PSA-ACT Complex Research Articles

Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling.

Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237-amino acid monomeric proteins, with 79% identity in primary structure. Twenty-five anti-PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence. Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I-IV). Binding region I was localized to amino acid residues 1-13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53-64, III (a and b) was localized to residues 80-91 (= kallikrein loop), and IV was localized to residues 151-164. Mabs binding to regions I and IIa were reactive with free PSA, PSA-ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA-ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA-ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.

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.

NONPROSTATIC SOURCES OF PROSTATE-SPECIFIC ANTIGEN

The name prostate-specific antigen has been given to a protein that now is known not to be prostate-specific; however, prostatic tissue does produces extremely high levels of PSA and secrets it into the seminal plasma. Seminal plasma contains about 1 million micrograms/L of PSA and is the richest source of PSA reported. The biologic fluid with the second highest PSA concentration, however, is nipple aspirate fluid from the female breast (up to about 5000 micrograms/L), and the third is milk from lactating women (up to 300 micrograms/L). Male serum PSA is usually less than 4 micrograms/L. In nonprostatic tissues, PSA exists mainly in its free molecular form, but PSA-ACT complex is also present in most of the fluids that contain PSA, such as breast secretions and amniotic fluid. The gene expression and protein production of PSA in nonprostatic tissues are under the regulation of steroid hormones via their receptors. Androgens, glucocorticoids, and progestins up-regulate the PSA gene expression, resulting in an increase of protein production. Estrogen by itself seems to have no effect on PSA regulation, but it can impair PSA production induced by androgen. It remains unknown whether PSA is enzymatically active and what is the physiologic role of PSA in nonprostatic tissues. It is speculated that PSA may be involved in the regulation of growth factors. Measuring PSA in breast cancer cytosol, breast-nipple aspirate fluid, and female serum may have potential clinical utilities, including breast cancer prognosis, breast cancer risk assessment, and evaluation of androgen excess. Further studies are needed to identify the exact function and regulation of PSA in nonprostatic tissues and to explore the clinical application of this protein.

Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies

To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the association targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also examined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with alpha 1-antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited > 90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT association by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA.

Two-dimensional gel electrophoresis detects prostate-specific antigen–α1-antichymotrypsin complex in serum but not in prostatic fluid

We investigated the interaction between prostate-specific antigen (PSA) and 1-antichymotrypsin (ACT) in prostatic secretions, identifying PSA and ACT in human serum, prostatic fluid, and seminal plasma by two-dimensional gel electrophoresis (2-D PAGE). Both PSA and ACT were detected in all three body fluids, but PSA-ACT complex was detected only in serum. Moreover, the 2-D PAGE Western blot staining profile for ACT from serum differed from that for prostatic fluid or seminal plasma. Incubation of prostatic fluid with purified ACT led to formation of PSA-ACT complex. Incubation of prostatic fluid with purified PSA, however, failed to form the complex, suggesting that the ACT in prostatic fluid was inactive or inhibited. Given that physiological concentrations of zinc inhibited the formation of PSA-ACT complex, we consider zinc a possible physiological inhibitor of the formation of the PSA-ACT complex. These results indicate that the failure to detect the PSA-ACT complex in prostatic fluid could be related to the inactivation of ACT, the presence of inhibitors (e.g., zinc), or simply the PSA:ACT ratio in the fluid.

Individual prostate-specific antigen (PSA) forms as prostate tumor markers

Prostate-specific antigen (PSA) is a kallikrein-like serine protease mainly expressed in the human prostate. It is responsible for the proteolysis of the gel-forming proteins in human semen. Two major extracellular protease inhibitors, alpha-1-antichymotrypsin (ACT) and alpha-2-macroglobulin (AMG) may inactivate PSA escaping from the prostate. The predominant immunodetected form of PSA in serum is complexed to ACT but PSA exists also in a free non-complexed form despite the large excess of inhibitors. The concentrations of PSA in serum are normally less than 4 μg/l, but elevated concentrations are found in a majority of patients with prostate cancer (CAP) and the analysis of PSA in serum has become invaluable in the detection and monitoring of patients with CAP. However, it is not an ideal tumor marker in the sense that there are CAP patients with normal PSA concentrations in serum and patients with benign hyperplasia of the prostate (BPH) with elevated PSA concentrations. Analysis of the various PSA forms in serum attracts much interest as there is a higher proportion of PSA in complex with ACT in patients with CAP than in those with BPH. Optimal combinations of monoclonal antibodies have been used to design sensitive noncross-reacting immunoassays for the detection of free PSA, PSA-ACT complexes and the detection of both free PSA and PSA complexes in an equimolar fashion (i.e. total PSA). Several studies have demonstrated that the analysis of the proportions of the free-to-total PSA in serum may increase the diagnostic specificity by 15–20% without significant loss in the sensitivity for detection of CAP.

Lyophilized PSA-ACT complex is stable

Lyophilized PSA-ACT complex is stable

Diagnostic value of molecular forms of prostate-specific antigen for female breast cancer

Objectives: To examine the diagnostic value of prostate-specific antigen (PSA) subfractions in the serum of female breast cancer patients. Design and Methods: PSA subfractions (free PSA, F-PSA; PSA bound to α 1-antichymotrypsin, PSA-ACT) were determined in the serum of patients with breast cancer and in the serum of healthy women. Serum was injected into a high-performance liquid chromatography column and all fractions were analyzed for PSA using a highly sensitive PSA immunofluorometric assay. We studied 3 normal male sera, 3 sera from prostate cancer patients who under-went radical prostatectomy (all for comparative purposes), 3 sera from healthy women, 3 sera from women with breast cancer obtained presurgically, and 7 sera from women with breast cancer, postsurgically. Results: All male sera contained mostly PSA-ACT complexes and very little free PSA. Sera from all healthy women also contained mostly PSA-ACT complexes and nondetectable or traces of free PSA. All 3 presurgical sera from patients with breast cancer contained predominantly free PSA. Patients who had surgical resection of the breast tumor and were in remission had postsurgical serum PSA subfractions similar to those of healthy women ( i.e., mostly PSA-ACT complexes). Conclusion: The serum PSA subfractions of breast cancer patients are substantially different from serum PSA subfractions of male patients, healthy females, and females who have apparently been treated successfully for breast cancer. These findings may form the basis for a serological diagnostic test for breast cancer.

Technicon Immuno 1 PSA assay measures both free and alpha-1-antichymotrypsin-complexed prostate-specific antigen on an equimolar basis.

In this study, we investigated the immunoreactivity of the Technicon Immuno 1 PSA assay, a monoclonal-polyclonal sandwich immunoassay, with free and ACT-complexed PSA. Assay calibrators prepared with free PSA (standard Immuno 1 calibrators) and calibrators consisting of 90% ACT-complexed and 10% free PSA (90:10 calibrators) yielded virtually identical PSA recoveries at all concentrations tested. Concentrations of total PSA at approximately 4 and 10 ng/mL, prepared in varying ratios of free PSA to PSA-ACT complex, recovered from 92-104% at 4 ng/mL and from 98-102% at 10 ng/mL. Additionally, an excellent correlation of serum total PSA values from a panel of 40 prostatic cancer patient samples was obtained using calibration curves generated with the standard Immuno 1 calibrators or the 90:10 calibrators. These results demonstrate that the Technicon Immuno 1 PSA assay measures free and ACT-complexed PSA on an equal molar basis.

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

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.

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

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.

Psa immunoreactivity detected in lncap cell medium, breast tumor cytosol, and female serum

We made an effort to identify a reliable source for obtaining large quantities of both free (PSA) and PSA-ACT complex for the preparation of the calibrator for the PSA assay. Using size exclusion chromatography, we found both free PSA and PSA-ACT complex in the conditioned cell medium of the LNCaP cell line, which was derived from a human metastatic adenocarcinoma of the prostate. An assay specific for PSA-ACT reacted only with the PSA-ACT complex from cells grown in serum-free medium, and not with the complex from the cell medium grown in 10% calf serum. We also found both free PSA and PSA-ACT complex in 15% of cytosols prepared from breast tumor tissues; the cytosol PSA concentrations ranged from 0.1 to 110 ng/ml. No correlation was found between cytosol PSA and concentrations of estrogen receptor, progestin receptor, epidermal growth factor receptor, cathepsin D, or the ectodomain of c-erbB-2 protein. Based on chromatographic characterizations and the slope of their dose-response curves, it appears that both free PSA and PSA-ACT complex found in the cytosols are similar to PSA complex from the cell medium and the serum of prostate cancer patients. Ectopic PSA was also detected in pooled sera from patients with breast, ovarian, pancreatic, and colon carcinoma. The PSA concentrations in these serum pools increased with the level of their dominant tumor marker. In any event, the LNCaP cell medium appears to be a reliable source for obtaining both free and ACT-complexed PSA of human tumor origin for the preparation of PSA assay calibrators.

A universal calibrator for prostate specific antigen (PSA).

We describe for the first time a protocol to purify an in vitro made PSA-ACT complex to apparent homogeneity 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. Purification of the PSA-ACT complex was highly reproducible. An extinction coefficient of 1.0 and 280 nm (L x g-1 x cm-1) was assigned to the PSA-ACT complex based on amino acid analysis. Molecular weight was assigned by taking cDNA of ACT (plus 26% carbohydrate) and the molecular weight of PSA (28,430) which totals 89,280. Two common calibrators were made consisting of 100% PSA-ACT complex and/or 90% PSA-ACT complex plus 10% free PSA (90:10 calibrator). The 90:10 calibrator is recommended as a universal calibrator for the international standardization of PSA immunoassays.

Development of a microplate elisa for free psa and psa–act complex in serum

An ELISA on microplate was established for the total serum PSA. We selected the monoclonal antibody for the assay from commercial sources making certain that it reacted with both free PSA and PSA-alpha 1-antichymotrypsin (PSA-ACT) complex from human serum with similar affinity (so-called "equimolar"). We also chose a test format with polyclonal anti-PSA antibodies coated on the well and monoclonal anti-PSA antibodies for quantification to gain higher test sensitivity. Two different sample volumes from each specimen, 5 and 50 microliters, were used for the assay in order not only to further increase test sensitivity and improve precision at both low and highly elevated PSA concentrations, but also to widen the assay concentration range (0-500 ng total PSA per ml). Using two sample volumes also reduces any hook effect and shortens the turn-around time because repeated determinations are usually required when specimens contain highly elevated PSA concentrations. The use of pooled sera containing approximately 95% PSA-ACT complex and 5% free PSA as a calibrator allows for a close matching of the calibrator with serum specimens in immunoreactivity and PSA composition. Moreover, our assay shows no hook effect up to 15,000 ng/ml. The within-day precision (% CV) in the critical concentration range of 4-12 ng/mL is approximately 5%. The PSA values obtained from this assay correlate well with that of the Hybritech kit (gamma = 0.998, slope equals to 1.033), indicating that this kit can replace the Hybritech Tandem E PSA kit for serum PSA determination in clinical laboratories.

Evaluation of free psa isoforms, psa complex formation, and specificity of anti‐psa antibodies by hplc and page‐immunoblotting techniques

Both high performance liquid chromatographic (HPLC) and polyacrylamide gel electrophoresis-immunoblotting (PAGE-immunoblotting) procedures have been established for the study of isoforms of free prostate-specific antigen (PSA) and the complex formation between free PSA and protease inhibitors, and for the evaluation of the specificities of various anti-PSA antibodies. We found multiple isoforms of free PSA on PAGE, which were all capable of forming complexes with protease inhibitors. The same isoform pattern can be produced from the original seminal fluid. The PSA isoforms differ from each other most likely in charge because they could be converted to one band on SDS-PAGE and to a single peak by gel filtration chromatography. We found it difficult to form large quantities of PSA complex when mixing free PSA from seminal fluid with protease inhibitors, regardless of whether the free PSA or the protease inhibitors were in excess. Except for the PSA-ACT complex, which was consistently detectable by both HPLC and PAGE-immunoblotting techniques after incubation, these two procedures disagreed in their detection of PSA-A2M and PSA-AT complexes. The PSA-A2M complex was usually observable by immunoblotting techniques but barely detectable on HPLC, whereas PSA-AT was totally invisible by immunoblotting but appeared as a peak in the HPLC elution profile. Mixing free PSA with serum clearly resulted in both PSA-ACT and PSA-A2M complexes. However, more PSA-ACT than PSA-A2M was formed; the result was also confirmed by using 125I-PSA for mixing. PSA could be separated into active and inactive PSA by DEAE Sepharose chromatography with a 14-fold difference in protease activity. The difference in enzymatic activity apparently had no effect on complex formation. All the anti-PSA antibodies examined in this study reacted with PSA isoforms, PSA-ACT and PSA-A2M complexes. We conclude that it would be almost impossible to establish an assay to measure all forms of PSA in the serum and to expect to produce precise and accurate PSA values.

ISSUES IN THE ASSESSMENT OF PSA IMMUNOASSAYS

The physician and laboratorian have several proven PSA assays from which to choose, and several more are likely to achieve FDA approval soon. Furthermore, an assay that is FDA approved offers considerable reassurance that the many aspects of assay design and manufacturing processes meet an acceptable standard. This will become even more critical as assays report values in the ultrasensitive range. We have attempted to review here some of the most important issues related to PSA assay performance and standardization. Standardization particularly is a complex problem for which solutions are just now appearing. Yet efforts must continue to minimize controversies, because many more assays will be forthcoming and because newer applications of PSA determinations will place more pressure on proper assay evaluations. For example, measurement of free PSA, PSA-ACT complexes, and the ratio of one to the other are intriguing possibilities for added clinical benefit. Also, ultrasensitive PSA assays add a new dimension to the detection of residual disease and may provide unique opportunities for the implementation of new therapies and their timely evaluation. In our opinion, the impact of PSA on the management of prostate cancer is still growing, and the opportunities for better and new assays are still great.

SIGNIFICANCE OF DIFFERENT MOLECULAR FORMS OF SERUM PSA: The Free, Noncomplexed Form of PSA Versus that Complexed to α1-Antichymotrypsin

Prostate specific antigen is an abundant prostate-derived serine protease in the seminal fluid. Low concentrations of the protein are normally released into blood, but above normal concentrations are frequently detected in prostate disease. The PSA-ACT complex is the predominant molecular form of serum PSA (up to approximately 95%) although complex formation is slow between the purified proteins in vitro. A free, noncomplexed form of PSA constitutes a minor fraction of the serum PSA, although serum ACT occurs in large molar excess. The free, noncomplexed form of serum PSA is reported to constitute a significantly smaller proportion of the PSA in untreated prostate cancer than in BPH. The molecular basis for this finding is unclear, but measurements of the proportion of the free form of serum PSA or the proportion of serum PSA-ACT may facilitate discrimination between prostate cancer and BPH.

Double-label time-resolved immunofluorometric assay of prostate-specific antigen and of its complex with alpha 1-antichymotrypsin

We have developed a procedure for simultaneous immunofluorometric assay (IFMA) of prostate-specific antigen (PSA) and its complex with alpha 1-antichymotrypsin (ACT). A PSA-specific monoclonal antibody, which captures both free PSA and the PSA-ACT complex, was used as solid-phase antibody. Total PSA immunoreactivity was measured with a Eu(3+)-labeled PSA antibody that reacted with both free PSA and PSA-ACT. PSA-ACT was assayed simultaneously with a Sm(3+)-labeled polyclonal ACT antibody as a tracer. As standard we used pooled serum in which most of the PSA occurred as the PSA-ACT complex. The assay range was 0.03-500 micrograms/L for total PSA and 0.16-450 micrograms/L for PSA-ACT. In comparison with the assay for total PSA, assay of the PSA-ACT/PSA ratio improved the clinical specificity for cancer by reducing the number of false-positive results in prostatic hyperplasia.