Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

scholarly journals Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

1996 ◽  
Vol 314 (2) ◽  
pp. 587-593 ◽  
Author(s):  
Antonio del CASTILLO-OLIVARES ◽  
Miguel A. MEDINA ◽  
Ignacio NÚÑEZ de CASTRO ◽  
Javier MÁRQUEZ
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Nadh Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumour Cells ◽  
Ammonium Sulphate Precipitation ◽  
Apparent Homogeneity

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis–Menten kinetics for the substrates, with apparent Km values of 4.3×10-5 M and 6.7×10-5 M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thiol-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

scholarly journals On Chip Immuno-Affinity Profiling of Cancer- and Benign Hyperplasia-Associated Free Prostate Specific Antigen

2011 ◽  
Vol 31 (2) ◽  
pp. 111-118 ◽  
Author(s):  
Maja M. Kosanovic ◽  
Sanja R. Goc ◽  
Goran S. Potpara ◽  
Miroslava M. Jankovic
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Seminal Plasma ◽  
Specific Antigen ◽  
Analytical Techniques ◽  
Molecular Forms ◽  
Striking Difference ◽  
Free Psa ◽  
Lower Molecular Mass ◽  
Benign Hyperplasia

Prostate specific antigen (PSA) exhibits pronounced heterogeneity in both primary structure and glycan composition, resulting in the existence of different molecular forms. Investigation of PSA structure is a demanding task facing limitations due to inadequate sensitivity of analytical techniques and low concentrations of the different forms. This study aimed to profile free PSA (fPSA), especially lower molecular mass species lacking detailed classification, in normal seminal plasma and in sera from subjects with benign hyperplasia (BPH) or cancer of the prostate (PCa) as samples of known clinical relevance. fPSA forms were separated from complex proteomes on chips with immobilized anti-fPSA antibody followed by detection using surface-enhanced laser desorption/ionization time of flight mass spectrometry. At least 39 fPSA-immunoreactive species, ranging from 3–29 kDa were detected in seminal plasma. General fPSA profiles in seminal plasma and sera were similar, but differed in the abundance and presence of particular peaks/clusters of the lower molecular mass species. No striking difference in fPSA forms was observed between BPH and PCa samples, but some distinct peaks varied in intensity and frequency within or between groups. Obtained data verify fPSA heterogeneity that might be important for better exploration of all their molecular and marker potentials.

scholarly journals Molecular forms of human prostate-specific antigen in urine of subjects with benign prostatic hyperplasia

2006 ◽  
Vol 58 (2) ◽  
pp. 77-82 ◽  
Author(s):  
Maja Kosanovic ◽  
Miroslava Jankovic
Keyword(s):  
Benign Prostatic Hyperplasia ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Structural Complexity ◽  
Exchange Chromatography ◽  
Prostatic Hyperplasia ◽  
Molecular Forms ◽  
Lectin Affinity Chromatography ◽  
Lectin Affinity

In the present study, we examined mollecular forms of urinary prostate-specific antigen (PSA), focusing on its structural complexity in general and specifically on its microheterogeneity in relation to benign prostatic hyperplasia (BPH). Gel filtration, ion-exchange chromatography, and lectin-affinity chromatography were used to characterize PSA. In comparing the binding pattern of PSA isoforms, moderate changes were observed in the relative abundance of distinct molecular subpopulations separated on lectin-affinity columns. They may be related to alteration in the position and type of linkage of fucose or sialic acid, as well as to modification of the trimannosyl core by branching of the PSA oligosaccharide chain.

Free and complexed prostate-specific antigen (PSA): in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and PSA-alpha 1-antichymotrypsin complex

1995 ◽  
Vol 41 (10) ◽  
pp. 1480-1488 ◽  
Author(s):  
K Pettersson ◽  
T Piironen ◽  
M Seppälä ◽  
L Liukkonen ◽  
A Christensson ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Solid Phase ◽  
Specific Antigen ◽  
Close Correlation ◽  
Cathepsin G ◽  
Free Psa ◽  
Total Psa ◽  
The Stability ◽  
Storage Buffer

Abstract Generation of 15 monoclonal antibodies (MAbs) allowed construction of epitope maps and specific two-site immunofluorometric assays for free prostate-specific antigen (PSA) and PSA complexed with alpha 1-antichymotrypsin (ACT). Close correlation of PSA concentrations obtained with the use of different assays of free PSA suggested extensive similarity in immunodetection of free PSA in serum. Assays of the PSA-ACT complex overestimated the concentration of PSA-ACT in serum because of nonspecific adsorbance of ACT or cathepsin G-complexed ACT to the solid phase. This interference was substantially decreased in the presence of heparin. In studying the stability of purified PSA and PSA-ACT complexes formed in vitro, we found that the free PSA was stable during storage for 4 weeks at 35 degrees C, whereas PSA-ACT complexes largely dissociated in these conditions. The instability of PSA-ACT complexes was counteracted by storage at low temperatures, by adjusting the pH of the storage buffer between 6.8 and 7.4, and through addition of 100-1000-fold molar excess of native ACT. The ease of calibration and the accuracy of free PSA assays in comparison with assays of the PSA-ACT complex suggest that measurements of free to total PSA most accurately reflect the inverse of the proportion of PSA complexed to ACT in serum.

scholarly journals Measurement of the Complex between Prostate-specific Antigen and α1-Protease Inhibitor in Serum

1999 ◽  
Vol 45 (6) ◽  
pp. 814-821 ◽  
Author(s):  
Wan-Ming Zhang ◽  
Patrik Finne ◽  
Jari Leinonen ◽  
Satu Vesalainen ◽  
Stig Nordling ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Protease Inhibitor ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Serum Samples ◽  
Free Psa ◽  
Total Psa ◽  
Healthy Females

Abstract Background: Prostate-specific antigen (PSA) occurs in serum both free and in complex with protease inhibitors. The complex with α1-antichymotrypsin (ACT) is the major form in serum, and the proportion of PSA-ACT is higher in prostate cancer (PCa) than in benign prostatic hyperplasia (BPH). PSA also forms a complex with α1-protease inhibitor (API) in vitro, and the PSA-ACT complex has been detected in serum from patients with prostate cancer. The aim of the present study was to develop a quantitative method for the determination of PSA-API and to determine the serum concentrations in patients with PCa and BPH. Methods: The assay for PSA-API utilizes a monoclonal antibody to PSA as capture and a polyclonal antibody to API labeled with a Eu-chelate as a tracer. For calibrators, PSA-API formed in vitro was used. Serum samples were obtained before treatment from 82 patients with PCa, from 66 patients with BPH, and from 22 healthy females. Results: The concentrations of PSA-API are proportional to the concentrations of total PSA. PSA-API comprises 1.0–7.9% (median, 2.4%) of total immunoreactive PSA in PCa and 1.3–12.2% (median, 3.6%) in BPH patients with serum PSA concentrations >4 μg/L. In patients with 4–20 μg/L total PSA, the proportion of PSA-API serum is significantly higher in BPH (median, 4.1%) than in PCa (median, 3.2%; P = 0.02). Conclusions: The proportion of PSA-API in serum is lower in patients with PCa than in those with BPH. These results suggest that PSA-API is a potential adjunct to total and free PSA in the diagnosis of prostate cancer.

scholarly journals Large discrepancy between prostate-specific antigen results from different assays during longitudinal follow-up of a prostate cancer patient

1996 ◽  
Vol 42 (4) ◽  
pp. 637-641 ◽  
Author(s):  
H L Van Duijnhoven ◽  
N C Péquériaux ◽  
J P Van Zon ◽  
M A Blankenstein
Keyword(s):  
Prostate Specific Antigen ◽  
Prostate Cancer Patient ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Correlation Study ◽  
2Nd Generation ◽  
Free Psa ◽  
Value Range ◽  
Selection Of

Abstract A case is presented of a patient with stage D prostatic carcinoma, from whom a serum sample proved to be an outlier in a correlation study performed with a 2nd-generation prostate-specific antigen (PSA) assay on the Immulite system (6.4 micrograms/L) and IMx (101 micrograms/L). Clearly, the PSA result reported by Immulite was falsely low. For nine longitudinal samples, Immulite results were approximately 20-fold lower than the IMx value (range of IMx results 5-275 micrograms/L). A selection of the samples was analyzed with the ACS:180, ES-600, and IMx (all > 180 micrograms/L); Immulite, DPC Coat-A-Count IRMA, Immuno 1, AIA-pack, and Tandem-R (all <70 micrograms/L); and Immulite free PSA assay (41 micrograms/L). Gel filtration demonstrated that apart from the alpha1-antichymotrypsin (ACT) complex, no other complexes were found. However, the sample consisted of 53% free PSA (IMx). Possibly, a change of conformation of the PSA molecule resulted in a decreased binding to ACT and a reduced affinity of the antibodies used in the affected assays.

Multiple forms of prostate-specific antigen in serum: differences in immunorecognition by monoclonal and polyclonal assays

1993 ◽  
Vol 39 (12) ◽  
pp. 2483-2491 ◽  
Author(s):  
A M Zhou ◽  
P C Tewari ◽  
B I Bluestein ◽  
G W Caldwell ◽  
F L Larsen
Keyword(s):  
Prostate Specific Antigen ◽  
Molecular Sieve ◽  
Specific Antigen ◽  
Prostatic Hyperplasia ◽  
Multiple Forms ◽  
Early Detection Of Disease ◽  
Peak Integration ◽  
Free Psa ◽  
Predominant Form

Abstract Prostate-specific antigen (PSA) in serum is primarily complexed with alpha 1-antichymotrypsin (alpha 1-ACT). However, 12-15% of prostate cancer (PCa) patients present with the predominant form being uncomplexed (free) PSA (Lilja et al., Clin Chem 1991;37:1618-24). We report that commercial immunoassays demonstrate variations in reactivity, especially to the uncomplexed form. We fractionated and analyzed commercial controls, PSA complexes prepared in vitro, and sera from patients with PCa or benign prostatic hyperplasia, using molecular sieve chromatography and Hybritech Tandem-R, Abbott IMx, and Ciba Corning ACS PSA assays. Peak integration of PCa samples demonstrated ACS:Tandem-R ratios of 1-1.3 for PSA/alpha 1-ACT complex. In contrast, ratios of uncomplexed peaks ranged from 2 to 4, suggesting a greater reactivity of the uncomplexed form in the ACS PSA assay. Discrepancies between assays, when PSA was measured in unfractionated sera, correlated directly with the percentage of the uncomplexed form. In controls, fractionation revealed the presence of uncomplexed PSA only, with ratios of ACS:Tandem-R and IMx:Tandem-R of 3:1 and 1.8:1, respectively. Immunoblots of PCa sera detected uncomplexed PSA (approximately 30 kDa) and PSA complexes of approximately 95 kDa (PSA/alpha 1-ACT) and > 200 kDa, indicative of alpha 2-macroglobulin. Maximal recognition of all forms of PSA may be important for early detection of disease progression.

scholarly journals Evaluation of Molecular Species of Prostate-Specific Antigen Complexed with Immunoglobulin M in Prostate Cancer and Benign Prostatic Hyperplasia

2013 ◽  
Vol 35 ◽  
pp. 847-855 ◽  
Author(s):  
Sanja Goč ◽  
Miroslava Janković
Keyword(s):  
Prostate Cancer ◽  
Benign Prostatic Hyperplasia ◽  
Prostate Specific Antigen ◽  
Molecular Species ◽  
General Pattern ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Prostatic Hyperplasia ◽  
Immunoglobulin M

This study was aimed at defining molecular species of prostate-specific antigen (PSA) in immune complexes with immunoglobulin M (IgM). Having in mind the oligoreactivity of IgM and its preference for carbohydrate antigens, there is the possibility that it can selectively recognize known PSA glycoisoforms. PSA-IgM complexes and free PSA fractions were separated from the sera of subjects with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) by gel filtration and subjected to on-chip immunoaffinity and ion-exchange chromatography. PSA-immunoreactive species were detected using surface-enhanced laser desorption/ionization time of flight mass spectrometry. The obtained spectra were analyzed for protein and glycan composition. The general pattern of the molecular species of PCa PSA and BPH PSA found in complexes with IgM was similar. It comprised major peaks at 17 kDa and minor peaks at 28 kDa, corresponding to the entire mature glycosylated PSA. The main difference was the presence of incompletely glycosylated 26.8 kDa species, having putative paucimannosidic structures, observed in PCa PSA-IgM, but not in BPH PSA-IgM. Characteristic PCa PSA-IgM glycoforms pose the question of the possible role of glycosylation as a framework for immune surveillance and may be of interest in light of recent data indicating mannose-containing glycans as cancer biomarker.

Fibrino(geno)lytic Properties of Purified Hementerin, a Metalloproteinase from the Leech Haementeria depressa

1998 ◽  
Vol 80 (07) ◽  
pp. 155-160 ◽  
Author(s):  
Ana Marisa Chudzinski-Tavassi ◽  
Eva Maria Kelen ◽  
Ana Paula de Paula Rosa ◽  
Stephane Loyau ◽  
Claudio Sampaio ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Exchange Chromatography ◽  
Single Chain ◽  
Terminal Sequence ◽  
Calcium Dependent ◽  
Amino Terminal ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Amino Terminal Sequence

SummaryThe fibrino(geno)lytic protein designated hementerin contained in crude extracts of the salivary complex of Haementeria depressa leeches was purified to apparent homogeneity by gel filtration, ion exchange chromatography and preparative SDS-PAGE. It is a single-chain 80 kDa, PhMeSO2F-resistant, calcium-dependent, metalloproteinase, which specifically degrades fibrin(ogen) through a plasminogen-independent pathway. The amino terminal sequence of 8 residues shows 80% similarity with hementin, another fibrino(geno)lytic protein purified from Haementeria ghilianii leeches. However, their activities differ somewhat in terms of kinetics and with regard to the structure of the fibrin(ogen) fragments they may produce. Cleavage by hementerin of fibrinogen Aα, γ and Bβ chains, in that order, produces 270 kDa to 67 kDa fragments which differ from those produced by plasmin. Hementerin was also able to degrade cross-linked fibrin although at a lower rate as compared to fibrinogen. In conclusion, hementerin is a plasminogen-independent fibrino(geno)lytic metalloproteinase that degrades fibrinogen faster than fibrin, prevents blood coagulation and destroys fibrin clots in vitro.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

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