Nicking enzyme-free strand displacement ampliﬁcation-assisted CRISPR-Cas-based colorimetric detection of prostate-specific antigen in serum samples

Abstract Background Previous studies have shown that the harmonization of prostate-specific antigen (PSA) assays remained limited even after the introduction of WHO International Standards. This information needs updating for current measuring systems (MS) and reevaluation according to established analytical performance specifications (APS) and the characteristics of antibodies used. Methods Total (tPSA) and free (fPSA) PSA were measured in 135 and 137 native serum samples, respectively, by Abbott Alinity i, Beckman Access Dxl, Roche Cobas e801, and Siemens Atellica IM MSs. Passing–Bablok regression and difference plots were used to compare results from each MS to the all-method median values. Agreement among methods was evaluated against APS for bias derived from biological variation of the 2 measurands. Results The median interassay CV for tPSA MSs (11.5%; 25–75th percentiles, 9.2–13.4) fulfilled the minimum APS goal for intermethod bias (15.9%), while the interassay CV for fPSA did not [20.4% (25–75th percentiles, 18.4–22.7) vs goal 17.6%]. Considering the all-method median value of each sample as reference, all tPSA MSs exhibited a mean percentage bias within the minimum goal. On the other hand, Alinity (+21.3%) and Access (−24.2%) were out of the minimum bias goal for fPSA, the disagreement explained only in minimal part by the heterogeneity of employed antibodies. Conclusions The harmonization among tPSA MSs is acceptable only when minimum APS are applied and necessitates further improvement. The marked disagreement among fPSA MSs questions the use of fPSA as a second-level test for biopsy referral.

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Miniature Single-Particle Immunoassay for Prostate-specific Antigen in Serum Using Recombinant Fab Fragments

Clinical Chemistry ◽

10.1093/clinchem/46.11.1755 ◽

2000 ◽

Vol 46 (11) ◽

pp. 1755-1761 ◽

Cited By ~ 12

Author(s):

Harri Härmä ◽

Piia Tarkkinen ◽

Tero Soukka ◽

Timo Lövgren

Keyword(s):

Prostate Specific Antigen ◽

Single Particle ◽

Binding Capacity ◽

Specific Antigen ◽

Genetically Engineered ◽

Covalent Attachment ◽

Serum Samples ◽

Fab Fragment ◽

Fab Fragments ◽

Total Psa

Abstract Background: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay. Methods: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-μm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer in a time-resolved mode. Results: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 μg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well assays for 21 serum samples: slopes for free and total PSA were 1.06 ± 0.03 and 1.03 ± 0.02, respectively (Sy|x = 0.084 and 0.057 μg/L), with intercepts of 0.013 ± 0.018 and 0.013 ± 0.017 μg/L (R >0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed. Conclusions: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected quantitatively.

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Measurement of Prostate-specific Antigen by Use of a Novel Blood Collection and Analytical System

Clinical Chemistry ◽

10.1093/clinchem/48.8.1272 ◽

2002 ◽

Vol 48 (8) ◽

pp. 1272-1278 ◽

Cited By ~ 4

Author(s):

Barbara R Grzeda ◽

Tuan Le Bui ◽

Cheryl N Warner ◽

Tracy L Pirucki ◽

Lisa M Dewey ◽

...

Keyword(s):

Prostate Cancer ◽

Prostate Specific Antigen ◽

Whole Blood ◽

Prostate Cancer Screening ◽

Specific Antigen ◽

Blood Collection ◽

Serum Samples ◽

Stabilizing Solution ◽

Professional Assistance ◽

Comparison Of The Results

Abstract Background: Prostate-specific antigen (PSA) is widely used in the detection and monitoring of prostate cancer. We developed a system for the self-collection and transport of capillary whole blood for PSA analysis, with the goal of reducing phlebotomy visits and, thus, increasing the access and utilization of PSA in prostate cancer screening and monitoring. Methods: The blood collection device [BIOSAFE Blood Transport System (BTSTM)] collects 70 μL of blood through a heparin-coated material into 200 μL of stabilizing solution. The diluted whole blood is used for measurement of PSA by a modified version of the Hybritech® Tandem-MP PSA Assay. Results were compared for matched samples of professionally and self-collected BTS blood and for matched BTS samples sera from blood collected by venipuncture. Imprecision for the whole-blood PSA measurement was estimated from analysis of whole-blood controls in duplicate, twice per day, over 20 days. Results: BTS samples (n = 140) collected by a qualified healthcare professional compared with serum samples yielded the regression equation: y =1.02x + 0.04 (Sy|x = 0.35; r = 0.99). Comparison of the results for samples (n = 128) collected by the patient without professional assistance with serum samples yielded: y = 1.08x + 0.02 (Sy|x = 0.31; r = 0.99). The between-run CVs at 0.069, 0.53, 2.9, and 10.7 μg/L were 21%, 6.0%, 3.5%, and 3.8%, respectively. PSA was stable in BTS samples stored for 21 days at 18–24 °C and for 7 days at 37 °C. Conclusion: The BIOSAFE BTS system allows accurate and convenient measurement of circulating PSA by a precise method for diluted whole blood.

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Silver nanoparticle@DNA tetrahedron-based colorimetric detection of HIV-related DNA with cascade strand displacement amplification

Journal of Materials Chemistry B ◽

10.1039/c9tb00274j ◽

2019 ◽

Vol 7 (16) ◽

pp. 2608-2612 ◽

Cited By ~ 5

Author(s):

Xiaoyi Ma ◽

Peng Miao

Keyword(s):

Silver Nanoparticles ◽

Silver Nanoparticle ◽

Colorimetric Detection ◽

Strand Displacement ◽

Colorimetric Analysis ◽

Strand Displacement Amplification ◽

Nicking Endonuclease ◽

Dna Tetrahedron

DNA tetrahedron-modified silver nanoparticles were constructed for the colorimetric analysis of HIV-related DNA with strand displacement polymerization and nicking endonuclease-aided cycles.

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Reference Reagents for Prostate-specific Antigen (PSA): Establishment of the First International Standards for Free PSA and PSA (90:10)

Clinical Chemistry ◽

10.1093/clinchem/46.9.1310 ◽

2000 ◽

Vol 46 (9) ◽

pp. 1310-1317 ◽

Cited By ~ 62

Author(s):

Brian Rafferty ◽

Peter Rigsby ◽

Matthew Rose ◽

Thomas Stamey ◽

Rose Gaines Das

Keyword(s):

Confidence Interval ◽

Prostate Specific Antigen ◽

Recombinant Dna ◽

Collaborative Study ◽

Specific Antigen ◽

International Standards ◽

Serum Samples ◽

International Standard ◽

Free Psa ◽

Total Psa

Abstract Background: Prostate-specific antigen (PSA) measurements in serum by immunoassay are widely used in the screening, diagnosis, and monitoring of patients with prostate cancer although the lack of common reference reagents has led in the past to wide differences in estimates. We report here the results of a WHO international collaborative study in which two preparations of PSA representative of the main immunoreactive components in serum, free PSA and PSA 90:10, and a preparation of recombinant DNA-derived PSA were assessed as potential standards for the calibration of diagnostic immunoassays for PSA. Methods: Coded vials of the candidate materials and serum preparations containing PSA in the clinically important range were provided to the 10 laboratories in the study, and participants were asked to perform PSA assays currently in use in their laboratories. Data from 89 immunoassays by 26 different method-laboratory combinations were contributed to the study and analyzed centrally at the National Institute for Biological Standards and Control. Results: Potency estimates of the preparations relative to the in-house calibrators were in good agreement with the target value of 1 μg of total PSA/vial, the preparation of free PSA giving 1.10 μg/vial (95% confidence interval, 0.99–1.21 μg/vial) and PSA 90:10, 1.11 μg/vial (95% confidence interval, 1.04–1.18 μg/vial). No immunoreactivity was detected in ampoules containing the recombinant material. Use of a common standard of PSA 90:10 significantly reduced the between-laboratory geometric coefficients of variation for serum samples included in the study and gave a much narrower range of potency estimates. Conclusions: The preparation of free PSA was established by WHO as the First International Standard for PSA (free) with an assigned content of 1 μg of total PSA per vial. In addition, the preparation of bound PSA was established as the First International Standard for PSA (90:10) with an assigned content of 1 μg of total PSA per vial.

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Integrated microfluidic systems with an immunosensor modified with carbon nanotubes for detection of prostate specific antigen (PSA) in human serum samples

Biosensors and Bioelectronics ◽

10.1016/j.bios.2007.11.003 ◽

2008 ◽

Vol 23 (7) ◽

pp. 1145-1151 ◽

Cited By ~ 82

Author(s):

Nancy V. Panini ◽

Germán A. Messina ◽

Eloy Salinas ◽

Héctor Fernández ◽

Julio Raba

Keyword(s):

Carbon Nanotubes ◽

Human Serum ◽

Prostate Specific Antigen ◽

Specific Antigen ◽

Microfluidic Systems ◽

Serum Samples ◽

Human Serum Samples

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Prostate Cancer Risk-Associated Single-Nucleotide Polymorphism Affects Prostate-Specific Antigen Glycosylation and Its Function

Clinical Chemistry ◽

10.1373/clinchem.2018.295790 ◽

2019 ◽

Vol 65 (1) ◽

pp. e1-e9 ◽

Cited By ~ 7

Author(s):

Srilakshmi Srinivasan ◽

Carson Stephens ◽

Emily Wilson ◽

Janaththani Panchadsaram ◽

Kerry DeVoss ◽

...

Keyword(s):

Prostate Cancer ◽

Prostate Specific Antigen ◽

Association Studies ◽

Specific Antigen ◽

Prostate Cancer Risk ◽

Genetic Association Studies ◽

Glycosylation Site ◽

Nucleotide Polymorphisms ◽

Serum Samples ◽

Single Nucleotide

Abstract BACKGROUND Genetic association studies have reported single-nucleotide polymorphisms (SNPs) at chromosome 19q13.3 to be associated with prostate cancer (PCa) risk. Recently, the rs61752561 SNP (Asp84Asn substitution) in exon 3 of the kallikrein-related peptidase 3 (KLK3) gene encoding prostate-specific antigen (PSA) was reported to be strongly associated with PCa risk (P = 2.3 × 10−8). However, the biological contribution of the rs61752561 SNP to PCa risk has not been elucidated. METHODS Recombinant PSA protein variants were generated to assess the SNP-mediated biochemical changes by stability and substrate activity assays. PC3 cell–PSA overexpression models were established to evaluate the effect of the SNP on PCa pathogenesis. Genotype-specific correlation of the SNP with total PSA (tPSA) concentrations and free/total (F/T) PSA ratio were determined from serum samples. RESULTS Functional analysis showed that the rs61752561 SNP affects PSA stability and structural conformation and creates an extra glycosylation site. This PSA variant had reduced enzymatic activity and the ability to stimulate proliferation and migration of PCa cells. Interestingly, the minor allele is associated with lower tPSA concentrations and high F/T PSA ratio in serum samples, indicating that the amino acid substitution may affect PSA immunoreactivity to the antibodies used in the clinical immunoassays. CONCLUSIONS The rs61752561 SNP appears to have a potential role in PCa pathogenesis by changing the glycosylation, protein stability, and PSA activity and may also affect the clinically measured F/T PSA ratio. Accounting for these effects on tPSA concentration and F/T PSA ratio may help to improve the accuracy of the current PSA test.

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Fifth-Generation Digital Immunoassay for Prostate-Specific Antigen by Single Molecule Array Technology

Clinical Chemistry ◽

10.1373/clinchem.2011.169540 ◽

2011 ◽

Vol 57 (12) ◽

pp. 1712-1721 ◽

Cited By ~ 43

Author(s):

David H Wilson ◽

David W Hanlon ◽

Gail K Provuncher ◽

Lei Chang ◽

Linan Song ◽

...

Keyword(s):

Prostate Cancer ◽

Radical Prostatectomy ◽

Prostate Specific Antigen ◽

Single Molecule ◽

Specific Antigen ◽

Analytical Performance ◽

Response To Treatment ◽

Serum Samples ◽

Limit Of Quantification ◽

Measurement Capability

BACKGROUND Measurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays. METHODS We developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients. RESULTS The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years. CONCLUSIONS The robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

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