Quantification Of Prostate Specific Antigen Research Articles

Measurement of prostate-specific antigen level as a biomarker for breast cancer by using mass signal amplification

Recently, it has been known that prostatespecific antigen (PSA) can be a useful biomarker for the diagnosis with breast cancer as well as that with prostate cancer. The level of PSA in patients of breast cancer is much lower than that in patients of prostate cancer, and therefore, the quantitative method of PSA with ultra-high sensitivity is required for the breast cancer diagnosis. In this paper, we introduce an efficient method for selective detection and quantification of PSA using gold nanoparticles (AuNPs), biochips, and mass spectrometry (MS). The PSA in a sample was captured by AuNPs and a biochip which present antibodies through a sandwich assay format. The AuNPs carried a huge number of reporter molecules, called amplification tag (Am-tag), and the presence of PSA in the sample was then verified through amplified mass signal of Am-tag by laser desorption/ionization timeof- flight (LDI-TOF) MS analysis to enable ultrasensitive detection of PSA. In addition, PSA in the sample was quantitated by using an internal standard (IS) which was prepared with AuNPs and deuterium-labeled Amtag (dAm-tag) which has a heavier molecular weight. Therefore, comparing the mass intensities between the Am-tag and the dAm-tag allowed quantification of PSA. This method was validated through the quantification of PSA in the female human serums with satisfactory results. We believe our method to detect and analyze PSA quantitatively and highly sensitively will be applied to patients’ serum for the diagnosis of breast cancer.

Hydrophilic interaction liquid chromatography as second dimension in multidimensional chromatography with an anionic trapping strategy: Application to prostate-specific antigen quantification

Liquid chromatography (LC) coupled with tandem mass spectrometry (MS–MS) in selected reaction monitoring mode (SRM) has become a widely used technique for the quantification of protein biomarkers in plasma and has already proven to give similar results compared to the conventional immunoassays. To improve the lack of insufficient sensitivity for quantification of low abundance protein, we propose a new two dimensional liquid chromatography (2D-LC-SRM) method for the quantitation of prostate specific antigen (PSA) in human plasma. The method centers on anion exchange cartridge between reversed-phase chromatography and hydrophilic interaction liquid chromatography (HILIC) in an on-line arrangement. The use of the anionic cartridge allows an easier online transfer of the analytes between both dimensions. Moreover, it provides an additional selectivity since the more basic peptides are not retained on this support. This setup has been applied to the quantification of prostate specific antigen (PSA) protein in plasma on a previous generation of mass spectrometer, which enabled a limit of quantification (LOQ) of 1ng/mL without any upfront immuno-depletion or intense off-line fractionation before the SRM analysis. The obtained LOQ is compatible with the required sensitivity for the clinically relevant plasma-based PSA tests.

Sandwich-type ELISA Impedimetric Immunosensor for Early Detection of Prostate-specific Antigen (PSA) in Human Serum

Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. A gold millielectrode (GME) functionalized with a mixed (16-MHA+EG3SH) self-assembled monolayer (SAM) was used to fabricate a point-of-care immunosensor for the detection of the prostate cancer biomarker: prostate-specific antigen (PSA) in human serum samples. An organic matrix (Amine-PEG3-biotin/Avidin) was used to reduce non-specific protein adsorption on the electrode surface. A 16-MHA/EG3SH/Amine-PEG3-biotin/Avidin GME was exposed to solutions with different sandwich-type immunocomplex (BtnAb-AgPSA-HRPAb) concentrations and its response was measured using the electrochemical impedance spectroscopy (EIS) technique. This approach was compared with regard to a reference ELISA immunoassay. The obtained results showed that the immunosensor proposed in this work qualifies for its pre-clinical application in the quantification of PSA in serum samples of a representative Mexican population.

Identification of a novel proteoform of prostate specific antigen (SNP-L132I) in clinical samples by multiple reaction monitoring.

Prostate specific antigen (PSA) is a well-established tumor marker that is frequently employed as model biomarker in the development and evaluation of emerging quantitative proteomics techniques, partially as a result of wide access to commercialized immunoassays serving as "gold standards." We designed a multiple reaction monitoring (MRM) assay to detect PSA proteoforms in clinical samples (n = 72), utilizing the specificity and sensitivity of the method. We report, for the first time, a PSA proteoform coded by SNP-L132I (rs2003783) that was observed in nine samples in both heterozygous (n = 7) and homozygous (n = 2) expression profiles. Other isoforms of PSA, derived from protein databases, were not identified by four unique proteotypic tryptic peptides. We have also utilized our MRM assay for precise quantitative analysis of PSA concentrations in both seminal and blood plasma samples. The analytical performance was evaluated, and close agreement was noted between quantitations based on three selected peptides (LSEPAELTDAVK, IVGGWECEK, and SVILLGR) and a routinely used commercialized immunoassay. Additionally, we disclose that the peptide IVGGWECEK is shared with kallikrein-related peptidase 2 and therefore is not unique for PSA. Thus, we propose the use of another tryptic sequence (SVILLGR) for accurate MRM quantification of PSA in clinical samples.

Abstract 3207: Application of an antibody-free, highly sensitive PRISM-SRM proteomics approach for monitoring low abundance proteins and protein isoforms in cell lines and tumor tissue.

Abstract Stable-isotope dilution coupled to targeted mass spectrometry, applying selected reaction monitoring (SRM), provides good selectivity, reproducibility, and sensitivity rendering it highly suitable for robust and high-throughput protein quantification. Historically, SRM sensitivity for measuring protein concentration in biofluids such as blood plasma has been limited to the low μg/mL level. Application of front-end sample preparation methods such as immunoaffinity depletion and strong cation exchange (SCX) chromatographic fractionation has improved the SRM detection limit to low ng/mL level. The ability to detect and quantify low abundance proteins present in blood plasma/serum at sub-ng/mL levels, however, relies on the availability of specific affinity reagents (e.g., antibodies that have the associated disadvantage of an expensive, lengthy, and high failure rate de novo process). To address this limitation, we have recently developed an antibody-free, targeted quantification capability that enables protein quantification of prostate-specific antigen and other exogenous proteins at 50-100 pg/mL levels in plasma/serum. Unlike the traditional SCX fractionation approach where all resulting fractions are analyzed by the second dimension LC-SRM, this new capability, PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing), uses high resolution. Citation Format: Jintang He, Xuefei Sun, Tujin Shi, Athena Schepmoes, Thomas Fillmore, Vladislav Petyuk, Fang Xie, Marina Gritsenko, Feng Yang, Naoki Kitabayashi, Sung Suk Chae, Mark Rubin, Javed Siddiqui, John Wei, Arul Chinnaiyan, Wei-Jun Qian, Richard Smith, Jacob Kagan, Sudhir Srivastava, Tao Liu, Karin Rodland, David Camp. Application of an antibody-free, highly sensitive PRISM-SRM proteomics approach for monitoring low abundance proteins and protein isoforms in cell lines and tumor tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3207. doi:10.1158/1538-7445.AM2013-3207

Role of magnetic Fe3O4 graphene oxide in chemiluminescent aptasensors capable of sensing tumor markers in human serum

Using π–π stacking interaction between magnetic Fe3O4 graphene oxide nanoparticles and impurities (e.g., biomolecules, free prostate specific antigen (PSA) aptamer-conjugated TEX615 that does not bind with PSA) in human serum, a rapid aptasensor with 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) detection was developed for the quantification of PSA, a tumor marker. The complexes, PSA bound with PSA aptamer-conjugated TEX615, were formed in human serum while the mixture (1 : 1 volume ratio) of PSA in 25% human serum diluted in Tris–HCl buffer (pH 7.5) and PSA aptamer-conjugated TEX615 in Tris–HCl buffer was incubated for 30 minutes. After the incubation, the impurities capable of reducing the efficiency of the aptasensor were removed using magnetic Fe3O4 graphene oxide nanoparticles. With the addition of ODI CL reagents (e.g., ODI, H2O2), finally, PSA in each sample was quantified within 1 second. The sensitivity of the aptasensor with good accuracy, precision, and recovery was as good as those of enzyme immunoassays with absorbance and chemiluminescence detections operated using time-consuming procedures such as multiple-time incubation and washing and expensive and intractable antibodies.

Clinical evaluation of a novel method for the measurement of prostate‐specific antigen, AccuPSATM, as a predictor of 5‐year biochemical recurrence‐free survival after radical prostatectomy: results of a pilot study

Study Type - Diagnostic (validating cohort) Level of Evidence 1b What's known on the subject? and What does the study add? Nadir Ultrasensitive PSA levels has some value for predicting BCR following RD. AccuPSA assays lower limit of PSA quantification of <0.01 pg/ml greatly enhances sensitivity and specificity of nadir PSA to predict BCR following RP. Our pilot study shows an AccuPSA of 3 pg/ml has a sensitory and specificity of 100% and 75% respectively for predicting 5 year BCR following RP. OBJECTIVES • To conduct a proof of concept study to evaluate a novel digital single molecule immunoassay (AccuPSA(TM) ) that detects prostate-specific antigen (PSA) a thousandfold more sensitively than current PSA detection methods. • To determine the ability of the AccuPSA(TM) assay to predict 5-year biochemical recurrence (BCR)-free survival after radical prostatectomy (RP). PATIENTS AND METHODS • A total of 31 frozen serum specimens were obtained from specimen logs maintained at New York University Langone Medical Center and the Johns Hopkins University School of Medicine on men who had undergone RP. Those men without evidence of BCR had a minimum of 5 years' PSA follow-up. • In all cases, preoperative and pathological information were available, as was a serum specimen 3-6 months after RP, with a PSA level of <0.1 ng/mL measured by conventional PSA methods at the time of serum collection. • Specimens were tested using the AccuPSA(TM) method. • A Cox proportional hazard model and Kaplan-Meier analysis were used to determine whether AccuPSA(TM) predicted the risk of BCR. RESULTS • Overall, 11/31 (35.5%) men developed BCR. • Mean AccuPSA(TM) nadir levels were significantly different (P < 0.001) between the non-BCR group (2.27 pg/mL) and the BCR group (46.99 pg/mL). • Using a multivariate Cox proportional hazard model, AccuPSA(TM) nadir level was a significant predictor of BCR-free survival (P < 0.01). • Kaplan-Meier analysis of up to 5 years follow-up showed that 100% of men with AccuPSA(TM) nadir values <3 pg/mL did not develop BCR, whereas 62.5% of men with values >3 pg/mL developed BCR (P= 0.00024). • The sensitivity, specificity, positive predictive value and negative predictive value of the AccuPSA(TM) method was 100%, 75%, 69% and 100%, respectively. CONCLUSIONS • AccuPSA(TM) assay predicts 5-year BCR- free survival after RP. • Identifying a reliable predictor of BCR soon after RP has important implications for frequency of PSA testing, selection of candidates for adjuvant therapy, and reassuring a large subset of men that they are not at risk of recurrence. • Larger studies are needed to validate these findings.

A novel multiplex assay combining autoantibodies plus PSA has potential implications for classification of prostate cancer from non-malignant cases

BackgroundThe lack of sufficient specificity and sensitivity among conventional cancer biomarkers, such as prostate specific antigen (PSA) for prostate cancer has been widely recognized after several decades of clinical implications. Autoantibodies (autoAb) among others are being extensively investigated as potential substitute markers, but remain elusive. One major obstacle is the lack of a sensitive and multiplex approach for quantifying autoAb against a large panel of clinically relevant tumor-associated antigens (TAA).MethodsTo circumvent preparation of phage lysates and purification of recombinant proteins, we identified B cell epitopes from a number of previously defined prostate cancer-associated antigens (PCAA). Peptide epitopes from cancer/testis antigen NY-ESO-1, XAGE-1b, SSX-2,4, as well as prostate cancer overexpressed antigen AMACR, p90 autoantigen, and LEDGF were then conjugated with seroMAP microspheres to allow multiplex measurement of autoAb present in serum samples. Moreover, simultaneous quantification of autoAb plus total PSA was achieved in one reaction, and termed the "A+PSA" assay.ResultsPeptide epitopes from the above 6 PCAA were identified and confirmed that autoAb against these peptide epitopes reacted specifically with the full-length protein. A pilot study was conducted with the A+PSA assay using pre-surgery sera from 131 biopsy-confirmed prostate cancer patients and 121 benign prostatic hyperplasia and/or prostatitis patients. A logistic regression-based A+PSA index was found to enhance sensitivities and specificities over PSA alone in distinguishing prostate cancer from nonmalignant cases. The A+PSA index also reduced false positive rate and improved the area under a receiver operating characteristic curve.ConclusionsThe A+PSA assay represents a novel platform that integrates autoAb signatures with a conventional cancer biomarker, which may aid in the diagnosis and prognosis of prostate cancer and others.

Arraying prostate specific antigen PSA and Fab anti‐PSA using light‐assisted molecular immobilization technology

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.

“Product Ion Monitoring” Assay for Prostate-Specific Antigen in Serum Using a Linear Ion-Trap

While numerous strategies exist for biomarker discovery, the bottleneck to product development and routine use at the clinic is in the verification phase of candidate biomarkers. The aim of this study was to establish a robust and high-throughput product ion monitoring (PIM) assay that is potentially capable of rapidly verifying candidates from discovery phase experiments. Using prostate-specific antigen (PSA), a model biomarker, and a routinely used mass spectrometer for discovery platforms, an ion trap (LTQ, Thermo), the utility of this instrument to perform PIM was explored. The proteotypic doubly charged intact peptide LSEPAELTDAVK ( m/ z 637) fragmenting to m/ z 943 (PAELTDAVK) was monitored. A limit of detection of 10 attomoles with a coefficient of variation (CV) of <20% was obtained for a purified recombinant PSA digest. Immunoextraction of endogenous PSA from serum using a monoclonal antibody on a 96-well microtiter plate, followed by PIM on the LTQ, enabled quantification of PSA down to less than 1 ng/mL with a limit of detection of 0.1 ng/mL and CVs < 20%. Mascot searching and ion ratio confirmation further supported the conclusion that the quantified moiety in serum was the PSA peptide. We conclude that this methodology could be adapted quickly and easily to other candidates, thus providing a much needed technology to bridge the gap between discovery and validation platforms.

Comparison of Chromogranin A, Insulin-Like Growth Factor 1 and Prostate-Specific Antigen Serum Markers in Prostate Adenocarcinoma and Benign Prostatic Hyperplasia

Background/Aim: To compare serum chromogranin A (CgA) and insulin-like growth factor 1 (IGF-1) with the classical prostate-specific antigen (PSA) marker in clinically localized prostate adenocarcinomas. Materials and Methods: This is a prospective single-center study that included 64 consecutive men with newly diagnosed clinically localized prostate adenocarcinoma and 20 consecutive men with histologically confirmed benign prostatic hyperplasia (BPH). A blood sample for the determination of serum total PSA, CgA and IGF-1 levels (RIA) was obtained from all cases. Analysis of variance was performed to evaluate their variations according to disease and the pathological characteristics of prostate adenocarcinoma. Results: Only serum PSA levels (p < 0.0001) and not IGF-1 (p = 0.5475) or CgA (p = 0.5043) were significantly higher in the prostate cancer (PCa) group as compared to the BPH group. A significant variance between BPH and PCa divided on the basis of pT stage was found for PSA levels (p < 0.0001) but not for CgA (p = 0.0869) and IGF-1 (p = 0.6883) levels. Dividing PCa on the basis of Gleason score, a significant variance was found for CgA (p = 0.0100) and for PSA (p < 0.001), but not for IGF-1 (p = 0.6895) levels. Conclusions: In our population the quantification of PSA and CgA serum levels and not of IGF-1 provides independent significant information in the diagnosis and aggressiveness of PCa, respectively.

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

This paper describes the development of an immunosensor coupled to glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (CNT-GCE) integrated with microfluidic systems for rapid and sensitive quantification of prostate specific antigen (PSA) in human serum samples. Mouse monoclonal (5G6) to PSA antibodies were immobilized on a rotating disk. PSA in the serum sample are allowed to react immunologically with the immobilized anti-tPSA and horseradish peroxidase (HRP) enzyme-labeled second antibodies specific to PSA. HRP, in the presence of hydrogen peroxide (H 2O 2) catalyzes the oxidation of 4- tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on CNT-GCE at −0.15 V. The electrochemical detection can be done within 1 min and total assay time was 30 min. The calculated detection limits for electrochemical detection and the ELISA procedure are 0.08 and 0.5 μg L −1, respectively and the intra- and inter-assay coefficients of variation were below 4.5%. The electrochemical immunosensor showed higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method, which shows potential for detecting PSA in clinical diagnosis.

Development and evaluation of three real-time immuno-PCR assemblages for quantification of PSA

Real-time PCR is a very sensitive technique to measure DNA concentrations. In real-time immuno-PCR, it is used as the detection system for quantification of proteins. Many ways to perform and assemble real-time immuno-PCR are possible. We have tested three different approaches for the detection of prostate specific antigen (PSA) and compared them with each other and with ELISA. We also demonstrate the applicability of real-time immuno-PCR to classify clinical PSA samples. Assemblage I is performed stepwise attaching the capture antibody to the vessel surface by adsorption and having the DNA-label linked to the detection antibody through biotin and streptavidin. In assemblage II, capture antibody is also adsorbed to the vessel surface but the detection antibody is pre-conjugated to the DNA-label. Assemblage III uses the pre-conjugated detection antibody/DNA-label but binds the capture antibody through biotin to surface immobilized streptavidin. We found assemblage II to be the most sensitive, with a detection limit of 4.8 × 10 5 PSA molecules. This can be compared to the detection limit of the ELISA, which is 5.7 × 10 7 molecules. Assemblage III was the most reproducible with a standard deviation (SD) of 0.21 cycles, while assemblage I was the least reproducible (SD = 0.45 cycles). The SD of assemblage II was 0.25 cycles. We conclude that using pre-conjugated detection antibody/DNA-label enhances both the sensitivity and the reproducibility of real-time immuno-PCR. Measurements of PSA in serum samples using real-time immuno-PCR correlated well with measurements performed with ELISA. The real-time immuno-PCR measurements were more sensitive and the dynamic range was larger than with the ELISA.

Simultaneous Quantification of Prostate-specific Antigen and Human Glandular Kallikrein 2 mRNA in Blood Samples from Patients with Prostate Cancer and Benign Disease

Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease. PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1-pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 10(2)-10(6) mRNA copies. PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P <0.0001, Mann-Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (< or =5) were negative for both PSA and hK2 (P = 0.02, Mann-Whitney U-test). The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.

Analytical performance of the Roche total and free PSA assays on the Elecsys 2010 immunoanalyzer

Recently, site-specific fucosylation of glycoproteins has attracted attention as it can be associated with several types of cancers including prostate cancer. However, individual glycoproteins, which might serve as potential cancer markers, often are very low-concentrated in complex serum matrices and distinct glycan structures are hard to detect by immunoassays. Here, we present a mass spectrometry-based strategy for the simultaneous analysis of core-fucosylated and total prostate-specific antigen (PSA) in human serum in the low ng/ml concentration range. Sample preparation comprised an immunoaffinity capture step to enrich total PSA from human serum using anti-PSA antibody coated magnetic beads followed by consecutive two-step on-bead partial deglycosylation with endoglycosidase F3 and tryptic digestion prior to LC-MS/MS analysis. The method was shown to be linear from 0.5 to 60 ng/ml total PSA concentrations and allows the simultaneous quantification of core-fucosylated PSA down to 1 ng/ml and total PSA lower than 0.5 ng/ml. The imprecision of the method over two days ranged from 9.7–23.2% for core-fucosylated PSA and 10.3–18.3% for total PSA depending on the PSA level. The feasibility of the method in native sera was shown using three human specimens. To our knowledge, this is the first MS-based method for quantification of core-fucosylated PSA in the low ng/ml concentration range in human serum. This method could be used in large patient cohorts as core-fucosylated PSA may be a diagnostic biomarker for the differentiation of prostate cancer and other prostatic diseases, such as benign prostatic hyperplasia (BPH). Furthermore, the described strategy could be used to monitor potential changes in site-specific core-fucosylation of other low-concentrated glycoproteins, which could serve as more specific markers (“marker refinement”) in cancer research.

Growth of human tumor xenografts in SCID mice quantified using an immunoassay for tumor marker protein in serum

The accurate measurement of the response of a tumor to a given treatment is critical to evaluating novel therapeutic modalities. An experimental design is reported here that can be generally applied to monitoring human tumor xenografts growing in immunodeficient mice. A human non-small cell lung tumor cell line was transfected with a mammalian expression vector containing the gene encoding human prostate specific antigen (PSA) and has been shown to grow progressively following the subcutaneous, intraperitoneal and intravenous inoculation of the tumor into severe combined immunodeficient (SCID) mice. The transfected human tumor cells produce PSA that accumulates in the sera of all tumor inoculated SCID mice. An enzyme-linked immunoassay using a rabbit polyclonal and a mouse monoclonal antibody specific for PSA was designed and tested for the detection and quantification of serum PSA in tumor-bearing mice. Over a 5-week period, the serum levels of PSA of mice inoculated subcutaneously with the tumor increased progressively, and the estimated tumor volumes correlated with the amount of PSA detected in the serum. Serum PSA levels correlated even better with total tumor mass following the intraperitoneal inoculation of tumor cells into SCID mice. Serum PSA levels fell rapidly following the surgical debulking of tumor xenograft, reaching background levels of PSA in the serum 1 week after tumor removal. Serum PSA levels were also observed in SCID mice inoculated intravenously with a PSA transfected human lung tumor cell line adapted to grow orthotopically in the lung. The transfection of human tumors with a tumor marker and the use of an immunoassay to detect this marker establish an experimental design that provides a reliable, non-invasive, accurate and simple approach to monitor and quantify the growth of human tumor xenografts in SCID mice.

PROSTATE-SPECIFIC ANTIGEN FORMS COMPLEXES WITH HUMAN alpha 2-MACROGLOBULIN AND BINDS TO THE alpha 2-MACROGLOBULIN RECEPTOR/LDL RECEPTOR-RELATED PROTEIN

Purpose To investigate the binding of the prostate-specific antigen (PSA) to human alpha 2-macroglobulin (alpha 2-M) and to alpha 1-antichymotrypsin (ACT). Materials and Methods Binding analysis was evaluated by electrophoresis, Western-blotting, enzyme-linked immunosorption assay (ELISA) and size exclusion chromatography. Quantification of PSA and of different forms of alpha 2-M was performed using commercial test kits. The cleavage site of PSA in alpha 2-M was analyzed by SDS-PAGE and microsequencing. Results Binding of PSA to alpha 2-M is initiated by the cleavage of the peptide bond between amino acids Tyr 686 and Glu 687 of the bait region indicating a chymotrypsin-like activity of the PSA. The PSA's proteolytic cleavage triggers the transformation of alpha 2-M as detected by conformation-specific monoclonal antibodies. Kinetic analysis revealed faster binding of PSA to alpha 2-M than to ACT. The PSA bound to alpha 2-M is caged by the inhibitor and thus escapes detection by antibodies. This results in an incorrect calculation of the level of PSA when released from prostate into the blood. Complexes of PSA-alpha 2-M and PSA-ACT were found to bind to the alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2-M-R/LRP) which may be the clearance receptor for PSA. Conclusions Quantifying free PSA and PSA-ACT complexes, as routinely done in managing prostate-associated diseases, does not represent the total secretion capacity of the prostate. The proteinase inhibitor alpha 2-M has to be considered as a main contributor to PSA complex formation in the blood.