PSA-ACT Complex Research Articles

PSA forms complexes with α1‐antichymotrypsin in prostate

PSA is the most useful prostate cancer marker. However, its levels are increased also in some non-malignant conditions. In circulation, the majority of PSA is complexed with protease inhibitors, including α(1) -antichymotrypsin (ACT). The proportion of the PSA-ACT complex is higher in patients with prostate cancer than in controls without cancer. The expression of ACT has been shown to be higher in prostate cancer than in benign prostatic hyperplasia. However, results regarding the extent which PSA forms complexes within the prostate and whether there are differences in complex formation between normal and malignant prostatic tissue are inconsistent and limited. We studied complex formation of PSA secreted by cultured human prostate tissues and in the tissue by in situ proximity ligation assay (PLA). Free, total and active PSA, and the PSA-ACT complex were determined in tissue culture media by immunoassays, immunoblotting, and chromatographic methods. The majority of PSA in tissue culture medium was free and enzymatically active. However, a significant proportion (1.6 ± 0.5%) of immunoreactive PSA was found to be complexed with ACT. Complex formation was confirmed by in situ PLA, which showed more intense staining of PSA-ACT in cancers with Gleason grade 3 than in adjacent benign tissues from the same patients. These results show that PSA forms complexes already within the prostate and that PSA-ACT levels are increased in moderately differentiated prostate cancer tissue. This may explain, at least partially, why the ratio of serum PSA-ACT to total PSA is increased in prostate cancer.

Detection of Complexes between Prostate-Specific Antigen and Protease Inhibitors in Plasma

Featured Article: Stenman UH, Leinonen J, Alfthan H, Rannikko S, Tuhkanen K, Alfthan O. A complex between prostate-specific antigen and α1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222–6.2 Prostate-specific antigen (PSA)3 had been in clinical use for several years when we encountered a problem with 2 samples that did not give expected results upon dilution. To explore this finding, we subjected the samples to gel filtration and found that a major part of immunoreactive PSA had a molecular size of about 90 kD rather than the expected size of 30 kD. Because PSA is a protease, we assumed that the 90-kD component was a complex between PSA and a protease inhibitor. We therefore developed assays for possible complexes using a PSA antibody for capture and, as tracer, antibodies to α1-protease inhibitor, α1-antichymotrypsin (ACT), inter-α-trypsin inhibitor, and α2-macroglobulin. PSA complexes with all these inhibitors were detected, but the PSA–ACT complex was by far the most abundant one. At comparable PSA concentrations, that of PSA–ACT was higher in …

Enhancement of sensitivity using hybrid stimulus for the diagnosis of prostate cancer based on polydiacetylene (PDA) supramolecules

In this study, hybrid stimulus was initially introduced to improve the sensitivity of PDA vesicle chip for detection of prostate-specific antigen-α1–antichymotrypsin (PSA–ACT) complex. The strategy of hybrid stimulus on PDA vesicle chip offers the amplification method of fluorescent signal which combines a primary response by the immune reaction of antigen–antibody and a secondary response by the mechanical pressure of pAb-conjugated magnetic beads. As the primary response result on PDA vesicle chip, the PSA–ACT complex in PBS buffer was detected at 10 ng/mL. However, this detection sensitivity was insufficient for diagnosis of prostate cancer because the normal human PSA concentration is less than 4.0 ng/mL. To solve this problem, polyclonal PSA antibody-conjugated magnetic beads were used as an amplifying agent after primary immunoresponse. As a result, the PSA–ACT complex concentrations (as low as 0.1 ng/mL) could be detected in the PBS buffer sample. Therefore, this result can be applied to various fields, such as the detection of cells, proteins, and DNA for sensitive and specific biosensing based on PDA supramolecules.

Enhancement of sensitivity and specificity by surface modification of carbon nanotubes in diagnosis of prostate cancer based on carbon nanotube field effect transistors

This paper presents a simple and sensitive method for the real-time detection of a prostate cancer marker (PSA-ACT complex) through label-free protein biosensors based on a carbon nanotube field effect transistor (CNT-FET). Herein, the CNT-FET was functionalized with a solution containing various linker-to-spacer ratios, the binding event of the target PSA-ACT complex onto the receptor detected by monitoring the gating effect caused by charges in the target PSA-ACT complex. Since the biosensors were used in a buffer solution, it was crucial to control the distance between the receptors through introduction of linkers and spacers so that the charged target PSA-ACT complex could easily approach the CNT surface within the Debye length to give a large gating effect. The results show that CNT-FET biosensors modified with only linkers could not detect target proteins unless a very high concentration of the PSA-ACT complex solution (∼500 ng/ml) was injected, while those modified with a 1:3 ratio of linker-to-spacer could detect 1.0 ng/ml without any pretreatment. Moreover, our linker and spacer-modified CNT-FET could successfully block non-target proteins and selectively detect the target protein in human serum. Significantly, this strategy can be applied to general antibody-based detection schemes and enables production of very simple and sensitive electronic biosensors to detect clinically important biomarkers for disease diagnosis.

Homogenous growth of gold nanocrystals for quantification of PSA protein biomarker

We report on another alternative sensing platform for the detection of protein biomarker (PSA–ACT complex) based on homogenous growth of Au nanocrystals in solution phase. The immuno-recognition event is translated into the gold nanoparticle growth signal which can be intuitively recognized by an unaided eye, or quantitatively measured by an UV–vis spectrophotometric analysis. Surface plasmonic signature and kinetics of the Au nanogrowth in the homogenous phase containing of HAuCl 4, AA, and CTAB have also been studied to provide suitable parameters for the immunoassay. As a result, detection limit of PSA–ACT complex was determined to be 10 fM. The result indicated that this is a very sensitive, robust, simple, and economic strategy to detect protein biomarkers, and it has great potential to detect other biological interactions.

P3-086: CSF levels of PSA and PSA-ACT complexes in Alzheimer's disease

Background: Alpha1-antichymotrypsin (ACT) is found in amyloid plaques of Alzheimer’s disease (AD), but not in other types of amyloid. AD patients have higher cerebrospinal fluid (CSF) levels of ACT than controls. The exact role of ACT in AD is not clear, ACT functions as a serine protease inhibitor and is able to form complexes with, and to inactivate serine proteases. Prostate specific antigen (PSA) is a serine protease mainly found complexed to ACT in serum. PSA is found in the human brain and CSF, therefore the aim of this study was to determine if PSA is also the target protease of ACT in AD brain and if PSA-ACT complex levels in CSF can be used as biomarker for AD. Methods: Levels of ACT and PSA-ACT in CSF and serum samples of AD patients (n 16), mild cognitive impaired (MCI) subjects (n 19) and controls (n 12) were determined by sandwich ELISA. Total PSA was detemined in a competitive immunoassay and albumin with nephelometry in an array protein system. Statistical analysis was performed using SPSS software 15.0 and Medcalc. Results: ACT-PSA complexes and total PSA, were detectable in CSF of most male, but of only very few women. PSA levels in CSF and serum highly correlated (r 0.88; p 0.001) and Passing and Bablock regression analysis showed no deviation from zero regarding the intercept. Moreover, no indications for the presence of PSA in brain tissue were found by immunohistochemical analysis. CSF levels of either ACT, PSA or PSA-ACT did not differ between AD, MCI and control groups. Conclusions: PSA in CSF is not locally produced in the brain, but derived from the blood. Furthermore, PSA-ACT complexes are not suitable as a biomarker in AD.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-α1-antichymotrypsin detection based on surface plasmon resonance

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-alpha(1)-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.

Ratio of prostate specific antigen minor molecular forms-to-total prostate specific antigen is constant regardless of the pathological condition of the prostate.

We assessed 3 molecular forms of prostate specific antigen (PSA) prospectively to determine the real proportion of the nonfree, nonalpha1-antichymotrypsin complex form of PSA-to-total PSA and evaluated the clinical significance of its various forms. We prospectively assessed 1,878 serum samples from 994 subjects for total PSA, free PSA and PSA-alpha1-antichymotrypsin complex (ACT). Nonfree, nonPSA-ACT PSA (minor form PSA) was calculated as the difference between total PSA and the sum of free PSA plus PSA-ACT complex. The proportion of the minor form PSA was approximately 20% to 25% of total PSA at any range of total PSA, whereas that of free PSA and PSA-ACT decreased and increased, respectively, in correlation with the increment of total PSA. There was no significant difference in the percent of minor form PSA in patients with prostatic carcinoma and those with benign prostatic status. The proportion of minor form PSA was constant, while the percent of free PSA and PSA-ACT increased and decreased, respectively, in accordance with total PSA regression after hormonal therapy for prostatic carcinoma. PSA-ACT was judged to be superior to total PSA for distinguishing prostatic carcinoma in men with PSA 2 to 10 ng./ml. by ROC analysis. Approximately a fourth of total PSA consists of minor forms of complexed PSA. The average proportion of minor form-to-total PSA was constant at various levels of total PSA and at any prostatic status of patients. PSA-ACT was superior to total PSA for the early detection of prostatic carcinoma.

Characterization of Novel Monoclonal Antibodies for Prostate-Specific Antigen (PSA) with Potency to Recognize PSA Bound to α2-Macroglobulin

Different molecular forms of prostate-specific antigen (PSA) have been used to differentiate between benign prostatic hyperplasia and prostate cancer. Detecting PSA bound to endogenous inhibitors such as alpha(1)-antichymotrypsin (ACT) and alpha(2)-macroglobulin (alpha(2)M) is often difficult because of epitope masking or sensitivity problems. Here we report the characterization of four novel mouse monoclonal antibodies (mabs) obtained by immunization with PSA-alpha(2)M complexes. Their ability to detect free PSA and PSA-inhibitor complexes was shown, and their epitopes were analyzed by phage display technology. The properties of the mabs were studied by competition and sandwich assays and by Western blotting. Epitope mapping was performed by screening of a phage display peptide library. All four mabs recognized free PSA, PSA-ACT, and PSA-alpha(2)M complexes, but to various degrees. With different combinations of mabs in competition experiments, antibodies were identified that enhance binding of other mabs to PSA, forming the molecular basis of a very sensitive assay for the detection of PSA and PSA-ACT complexes. Mabs with highest reactivity for PSA-alpha(2)M were selected to establish an immunoassay for that complex. Western blot analysis revealed that all mabs recognized conformational epitopes of PSA. These findings were supported by phage display results demonstrating mimotopes in the PSA molecule. The results presented here could aid in the further development of clinically relevant assays for PSA and PSA-alpha(2)M complexes.

Three immunoassays based on monoclonal antibodies specific for prostate specific antigen (PSA), alpha-1-antichymotrypsin (ACT), and the PSA-ACT complex.

Prostate specific antigen (PSA) has been widely used as a biomarker for the screening and diagnosis of prostate cancer. PSA in serum predominantly exists as a complex with alpha-1-antichymotrypsin (ACT), and measurement of free PSA and the PSA-ACT complex may improve the utility of the serum PSA assay for differential diagnosis of prostate cancer and non-malignant prostate diseases, such as benign prostatic hyperplasia (BPH). Monoclonal antibodies (MAbs) against PSA, ACT, and the PSA-ACT complex were produced by immunizing mice with an incubated mixture of PSA and ACT, and characterized by Western blot analyses and several enzyme-linked immunosorbant assay (ELISA) methods. The MAbs produced in this study are capable of distinguishing the PSA-ACT complex from free PSA and ACT. Four MAbs have been selected and utilized to construct three ELISA systems for the separate measurements of free PSA, the PSA-ACT complex, and total PSA. The three PSA assay systems developed in this study can specifically measure free PSA, total PSA, and the PSA-ACT complex with equal molar sensitivity. It is expected that these PSA assay systems could be useful in the diagnosis of prostate cancer.

Ratio Of Prostate Specific Antigen Minor Molecular Forms-To-Total Prostate Specific Antigen Is Constant Regardless Of The Pathological Condition Of The Prostate

We assessed 3 molecular forms of prostate specific antigen (PSA) prospectively to determine the real proportion of the nonfree, nonalpha1-antichymotrypsin complex form of PSA-to-total PSA and evaluated the clinical significance of its various forms. We prospectively assessed 1,878 serum samples from 994 subjects for total PSA, free PSA and PSA-alpha1-antichymotrypsin complex (ACT). Nonfree, nonPSA-ACT PSA (minor form PSA) was calculated as the difference between total PSA and the sum of free PSA plus PSA-ACT complex. The proportion of the minor form PSA was approximately 20% to 25% of total PSA at any range of total PSA, whereas that of free PSA and PSA-ACT decreased and increased, respectively, in correlation with the increment of total PSA. There was no significant difference in the percent of minor form PSA in patients with prostatic carcinoma and those with benign prostatic status. The proportion of minor form PSA was constant, while the percent of free PSA and PSA-ACT increased and decreased, respectively, in accordance with total PSA regression after hormonal therapy for prostatic carcinoma. PSA-ACT was judged to be superior to total PSA for distinguishing prostatic carcinoma in men with PSA 2 to 10 ng./ml. by ROC analysis. Approximately a fourth of total PSA consists of minor forms of complexed PSA. The average proportion of minor form-to-total PSA was constant at various levels of total PSA and at any prostatic status of patients. PSA-ACT was superior to total PSA for the early detection of prostatic carcinoma.

Inhibition of prostate-specific antigen (PSA) by alpha(1)-antichymotrypsin: salt-dependent activation mediated by a conformational change.

Prostate-specific antigen (PSA) and its SDS-stable complex with the serine proteinase inhibitor (serpin) alpha(1)-antichymotrypsin (ACT), which is the dominant form of PSA in serum, are in widespread use as markers for the diagnosis of prostate cancer, and there is increasing evidence for the involvement of PSA proteinase activity itself in the development of prostate and other cancers. However, both the formation and degradation of the PSA-ACT complex, denoted PSA*ACT* to indicate substantial changes in the structure of both proteins on complex formation, have been incompletely studied. Here we determine rate and equilibrium constants for the steps involved in PSA*ACT* formation and demonstrate that (a) the effects of added NaCl, polyamines, and Zn(2+) on this process parallel their effects on PSA catalytic activity [Hsieh, M.-C., and Cooperman, B. S. (2000) Biochim. Biophys. Acta 1481, 75-87], (b) the effect of added NaCl in dramatically increasing the rate of ACT inhibition of PSA correlates with salt-induced changes in PSA conformation, and (c) the PSA*ACT* complex is subject to proteolysis by human neutrophil elastase. Possible clinical implications of these findings are considered.

ANALYSIS OF PROSTATE SPECIFIC ANTIGEN AND α1-ANTICHYMOTRYPSIN INTERACTION USING ANTIPEPTIDE MONOCLONAL ANTIBODIES

The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs). Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane. Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA. The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.

EXAMINATION OF THE 3 MOLECULAR FORMS OF SERUM PROSTATE SPECIFIC ANTIGEN FOR DISTINGUISHING NEGATIVE FROM POSITIVE BIOPSY: RELATIONSHIP TO TRANSITION ZONE VOLUME

We evaluated the relative usefulness of total, free and complexed serum prostate specific antigen (PSA), and their ratios for distinguishing positive from negative biopsy of prostates in a university referral practice. We compared 90 consecutive men who had 2 sets of 6 negative systematic biopsies with 70 who had at least 5 mm. of prostate cancer in systematic biopsies during the same period at our institution. Total prostate and transition zone volumes were determined by transrectal ultrasound. The Bayer, DPC and Hybritech assays were performed to measure total, free and complexed serum PSA. Receiver operating characteristics curves were constructed for all forms of serum PSA and their ratios as well as prostate size to distinguish true positive (sensitivity) from false-positive (1 minus specificity) fractions. Complexed PSA was only marginally better than total serum PSA. Free-to-total, complexed-to-total and prostate size had highly significant areas under the curves of greater than 80%. Free PSA only was better than complexed or total PSA. When factored by prostate volume, total PSA performed as well as the PSA ratios, and transition zone volume was consistently better than total prostate volume. DPC free-to-total ratios were equivalent to Hybritech ratios in all respects. Complexed PSA is only marginally better than total PSA for distinguishing negative from positive biopsy of prostates. It is inferior to free PSA and far less useful than free-to-total or complexed-to-total ratios. Prostate size is a decisive variable in men in whom we avoided the expected 25% false-negative biopsy rate in terms of specificity and hopefully avoided insignificant cancer in terms of sensitivity. In the future the performance of PSA serum markers should be related to a transition zone volume of less than 20, 20 to 60 and greater than 60 gm. when comparing assays to each other.

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

Prostate-specific antigen (PSA) occurs in serum both free and in complex with protease inhibitors. The complex with alpha1-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 alpha1-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. 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. 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 microgram/L. In patients with 4-20 microgram/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). 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.

Epitope Mapping and Affinity Estimation of 83 Antibodies against Prostate-Specific Antigen

Eighty-three antibodies submitted to the ISOBM TD-3 Workshop on prostate-specific antigen (PSA) were characterized by cross-inhibition studies, immunometric assay and affinity estimation with free or complexed PSA (PSA-α₁-antichymotrypsin, PSA-ACT). Nine antibodies did not bind PSA or PSA-ACT when coated onto microtiter plates or in solution. Another 3 antibodies bound the antigens only when in solution and were therefore omitted from the cross-inhibition experiments. Dissociation constants (K_d) were estimated from the concentration of free antibody needed to achieve half-maximal binding of the antigen. K_d values for PSA and PSA-ACT ranged from 2 × 10^–12 to >10^–8 mol/l. Antibodies were classified into 6 main groups according to their reactivity. Group 1 comprised 15 antibodies (#25, 26, 33, 68, 73, 77, 78, 80, 85, 209, 213, 216, 223, 230, and 262) specific for free PSA. These antibodies had >80% cross-inhibition and showed high affinity for PSA with minimal or no affinity for the PSA-ACT complex. Group 2 comprised antibodies that reacted with both free PSA and PSA-ACT. Three subgroups were defined: group 2a (#40), group 2b (#32) and group 2c (#35, 37, 63, 90, 215 and 226). Group 3a antibodies (#31, 36, 37, 57, 64, 66, 72, 82, 84, 212, 224, 229, 257 and 260) were closely related to those of group 2, with two exceptions in group 3b (#88 and 89). Group 4 contains antibodies with binding patterns similar to those represented by groups 3b and 6b. These antibodies could be divided into two subgroups: group 4a (#30, 38, 51, 217, and 220) and group 4b (#74). Group 5 was more heterogeneous, with distinct inhibition patterns: group 5a (#50, 54, 76, 81, 207, and 222); group 5b (#41), and group 5c (#28 and 86). Group 6 antibodies bind epitopes on both free PSA and PSA-ACT, but have epitopes unrelated to those represented in groups 1–3: group 6a contains 15 antibodies (#24, 27, 29, 34, 55, 56, 65, 79, 210, 214, 218, 221, 225, 258 and 261), and group 6b 2 antibodies (#67 and 75). These 6 groups represent the major immunodominant regions, one of which is exposed only on free PSA. Our classification could provide a useful guide in choosing antibodies for future PSA assays.

MONOCLONAL ANTIBODIES 2F5 AND 4G10 AGAINST PROSTATE SPECIFIC ANTIGEN (PSA) COMPLEXED TO alpha 1-ANTICHYMOTRYPSIN

Objectives We have shown that an immunoassay (Chugai) for the PSA-ACT complex in serum has 2 to 3 times better specificity than total PSA at sensitivities of 85 to 97% in distinguishing biopsy positive men from biopsy negative men undergoing transrectal ultrasound (TRUS) examination. To increase the specificity of PSA immunoassay for prostate cancer, we produced specific antibodies exclusively against our purified PSA-ACT complex for development of assay kits for PSA-ACT complex. Methods PSA-ACT complex was used as antigen to immunize BALB/c mice. PSA-ACT complex, free PSA, and ACT were used for hybridoma screening. To characterize the new monoclonal antibodies, we used Western blot, immunohistochemistry, and an in house immunoassay. Results Two monoclonal antibodies, 2F5 and 4G10 were produced exclusively against PSA-ACT complex without any immunoreactivity to ACT or PSA alone. Western blot analysis indicated that 2F5 and 4G10 recognize conformation-dependent epitopes on PSA-ACT complex. Immunohistochemistry studies showed that 2F5 reacted with prostate cancer in about 30% of the cancer cells (sensitivity), but almost never stained normal prostate glands in the peripheral or transition zone tissue (about 100% specificity). Our in-house assay showed that 2F5 can be used as a tracer antibody specifically to detect PSA-ACT complex. Conclusions Using monoclonal antibody 2F5 as tracer antibody, we have developed a PSA immunoassay exclusively against PSA-ACT complex. This assay should maximize specificity in distinguishing BPH from prostate cancer.

Monoclonal antibodies 2F5 and 4G10 against prostate specific antigen (PSA) complexed to alpha1-antichymotrypsin.

We have shown that an immunoassay (Chugai) for the PSA-ACT complex in serum has 2 to 3 times better specificity than total PSA at sensitivities of 85 to 97% in distinguishing biopsy positive men from biopsy negative men undergoing transrectal ultrasound (TRUS) examination. To increase the specificity of PSA immunoassay for prostate cancer, we produced specific antibodies exclusively against our purified PSA-ACT complex for development of assay kits for PSA-ACT complex. PSA-ACT complex was used as antigen to immunize BALB/c mice. PSA-ACT complex, free PSA, and ACT were used for hybridoma screening. To characterize the new monoclonal antibodies, we used Western blot, immunohistochemistry, and an in house immunoassay. Two monoclonal antibodies, 2F5 and 4G10 were produced exclusively against PSA-ACT complex without any immunoreactivity to ACT or PSA alone. Western blot analysis indicated that 2F5 and 4G10 recognize conformation-dependent epitopes on PSA-ACT complex. Immunohistochemistry studies showed that 2F5 reacted with prostate cancer in about 30% of the cancer cells (sensitivity), but almost never stained normal prostate glands in the peripheral or transition zone tissue (about 100% specificity). Our in-house assay showed that 2F5 can be used as a tracer antibody specifically to detect PSA-ACT complex. Using monoclonal antibody 2F5 as tracer antibody, we have developed a PSA immunoassay exclusively against PSA-ACT complex. This assay should maximize specificity in distinguishing BPH from prostate cancer.

Variation in the Quantitation of Prostate-specific Antigen in Reference Material: Differences in Commercial Immunoassays,

Prostate-specific antigen (PSA) is a widely used biochemical marker for prostate cancer (1). PSA, a serine protease, forms complexes with serine protease inhibitors such as α2-macroglobulin and α1-antichymotrypsin (ACT) (2). The major immunoreactive forms of serum PSA are PSA-ACT complex and free PSA. PSA complexed to α2-macroglobulin is not measured by current immunoassays (3). Because the proportion of PSA complexed to ACT is higher in men with prostate cancer than in men with benign prostatic hyperplasia (BPH) (3)(4), the use of a ratio of free-to-total PSA may improve discrimination between men with BPH and prostate cancer (5)(6). Results obtained with different PSA immunoassays are not interchangeable (7)(8)(9)(10). The variation among immunoassays reflects, in large part, the specificity of different PSA antibodies (7). To a lesser extent, the composition of the calibrator, the PSA values assigned to the calibrator, the test diluent, and the physical design of the immunoassays contribute to intermethod differences (6)(7). Assays using a monoclonal-monoclonal sandwich format yield, in general, an equimolar response to free and ACT-complexed PSA, whereas monoclonal-polyclonal formats tend to yield higher values for samples containing greater proportions of free PSA (8). We have recently described the Bayer Immuno 1TM PSA Assay (Bayer Corporation), which uses a monoclonal-polyclonal antibody format that yields an equimolar response to varying proportions of free and complexed PSA (11). This has now been found to be a result of the unique binding properties of the monoclonal antibody used for the capture of PSA (manuscript in submission). Proficiency testing among clinical laboratories has indicated large intermethod variability in the quantitation of total PSA (12). The aim of this study was to determine the magnitude and cause of this intermethod variation. Here we report our multi-site analysis of …

Molecular mass determination for prostate-specific antigen and alpha 1-antichymotrypsin complexed in vitro.

The results from molecular mass determinations and amino acid analyses for prostate-specific antigen (PSA), alpha 1-antichymotrypsin (ACT) and PSA-ACT complexed in vitro are reported. Molecular masses for two separate PSA-ACT lots were determined by matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) coupled to a time-of-flight (TOF) detector. Interestingly, both PSA-ACT lots contained two predominant protein species: 78,095 +/- 138 Da (approx. 67% of total protein) and 82,519 +/- 104 Da. Because a heterogneous population was observed and the masses were less than expected on the basis of less sensitive techniques, molecular masses for the individual PSA and ACT components were determined. As expected, PSA possessed a single molecular mass (27,755.8 Da). Each ACT raw material lot, however, also contained two predominant species: 55,106 +/- 111 and 51,414 +/- 32 Da. To assess the amino acid composition, each PSA, ACT and PSA-ACT lot was subjected to acid hydrolysis for 24, 48 and 72 h followed by amino acid analysis. Experimental results, expressed as mole percentages for each measurable residue and the number of residues per mole of protein, were compared with both the predicted and previously published values and were as expected. These more accurate molecular mass values for ACT and purified PSA-ACT complex should be considered in preparing and characterizing international standard preparations.