Model systems are described for the measurement of testosterone and cortisol. Immunometric assays of human thyrotropin (hTSH) and luteotropin (LH) were performed with monoclonal antibodies, by either a one-step (hTSH) or two-step (LH) incubation procedure. These assays, which exploit the specific activity of the label, give a very high sensitivity and good reproducibility. The standard curves are linear and the dynamic range is at least 1000-fold. Because of the properties of the europium label and the simple assay design, the immunoassays based on time-resolved fluorescence are expected to gain wide application both in research and in routine determinations.

Photochemical Characterization of Up-Converting Inorganic Lanthanide Phosphors as Potential Labels

We have characterized commercially available up-converting inorganic lanthanide phosphors for their rare earth composition and photoluminescence properties under infrared laser diode excitation. These up-converting phosphors, in contrast to proprietary materials reported earlier, are readily available to be utilized as particulate reporters in various ligand binding assays after grinding to submicron particle size. The laser power density required at 980 nm to generate anti-Stokes photoluminescence from these particulate reporters is significantly lower than required for two-photon excitation. The narrow photoluminescence emission bands at 520–550 nm and at 650–670 nm are at shorter wavelengths and thus totally discriminated from autofluorescence and scattered excitation light even without temporal resolution. Transparent solution of colloidal bead-milled up-converting phosphor nanoparticles provides intense green emission visible to the human eye under illumination by an infrared laser pointer. In this article, we show that the unique photoluminescence properties of the up-converting phosphors and the inexpensive measurement configuration, which is adequate for their sensitive detection, render the up-conversion an attractive alternative to the ultraviolet-excited time-resolved fluorescence of down-converting lanthanide compounds widely employed in biomedical research and diagnostics.

Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence.

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.

Comparison of analysis of the different prostate-specific antigen forms in serum for detection of clinically localized prostate cancer

OBJECTIVES To compare different forms and ratios of serum prostate-specific antigen (PSA) to determine which form or ratio provides optimal diagnostic specificity and sensitivity in distinguishing between benign prostatic hyperplasia (BPH) and clinically localized prostate cancer. METHODS Serum samples were obtained from 47 patients with BPH and 39 with clinically localized prostate cancer. Patients with BPH underwent either transurethral resection of the prostate or transurethral microwave thermotherapy. Patients with prostate cancer, all of whom had no metastases on radionucleotide bone scans and no pelvic lymph node involvement, underwent either radical external beam radiation therapy or radical retropubic prostatectomy. All patients had pretreatment serum PSA levels between 1 and 20 ng/mL. The different forms of serum PSA (free PSA [PSA-F], PSA complexed to alpha 1-antichymotrypsin [PSA-ACT], and total PSA [PSA-T]) were measured using different monoclonal antibodies against PSA and ACT and immunofluorometric assay techniques. Furthermore, three ratios (PSA-F/PSA-T, PSA-ACT/PSA-T, and PSA-F/PSA-ACT) were calculated. RESULTS By receiver operating characteristic curve analysis, the performance of the different forms and ratios were compared. The PSA-F/PSA-T ratio had the greatest area under the curve (AUC, 0.776), significantly larger than that for PSA-T (0.612; P = 0.024). For PSA-ACT/PSA-T, the AUC was 0.695 (P = 0.283 versus PSA-T) and 0.773 for PSA-F/PSA-ACT (P = 0.051 versus PSA-T). At a cutoff level < 0.17, PSA-F/PSA-T had a sensitivity of 79%, a specificity of 66%, and a positive predictive value of 66% compared with 74%, 38%, and 50%, respectively, for PSA-T at a cutoff level > 4.0 ng/mL. CONCLUSIONS The PSA-F/PSA-T ratio gives the best diagnostic performance compared with that for other forms and ratios of PSA and will reduce the number of prostatic biopsies in patients with BPH.

Highly sensitive immunoassay of free prostate-specific antigen in serum using europium(III) nanoparticle label technology

BACKGROUND Recent proceedings in utilization of europium(III) chelate-dyed polystyrene nanoparticles as labels have combined the advantages of an enhanced monovalent binding affinity and a high specific activity of nanoparticle-antibody bioconjugate. Our objective was to evaluate the performance of the nanoparticle label technology with biological samples in an immunoassay of free prostate-specific antigen (PSA-F) using a standard microtitration well platform. METHODS Long-lifetime luminescent europium(III)-chelate nanoparticles, 107 nm in diameter, were coated with a PSA-F specific monoclonal antibody. The two-step noncompetitive immunoassay was performed in a microtitration well coated with a second monoclonal antibody. The signal of the surface-bound nanoparticle-antibody bioconjugates was measured directly from the bottom of the well using a standard time-resolved plate fluorometer. RESULTS The detection limit (mean + 2SD) of the nanoparticle-based PSA-F assay was 0.21 ng/l using a 20-microl sample volume. The assay response was linear up to 5 microg/l, and the functional sensitivity was approximately 0.5 ng/l. The within-run imprecision for spiked serum samples at concentrations 0.0005-0.5 microg/l was 6.4-21.8%, and the within-run and between-run imprecisions for serum samples at concentrations 0.2-2.5 microg/l were 3.4-7.2% and 4.4-7.6%, respectively. The concentrations obtained from serum samples correlated well with the reference immunoassay; slope = 1.018 +/- 0.018; intercept = 0.012 +/- 0.021 microg/l; S(y/x) = 0.112 microg/l; r = 0.993; n = 51. CONCLUSIONS The developed method demonstrated acceptable performance characteristics allowing clinical studies utilizing patient samples with extremely low concentrations of PSA-F. The present assay detected PSA-F in most of samples from prostatectomized men and in few samples from healthy women that were nondetectable according to the reference immunoassay.

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.