Polymer–protein-enhanced fluoroimmunoassay for prostate-specific antigen

Prostate-specific antigen (PSA) is a serum glycoprotein overproduced in prostate cancer, the total of which is comprised of two major forms, free and complexed. The common method for measuring of PSA is an Enzyme-linked immunosorbent assay (ELISA). Limits of detection using commercial PSA ELISA kits for free and total PSA were determined in our laboratory to be 1 and 10 ng/mL, respectively. A value of 0.10 was obtained for the free to total PSA ratio, a ratio of 0.25 being indicative of prostate cancer. A possible improvement in the sensitivity and detection limits of free and total PSA may be achieved by the ALYGNSA system (Clarizia et al., Anal Bioanal Chem 393:1531–1538, 2009). This fluorescent assay system employed a selective polymer biolinker system proven to enhance primary antibody orientation. Using this system, free and total PSA detection limits of 0.13 and 0.63 ng/mL, respectively, were realized. This amounted to an 8- and 15-fold improvement in detection limits for free and total PSA, respectively. A free to total PSA ratio of 0.20 was maintained in this study and may be useful for definitive diagnosis of prostate, as well as, other cancers.     

Differential diagnosis of prostate cancer and benign prostate hyperplasia using two-dimensional electrophoresis.

Prostate specific antigen (PSA) is a protease which is characteristic of the prostate. It is widely used as a serum marker for the early diagnosis of prostate cancer (PCa). Nevertheless, for concentrations between 4 and 10 ng/mL, PSA does not enable PCa to be distinguished from benign diseases, such as benign prostate hyperplasia (BPH). In sera, the use of a ratio between free PSA (PSA uncomplexed with protease inhibitor) and total PSA (free PSA and PSA bound to alpha-1 anti-chymotrypsin) enables the "gray zone" to be reduced, but an important proportion of patients are still wrongly classed. Using two-dimensional electrophoresis, we demonstrated using 52 PCa and 40 BPH well-documented clinical cases that BPH sera show a significantly greater percentage of low-molecular-weight free PSA elements (IwPSA) than PCa sera. In our study, the use of a ratio between IwPSA and standard free PSA enables the correct diagnosis of 100% of PCa and 82.5% of BPH cases as against when 73.1% and 42.5% respectively were correctly diagnozed using the total PSA and the free/total PSA ratio. This important finding may be related to differences in the mechanism secreting PSA from the prostate into the bloodstream. We have shown how a tissue marker may be turned into a powerful tumor marker by events probably unrelated to its expression.     

Determination of Prostate-Specific Antigen in Serum and a Reference Material by On-Chip Immunoaffinity Chromatography

Glycoprotein tumor markers are striking examples of heterogeneous analytes. The complexity of their structural forms in biological fluids is generally not reflected in reference materials. Therefore, they are not specified to consist of a distinct form, but rather to contain a mixture of molecular species. In this study, the question of the heterogeneity of free prostate-specific antigen (free PSA) is addressed in reference materials to define the immunoreactive molecular species and compare them to those in clinical serum. The reference material for free PSA and serum samples of subjects with benign prostatic hyperplasia and prostate cancer was examined for immunoreactivity to epitope I-specific anti-free PSA antibody using on-chip immunoaffinity chromatography in combination with mass spectrometry for the determination of bound forms. The mass spectra of the reference material for free PSA and clinical serum, obtained by on-chip immunoaffinity chromatography, were similar. The cluster of major free PSA-immunoreactive peaks at 28–29 kDa corresponding to the mature glycosylated PSA molecule overlapped in both analytes. However, the reference material displayed a more restricted pattern of low molecular mass species corresponding to nicked PSA fragments or PSA degradation products. The PSA concentration in clinical serum seems to consist of more species than equivalent concentrations of reference material. Regarding analysis of heterogeneous proteins, immunoaffinity capture combined with mass-specific detection represents a rapid means for selective detection of distinct molecular species, exceeding the analytical performance of current formats of immunoassays.     

Electron paramagnetic resonance, photo-acoustic spectroscopy and thermally stimulated luminescence for identification of thermally stimulated processes in gamma-irradiated LaBa2F7

Prostate specific antigen (PSA) is a reliable biochemical marker used in screening for prostate carcinoma. Immunoradiometric assays (IRMA) are generally used for the estimation of total/free PSA in serum samples. Radiolabeled antibody, an important reagent of IRMA was prepared and characterized using an in-house anti-PSA monoclonal antibody (Mab), Mab-2S. Mab-2S was radiolabeled with ¹²⁵I and characterized for immunoreactivity and radiochemical purity. The usability of the radioiodinated Mab as tracer in IRMA was ascertained using authentic reagents for IRMA of PSA.     

Electrophoretic fingerprint metallothionein analysis as a potential prostate cancer biomarker

Prostate-specific antigen (PSA) is a routinely used marker of prostate cancer; however, the cut-off values for unambiguous positive/negative prostate cancer diagnoses are not defined. Therefore, despite the best effort, certain percentage of misdiagnosed cases is being recorded every year. For this reason, search for more specific diagnostic markers is of great interest. In this study, systematic comparison of PSA and metallothionein (MT) levels in blood serum of 46 prostate cancer-diagnosed patients is presented. It is clearly demonstrated that PSA levels vary significantly and despite normal total PSA values in the range of 0 - 4?ng/mL were obtained in over 36.9% of cases, positive prostate cancer was diagnosed by biopsy. In contrary, MT levels were considerably elevated in all tested samples and no significant variations were observed. These results are indicating the potential of MT as an additional prostate cancer marker reducing, in combination with PSA, the probability of false positive/negative diagnosis. To increase the throughput of the screening, chip-based capillary electrophoresis was suggested as a rapid and effective method for the fingerprinting analysis of prostate cancer from diseased blood sera.     

Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers

We describe herein the combination of electrochemical immunosensors using single-wall carbon nanotube (SWNT) forest platforms with multi-label secondary antibody-nanotube bioconjugates for highly sensitive detection of a cancer biomarker in serum and tissue lysates. Greatly amplified sensitivity was attained by using bioconjugates featuring horseradish peroxidase (HRP) labels and secondary antibodies (Ab(2)) linked to carbon nanotubes (CNT) at high HRP/Ab(2) ratio. This approach provided a detection limit of 4 pg mL(-)(1) (100 amol mL(-)(1)), for prostate specific antigen (PSA) in 10 microL of undiluted calf serum, a mass detection limit of 40 fg. Accurate detection of PSA in human serum samples was demonstrated by comparison to standard ELISA assays. PSA was quantitatively measured in prostate tissue samples for which PSA could not be differentiated by the gold standard immunohistochemical staining method. These easily fabricated SWNT immunosensors show excellent promise for clinical screening of cancer biomarkers and point-of-care diagnostics.     

A bio-barcode assay for on-chip attomolar-sensitivity protein detection

Functionalized nanoparticles hold great promise in realizing highly sensitive and selective biodetection. We report a single disposable chip which is capable of carrying out a multi-step process that employs nanoparticles--a bio-barcode assay (BCA) for single protein marker detection. To illustrate the capability of the system, we tested for the presence of prostate specific antigen (PSA) in buffer solution and goat serum. Detection was accomplished at PSA concentrations as low as 500 aM. This corresponds to only 300 copies of protein analytes using 1 microL total sample volume. We established that the on-chip BCA for PSA detection offers four orders of magnitude higher sensitivity compared to commercially available ELISA-based PSA tests.     

Surface plasmon resonance application in prostate cancer biomarker research

Prostate cancer (PCa) diagnostics can be effectively addressed using sensor-based approaches. Proper selection of biomarkers to be included in biosensors for accurate detection becomes the need of the hour. Such biosensor and biochip technologies enable fast and efficient determination of proteins and provide a remarkable insight into the changes in the protein structure, such as aberrant glycosylation, which can increase the performance, sensitivity and specificity of clinic assays. However, for a thorough comprehension of such complex protein modifications, it is crucial to understand their biospecific interactions. Surface plasmon resonance (SPR), one of the most rapidly developing techniques for measuring real-time quantitative binding affinities and kinetics of the interactions of antigens and antibodies, was chosen as an appropriate tool for this purpose. Herein, experiments on the interactions of antibodies specific against different epitopes of free and complexed prostate-specific antigen (PSA), a prominent PCa biomarker, are presented with two main aims: (i) to continue as lectin glycoprofiling studies and; (ii) to be used in microfluidic immunoassay-based platforms for point-of-care devices. Various PSA-specific antibodies were covalently immobilized on the biochip surface via amine coupling, and free or complexed PSA was injected into the dual-flow channels of the SPR device. Kinetic parameters and affinity constants of these interactions, as well as cross-reactivities of the used antibodies were determined. The sandwich assay for PSA determination was developed employing both primary and secondary anti-PSA antibodies. Sensitivity of the assay was 3.63 nM^?1, the detection limit was 0.27 nM and the SPR biosensor response towards free PSA was linear up to 25 nM. All these findings are essential for proper design of a selective, sensitive, and highly reliable biosensor for PCa diagnosis as a lab-on-chip device.     

Kinetic Analysis of Prostate-Specific Antigen Interaction with Monoclonal Antibodies for Development of a Magnetic Immunoassay Based on Nontransparent Fiber Structures

Prostate cancer is the second most common cancer diagnosed in men worldwide. Measuring the prostate-specific antigen (PSA) is regarded as essential during prostate cancer screening. Early diagnosis of this disease relapse after radical prostatectomy requires extremely sensitive methods. This research presents an approach to development of an ultrasensitive magnetic sandwich immunoassay, which demonstrates the limit of PSA detection in human serum of 19 pg/mL at a dynamic range exceeding 3.5 orders of concentration. Such attractive performance stems, inter alia, from the kinetic analysis of monoclonal antibodies (mAbs) against free PSA to select the mAbs exhibiting best kinetic characteristics and specificity. The analysis is carried out with a label-free multiplex spectral-correlation interferometry compatible with inexpensive single-use glass sensor chips. The high sensitivity of developed PSA immunoassay is due to electronic quantification of magnetic nanolabels functionalized by the selected mAbs and three-dimension porous filters used as an extended solid phase. The assay is promising for PSA monitoring after radical prostatectomy. The proposed versatile approach can be applied for the rational design of highly sensitive tests for detection of other analytes in many fields, including in vitro diagnostics, veterinary, food safety, etc.     

Multiplex immunoassays on size-categorized individual beads using time-resolved fluorescence

Miniaturized multiplex immunoassays were studied on individual beads to detect analytes in the same reaction mixture. With this approach hands-on time, cost, and amount of reagents as well as waste produced in the assays were reduced. Particles were categorized according to size for immunoassays of prostate specific antigen (PSA) and acute myocardial infarction (AMI) markers. Additionally, free and dual PSA assays were carried out on a single bead. The analyte concentration was detected directly on the surface of the beads using stable, intrinsically fluorescent europium and terbium chelates, and time-resolved fluorometry. Less than 0.4 ng ml⁻¹ PSA (corresponding to ca. 10 amol) was detected in free, dual and multiplex PSA assays and 0.1 and 2.4 ng ml⁻¹ of myoglobin (Mb) and creatine kinase MB (CK-MB), respectively, were monitored in the multiplex AMI marker assay. The effect of bead size and material on free PSA detection was also investigated. The beads were detected three times under different conditions; in liquid, after drying and after dissociating europium ions from the chelate into the DELFIA^® fluorescence enhancement solution. The same detection sensitivity was found for the dissociative and non-dissociative methods indicating that the current labeling technology for the surface detection is comparable to the commercial DELFIA system.     

An overview of the health effects of isoflavones with an emphasis on prostate cancer risk and prostate-specific antigen levels

Soybean isoflavones possess hormonal and nonhormonal properties that may reduce the risk of coronary heart disease, osteoporosis, and certain cancers, and alleviate hot flashes in menopausal women. Among the various cancers whose risk may be reduced by isoflavones, there is particular interest in prostate cancer. Eleven trials have examined the effects of isoflavones on serum prostate-specific antigen (PSA) levels. The dose of isoflavones in these trials from supplements or soy protein ranged from 60 to 900 mg/day (typical Japanese intake is 30-50 mg/day), subject number/group ranged from 8 to 62, and study duration from 20 days to 1 year. Isoflavones did not affect serum PSA in healthy subjects. In contrast, in 4 of 8 trials involving prostate cancer patients, isoflavones significantly favorably affected PSA although in no studies was there an absolute decrease in PSA concentrations. The mechanism by which isoflavones affect PSA could not be determined from the existing research, although hormonal changes do not seem to be a factor. The clinical evidence is sufficiently encouraging to justify considering additional Phase II and III clinical trials investigating the efficacy of soy isoflavones in different populations of prostate cancer patients alone and in combination with other treatments.     

Selection of 2′F-modified RNA aptamers against prostate-specific antigen and their evaluation for diagnostic and therapeutic applications

Currently, prostate-specific antigen (PSA) is considered to be the most sensitive marker available for prostate cancer detection and for monitoring of disease progression. In addition to its importance as a tumor marker, PSA has a role in the biological activity of cancer growth and proliferation. Therefore, the inhibition or activation of its biological activity may be used in prostate cancer therapy. Here, we describe the isolation and characterization of new 2′F-modified RNA aptamers directed against PSA. Binding studies demonstrate the ability of these new aptamers to specifically recognize their target with dissociation constants in the nanomolar range. In order to demonstrate the functionality of the selected aptamers, an apta-PCR approach was used for the quantitative detection of PSA, achieving a limit of detection of 11 nM. Furthermore, the potential use of the selected aptamers in therapeutics was demonstrated with the 2′F-modified aptamers being highly stable in human serum and having the ability to moderate the activity of PSA, which will be explored for the treatment of prostate cancer.     

Electrochemical Immunoassay for Free Prostate Specific Antigen (f‐PSA) Using Magnetic Beads

Prostate specific antigen (PSA) is a prominent marker for the prostate carcinoma. It is found in human blood in free (f‐PSA) and complex forms. These two forms together are called total PSA (t‐PSA). Estimation of both forms is essential to predict malignancy. In this study we report a unique and effective technique of electrochemical detection of f‐PSA using magnetic beads on a three‐electrode screen‐printed sensor. A magnetic bead enzyme linked immunosorbent assay (ELISA) was performed in a cuvette. Following the immunoassay, magnetic beads were recovered by a magnetic concentrator and transferred on the working electrode of the 3‐electrode assembly. The amperometric response, a measure of the amount of residual enzyme activity on the beads and hence the concentration of analyte in solution, was determined by addition of enzyme substrate. The device has a detection limit of <0.1 ng mL^?1 f‐PSA and a linear range of 0 to 1 ng mL^?1 f‐PSA.     

核磁共振联合超声融合成像引导下前列腺靶向穿刺对PI-RADS评分≥3分的前列腺癌患者的诊断价值

为了探讨核磁共振(MRI)联合超声融合成像引导下前列腺靶向穿刺(MR-TRUS)对PI-RADS评分≥3分的前列腺癌患者的诊断价值,本研究选取了进行前列腺癌筛查且MRI检查PI-RADS评分≥3分的患者100例,均行MRI与超声融合靶向穿刺和传统穿刺.并对上述100名患者进行前列腺特异性抗原(PSA)检测,按4＜PSA...     

HPLC for stress-free screening of potential prostate cancer marker catechol estrogens in urine using a diamond-electrode electrochemical and a fluorescence detector

Improvement of the sensitivity and specificity of a simultaneous stress-free screening method for catechol estrogens as a potential prostate cancer marker in urine has been accomplished by HPLC with a diamond-electrode electrochemical detector and a fluorescence detector. Since taking urine samples generates less stress (or pain) than the drawing of blood, the method can readily be applied to almost any patient, and will also assist in improving the sensitivity and specificity of the prostatic specific antigen test. Catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 2-hydroxyestriol) and estrogens (estrone, estradiol, estriol) were separated on an Inertsil ODS-II column with acetonitrile-potassium dihydrogen phosphate (pH 3.0). The diamond-electrode electrochemical detector used had the great advantage of being a maintenance-free system, and could sequentially analyze hundreds of samples. Fluorescence detection improved the sensitivity 10-500 times (e. g., the LOD of 2-hydroxyestriol was improved 250 times) compared to previous electrochemical detection reports, and dual detection improved peak identification in the urine samples. The proposed method was applied to the simultaneous determination of catechol estrogens in spiked urine in a preliminary study on estrogens and PSA values in biopsy and prostate cancer patients.     

Potent and selective peptidyl boronic acid inhibitors of the serine protease prostate-specific antigen

Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a K_i for PSA of 65 nM. The inhibitor had a 60-fold higher K_i for chymotrypsin. A validated model of PSA's catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme.     

Immunoaffinity chromatographic isolation of prostate-specific antigen from seminal plasma for capillary electrophoresis analysis of its isoforms

Prostate-specific antigen (PSA) concentration in serum has been the biomarker employed for prostate cancer diagnosis in the last two decades. However, new more specific biomarkers allowing a better differentiation of cancer from non-malignant prostate diseases are necessary. Glycosylation of PSA gives rise to different forms of the protein which can be separated into several isoforms by analytical techniques, such as CE. Because PSA glycosylation is influenced by pathological conditions, the CE pattern of PSA isoforms could be different in prostate cancer than in non-malignant prostate diseases. To study this CE pattern of PSA, prior purification of the protein from the biological fluid is mandatory. In this study an immunoaffinity chromatography method which allows PSA purification without altering the CE pattern is developed. An in-house prepared column produced with commercial anti-PSA antibodies is employed. The use of 1 M propionic acid as elution agent provides higher than 40% recovery of high purity PSA. CE analysis of PSA immunopurified from seminal plasma of a healthy individual shows the same 8 peaks as the commercially available PSA standard. Sample preparation only requires dilution with phosphate buffered saline prior to immunoaffinity purification. High repeatability for the sample preparation step was achieved (RSD% for percentage of corrected peak area in the range 0.6–5.3 for CE analysis of three independently purified seminal plasma aliquots compared to range 0.8–4.9 for a given aliquot analyzed three times by CE). IAC of five microliters seminal plasma provided enough PSA to achieve signal/noise ratio larger than 5 for the smallest CE isoforms.     

Serum-based ALYGNSA immunoassay for the prostate cancer biomarker, total prostate-specific antigen (tPSA)

Prostate-specific antigen (PSA) is a serum glycoprotein overproduced by the prostate in prostate cancer (≥4 ng/mL in the bloodstream). An immunoassay for total PSA (tPSA) was developed using the ALYGNSA method to enhance capture antibody orientation and a limit of detection of 0.63 ng/mL was reported, a limit 15-fold lower than a commercial tPSA ELISA assay. This ALYGNSA assay, however, was performed using only buffer-based proteins and blocking agents (Mackness et al., Anal Bioanal Chem 396:681–686, 2010). To improve the clinical application of this system, a serum-based tPSA ALYGNSA was developed employing human serum. This assay also resulted in a limit of detection of 0.63 ng/mL of tPSA protein. The findings reported here provide support for the clinical application of this assay for diagnosis, progression, treatment, and possible recurrence of prostate cancer.     

Sensitivity improvement of a sandwich-type ELISA immunosensor for the detection of different prostate-specific antigen isoforms in human serum using electrochemical impedance spectroscopy and an ordered and hierarchically organized interfacial supramolecular architecture

A gold millielectrode (GME) functionalized with a mixed (16-MHA + EG₃SH) self-assembled monolayer (SAM) was used to fabricate an indirect enzyme-linked immunosorbent assay (ELISA) immunosensor for the sensitive detection of prostate-specific antigen (PSA), a prostate cancer (PCa) biomarker, in human serum samples. To address and minimize the issue of non-specific protein adsorption, an organic matrix (amine-PEG₃-biotin/avidin) was assembled on the previously functionalized electrode surface to build up an ordered and hierarchically organized interfacial supramolecular architecture: Au/16-MHA/EG₃SH/amine-PEG₃-biotin/avidin. The electrode was then exposed to serum samples at different concentrations of a sandwich-type immunocomplex molecule (^BtnAb-Ag_PSA-^HRPAb), and its interfacial properties were characterized using electrochemical impedance spectroscopy (EIS). Calibration curves for polarization resistance (R_P) and capacitance (1/C) vs. total and free PSA concentrations were obtained and their analytical quality parameters were determined. This approach was compared with results obtained from a commercially available ELISA immunosensor. The results obtained in this work showed that the proposed immunosensor can be successfully applied to analyze serum samples of patients representative of the Mexican population.     

Designing nanomaterial-enhanced electrochemical immunosensors for cancer biomarker proteins

Detection of multiple cancer biomarker proteins in human serum and tissue at point-of-care is a viable approach for early cancer detection, but presents a major challenge to bioanalytical device development. This article reviews recent approaches developed in our laboratories combining nanoparticle decorated electrodes and multilabeled secondary antibody labeled particles to achieve high sensitivity for the detection of cancer biomarker proteins. Two nanomaterial-based sensor platforms were used: (a) upright single wall carbon nanotube forests and (b) layers of densely packed 5 nm gold nanoparticles. Both platforms feature pendant carboxylate groups for easy attachment of enzymes or antibodies by amidization. In quality performance tests, the biocatalytic responses for determination of hydrogen peroxide of AuNP layers with attached horseradish peroxidase (HRP) on electrodes gave somewhat better detection limit and sensitivity than single wall carbon nanotube (SWNT) forest platforms with HRP attached. Evaluation of these sensors as platforms for sandwich immunoassays for cancer biomarker prostate specific antigen (PSA) in serum showed that both approaches gave accurate results for human serum samples from cancer patients. The best detection limit (0.5 pg mL^− 1) and sensitivity were obtained by combining the AuNP immunosensors with binding of 1 μm diameter magnetic particles decorated with secondary antibodies and 7500 HRP labels.