scholarly journals Europium Nanoparticles and Time-resolved Fluorescence for Ultrasensitive Detection of Prostate-specific Antigen

2001 ◽  
Vol 47 (3) ◽  
pp. 561-568 ◽  
Author(s):  
Harri Härmä ◽  
Tero Soukka ◽  
Timo Lövgren
Keyword(s):  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
High Specific Activity ◽  
Microtiter Plate ◽  
Detection Sensitivity ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Biochemical Assays ◽  
Microtiter Plate Format

Abstract Background: Nanoparticle-based detection technologies have the potential to improve detection sensitivity in miniature as well as in conventional biochemical assays. We introduce a detection technology that relies on the use of europium(III) nanoparticles and time-resolved fluorometry to improve the detection limit of biochemical assays and to visualize individual molecules in a microtiter plate format. Methods: Streptavidin was covalently coated on 107-nm nanoparticles containing >30 000 europium molecules entrapped with β-diketones. In a model assay system, these nanoparticles were used to trace biotinylated prostate-specific antigen (PSA) in a microtiter plate format. Results: The detection limit (mean + 3 SD of the zero calibrator) of biotinylated PSA was 0.38 ng/L, corresponding to 10 fmol/L or 60 zeptomoles (60 × 10−21 moles) of PSA. Moreover, single nanoparticles, representing individual PSA molecules, were visualized in the same microtiter wells with a time-resolved fluorescence microscope using a ×10 objective. Single nanoparticles, possessing high specific activity, were also detected in solution by a standard time-resolved plate fluorometer. Conclusions: The universal streptavidin-coated europium(III) nanoparticle label is suitable for detection of any biotinylated molecule either in solution or on a solid phase. The europium(III) nanoparticle labeling technology is applicable to many areas of modern biochemical analysis, such as immunochemical and multianalyte DNA-chip assays as well as histo- and cytochemistry to improve detection sensitivities.

Sensitive miniature single-particle immunoassay of prostate-specific antigen using time-resolved fluorescence

2003 ◽  
Vol 482 (2) ◽  
pp. 157-164 ◽  
Author(s):  
Harri Härmä ◽  
Anne-Maria Pelkkikangas ◽  
Tero Soukka ◽  
Petri Huhtinen ◽  
Saila Huopalahti ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Single Particle ◽  
Specific Antigen ◽  
Time Resolved Fluorescence ◽  
Time Resolved

scholarly journals Supersensitive Time-resolved Immunofluorometric Assay of Free Prostate-specific Antigen with Nanoparticle Label Technology

2001 ◽  
Vol 47 (7) ◽  
pp. 1269-1278 ◽  
Author(s):  
Tero Soukka ◽  
Janika Paukkunen ◽  
Harri Härmä ◽  
Stefan Lönnberg ◽  
Hanne Lindroos ◽  
...  
Keyword(s):  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Binding Affinity ◽  
Specific Activity ◽  
Solid Phase ◽  
Specific Antigen ◽  
Nonspecific Binding ◽  
Time Resolved ◽  
Detection Range ◽  
Assay Time

Abstract Background: The extreme specific activity of the long-lifetime fluorescent europium(III) chelate nanoparticles and the enhanced monovalent binding affinity of multivalent nanoparticle-antibody bioconjugates are attractive for noncompetitive immunoassay. Methods: We used a noncompetitive, two-step immunoassay design to measure free prostate-specific antigen (PSA). Europium(III) chelate nanoparticles (107 nm in diameter) were coated with a monoclonal anti-PSA antibody (intrinsic affinity, 6 × 109 L/mol). The nanoparticle-antibody bioconjugates had an average of 214 active binding sites per particle and a monovalent binding affinity of 7 × 1010 L/mol. The assay was performed in a low-fluorescence microtitration well passively coated with an another monoclonal anti-PSA antibody (affinity, 2 × 1010 L/mol), and the europium(III) fluorescence was measured directly from the bottom of the well by a standard time-resolved microtitration plate fluorometer. Results: The detection limit (mean + 2 SD) was 0.040 ng/L (7.3 × 105 molecules/mL), and the dynamic detection range covered four orders of magnitude in a 3-h total assay time. The imprecision (CV) over the whole assay range was 2–10%. The detection limit of the assay was limited by the fractional nonspecific binding of the bioconjugate to the solid phase (0.05%), which was higher than the nonspecific binding of the original antibody (<0.01%). Conclusions: The sensitivity of the new assay is equal to that of the ambient-analyte, microspot immunoassay and will be improved by use of optimized, high binding-site density nanoparticle-antibody bioconjugates with reduced nonspecific binding and improved monovalent binding affinity.

scholarly journals Analysis of Urinary Prostate-Specific Antigen Glycoforms in Samples of Prostate Cancer and Benign Prostate Hyperplasia

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Chun-Jen Hsiao ◽  
Tzong-Shin Tzai ◽  
Chein-Hung Chen ◽  
Wen-Horng Yang ◽  
Chung-Hsuan Chen
Keyword(s):  
Prostate Cancer ◽  
Prostate Specific Antigen ◽  
Benign Prostate Hyperplasia ◽  
Clinical Sample ◽  
Specific Antigen ◽  
Ion Accumulation ◽  
Detection Sensitivity ◽  
Prostate Hyperplasia ◽  
Liquid Chromatography Tandem Mass ◽  
Benign Prostate

Glycans of prostate-specific antigen (PSA) in prostate cancer were found to be different from that in benign disease. It is difficult to analyze heterogeneous PSA glycoforms in each individual specimen because of low protein abundance and the limitation of detection sensitivity. We developed a method for prostate cancer diagnosis based on PSA glycoforms. Specific glycoforms were screened in each clinical sample based on liquid chromatography-tandem mass spectrometry with ion accumulation. To look for potential biomarkers, normalized abundance of each glycoform in benign prostate hyperplasia (BPH) and in prostate cancer was evaluated. The PSA glycoform, Hex5HexNAc4NeuAc1dHex1, and monosialylated, sialylated, and unfucosylated glycoforms differed significantly between the prostate cancer and BPH samples. The detection sensitivity (87.5%) and specificity (60%) for prostate cancer identification are higher than those of the serum PSA marker. As low as 100 amol PSA could be detected with the ion accumulation method which has not been reported before. The improved detection specificity can help reduce unnecessary examinations.

Prostate-specific antigen in milk of lactating women

1995 ◽  
Vol 41 (1) ◽  
pp. 54-58 ◽  
Author(s):  
H Yu ◽  
E P Diamandis
Keyword(s):  
Breast Milk ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Normal Breast ◽  
Specific Marker ◽  
Time Resolved ◽  
Lactating Women ◽  
Highly Sensitive ◽  
Mother’S Age ◽  
Total Psa

Abstract Prostate-specific antigen (PSA) is believed to be a highly specific marker for normal and cancerous prostatic tissue. We recently found that 30-40% of breast tumors produce PSA. Other data from our group suggest that normal breast can also produce PSA under conditions of stimulation by steroid hormones. In addition, we detected PSA in amniotic fluid. Here we report the presence of PSA in breast milk of lactating women. PSA concentrations in breast milk were quite variable, ranging from < 0.01 microgram/L in 4 of 38 milks to 350 micrograms/L; the median was 0.47 microgram/L. PSA concentration in breast milk was not correlated with mother's age or the sex of the newborn. It did tend to decrease with increasing time postdelivery, but was still detectable 2 weeks postdelivery. PSA in milk was equally measurable by a highly sensitive PSA assay based on time-resolved fluorometry and by the IMx automated PSA method. As confirmed by Western blot analysis, PSA in milk was present predominantly in its 33-kDa form; the PSA-alpha 1-antichymotrypsin complex (100 kDa) was also present but its concentration was < 25% of total PSA. We conclude that the female breast can produce PSA and that PSA is secreted into the milk during lactation; however, the biological role of PSA in milk is unknown. These and other data presented by our group suggest that PSA, a serine protease, may play a role in control of growth in mammary and other tissues through regulation of growth factors, cytokines, and growth-factor-binding proteins.

scholarly journals High-Throughput Screen for Inhibitors of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase by Surrogate Ligand Competition

2003 ◽  
Vol 8 (3) ◽  
pp. 332-339 ◽  
Author(s):  
Elizabeth B. Gottlin ◽  
R. Edward Benson ◽  
Scott Conary ◽  
Brett Antonio ◽  
Kellie Duke ◽  
...  
Keyword(s):  
High Throughput ◽  
Binding Sites ◽  
Biosynthetic Pathway ◽  
Enzyme Assay ◽  
High Throughput Screen ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Biochemical Assays ◽  
Key Enzyme ◽  
Nadph Oxidation

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function. ( Journal of Biomolecular Screening 2003:332-339)

scholarly journals Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA

1999 ◽  
Vol 45 (9) ◽  
pp. 1397-1407 ◽  
Author(s):  
Alice Ylikoski ◽  
Minna Sjöroos ◽  
Åke Lundwall ◽  
Matti Karp ◽  
Timo Lövgren ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Internal Standard ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

Abstract 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 106 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 ∼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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