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

Luminescence ◽  
2000 ◽  
Vol 15 (6) ◽  
pp. 351-355 ◽  
Author(s):  
Harri H�rm� ◽  
Tero Soukka ◽  
Stefan L�nnberg ◽  
Janika Paukkunen ◽  
Piia Tarkkinen ◽  
...  
2001 ◽  
Vol 47 (3) ◽  
pp. 561-568 ◽  
Author(s):  
Harri Härmä ◽  
Tero Soukka ◽  
Timo Lövgren

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.


2003 ◽  
Vol 482 (2) ◽  
pp. 157-164 ◽  
Author(s):  
Harri Härmä ◽  
Anne-Maria Pelkkikangas ◽  
Tero Soukka ◽  
Petri Huhtinen ◽  
Saila Huopalahti ◽  
...  

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Chun-Jen Hsiao ◽  
Tzong-Shin Tzai ◽  
Chein-Hung Chen ◽  
Wen-Horng Yang ◽  
Chung-Hsuan Chen

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.


1995 ◽  
Vol 41 (1) ◽  
pp. 54-58 ◽  
Author(s):  
H Yu ◽  
E P Diamandis

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.


Weed Science ◽  
1994 ◽  
Vol 42 (4) ◽  
pp. 517-522 ◽  
Author(s):  
Michael P. Anderson ◽  
Curtis N. Bensch ◽  
Jimmy F. Stritzke

This paper reports on a microtitre plate version of the 2,6-dichlorophenol-indophenol (DCPIP) reduction assay. DCPIP reduction rates of alfalfa thylakoid membranes were determined by measuring absorbance at 600 nm before and after a 1 min illumination period. The membranes were near light-saturated at intensities above 600 μE m–2s–1. Saturation of thylakoid membranes with DCPIP occurred above 120 μM. DCPIP reduction rates increased linearly with chlorophyll concentrations from 0.25 to 2 μg. The rate of DCPIP reduction was linear throughout a 2 min illumination period (R2= 0.99). The assay was sensitive enough to terbacil to differentiate between a 20% change in the I50concentration. DCPIP reduction rates were sensitive to concentrations of terbacil as low as 100 nM. It only takes 13 minutes to load and read 96 samples using the microtitre plate assay compared to 5.5 h using the conventional procedure.


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