A dual-monoclonal sandwich assay for prostate-specific membrane antigen: levels in tissues, seminal fluid and urine.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a 750-residue integral membrane glycoprotein and the target of an in-vivo imaging agent for metastatic prostate carcinoma (PCa). PSMA expression in normal and diseased prostatic tissues has previously been demonstrated by immunohistochemical techniques. In order to quantify PSMA levels in tissue homogenates and physiological fluids, we have developed a dual monoclonal antibody (mAb) sandwich assay which detects the antigen at a sensitivity <1 ng/mL and which is linear across the working range 0-50 ng/mL. METHODS: The assay involves capture of the PSMA by a biotinylated mAb (7E11) immobilized onto a streptavidin-coated microtiter plate; this mAb binds to the N-terminus of the antigen. The captured PSMA is detected by an Eu-labelled mAb (PEQ226) which binds in the region corresponding to Residues 134-437 of the antigen. PSMA was purified from LNCaP cells by immunoaffinity chromatography, and used as a calibrator, based on its concentration by the bicinchoninic acid (BCA) protein assay. RESULTS: The assay was applied to a panel of normal and tumor tissues. Levels were highest in the prostate tissues (292-4254 ng/mg protein). Low levels (21-51 ng/mL) were observed in membranes from ovary and breast, and neglible levels (1-10 ng/mg) in membranes from skin, liver, intestine, and kidney. Levels in the corresponding cytosol fractions were 20-to 50-fold lower. The average PSMA level in seminal fluid from 21 donors was 9, 012 ng/mL. On average, levels in normal-male urine (3.47 ng/mL) were ten-fold higher than in normal-female urine (0.3 ng/mL). CONCLUSIONS: This report is the first to describe absolute quantitation of PSMA in tissues and fluids. Congruent with earlier tissue studies based on immunohistochemical staining and Western-blot analysis, prostate tissue membranes expressed the highest levels of PSMA.

Western blotting analysis of antibodies to prostate-specific antigen: specificities for prostate-specific antigen and prostate-specific antigen fragments.

Approximately 40% of the prostate-specific antigen (PSA) purified from seminal fluid comprises cleaved or fragmented forms of PSA. These fragments are observed by reduced SDS-PAGE and have been identified in preparations of purified seminal plasma. The comparative analysis of 53 antibodies, submitted to an international PSA Workshop, with different PSA variants was studied using SDS-PAGE and Western blotting. Six different patterns of reactivity were observed which may reflect different epitopes recognized by this panel of antibodies. Additional antibody studies to nonreduced intact PSA suggest the epitopes are conformation-dependent.

Dual monoclonal antibody immunoassay for free prostate-specific antigen.

Prostate-specific antigen (PSA) is the most important tumor marker for early detection and monitoring of prostate cancer (PCa) patients. PSA is also elevated in many patients with benign prostatic hyperplasia (BPH). The study of the serum PSA forms, free PSA (f-PSA) and PSA complexed with alpha 1-antichymotrypsin (PSA-ACT), may improve the discrimination between PCa and BPH. An immunoassay specific for f-PSA is reported with very low cross-reactivity (0.7%) to PSA-ACT. Serum specimens from BPH and PCa patients (determined by biopsy) with PSA levels from < 1 to > 100 ng/ml were tested. No f-PSA was detected in serum specimens from normal females (N = 50). Low levels (0-0.3 ng/ml) were detected in specimens from healthy males (N = 60). In specimens from PCa and BPH patients, the f-PSA to total PSA ratio (f/t) was found to range from 1% to higher than 60%. While maintaining an 80% sensitivity for cancer, the f/t ratio improved specificity to approximately 80%, as compared to 55% for total PSA alone. The receiver operating characteristics (ROC) curve analysis of the f/t ratio displayed a greater area under the plot (0.84) compared to total PSA alone (0.745). The results demonstrate that the f/t ratio significantly increases specificity for PCa detection compared to total PSA alone, showing the potential clinical value of the f-PSA immunoassay.

Molecular forms of prostate-specific antigen and the human kallikrein gene family: a new era.

Without question, much has been learned about the glycoprotein PSA in recent years. By increasing our understanding of this tumor marker's biochemical and physiologic properties, we will be able to improve its clinical utility. The discovery of the various molecular forms of PSA represents a significant advancement. Knowing the concentration and ratio of these PSA forms will be valuable in deciding which patients require further evaluation with transrectal ultrasound and prostate biopsy and which men can be monitored safely without undergoing further invasive testing. This information will be most valuable in treating the patient with a mildly elevated serum PSA level. Although assays are not yet available to detect specifically hK2, the striking similarities of hK2 to PSA, including selective expression in the prostate, suggest that this marker may also prove useful in prostate cancer management. Indeed, a new era of PSA testing has been entered, and the entire field of prostate cancer will benefit.

Dopamine-beta-hydroxylase: structural comparisons of membrane-bound versus soluble forms from adrenal medulla and pheochromocytoma.

Dopamine-beta-hydroxylase (DBH) in membrane-bound (mDBH) and water-soluble (sDBH) forms was isolated from chromaffin granules of bovine adrenal medullae and a human pheochromocytoma tumor. sDBH was purified by concanavalin A-agarose column chromatography followed by DEAE-Sepharose column chromatography. The final bovine preparation had a specific activity of 16.27 IU/mg; the human preparation had a specific activity of 9.16 IU/mg. mDBH was isolated in enzymatically inactive form by preparative polyacrylamide gel electrophoresis. The proteins were subjected to amino acid analysis, as well as digestion with trypsin, followed by separation of the resulting peptides by two-dimensional TLC/electrophoresis. No intraspecies differences between sDBH and mDBH were found from comparisons of amino acid composition or peptide maps. Thus the basis of the difference between sDBH and mDBH cannot easily be explained by differences in primary structure, within the resolution of these techniques.

Human chromogranin A. Purification and characterization from catecholamine storage vesicles of human pheochromocytoma.

Chromogranin A is the quantitatively major soluble protein in catecholamine storage vesicles of the adrenal medulla and sympathetic nerve, and has been a useful index of exocytosis during sympathoadrenal neurosecretion. To probe human catecholamine storage and release, we isolated chromogranin A from chromaffin tissue in human pheochromocytoma, and compared it to chromogranin A isolated from chromaffin tissue in bovine adrenal medulla. The preparation included catecholamine storage vesicle isolation by sucrose gradient centrifugation, removal of dopamine-beta-hydroxylase by affinity chromatography on Concanavalin A-Sepharose, and preparative polyacrylamide gel electrophoresis. Human and bovine chromogranin A displayed considerable interspecies homology. Human chromogranin A is a 68,000 dalton monomeric protein with an unusual amino acid composition (31.53 weight % glutamic acid); an acidic, microheterogeneous isoelectric point (4.57-4.68); a characteristic tryptic digest peptide map; and marked dissimilarity to dopamine-beta-hydroxylase in all properties studied. A new probe of human sympathoadrenal function is available in chromogranin A.

Human catecholamine storage vesicle proteins.

Catecholamines in the adrenal medulla and sympathetic nerve are stored in vesicles, along with numerous proteins and peptides; both catecholamines and proteins are released by and are markers of exocytosis during sympathoadrenal neurosecretion. The proteins include the enzyme dopamine-beta-hydroxylase and the chromogranins, a group of molecules of largely undetermined function. We have isolated catecholamine storage vesicles (chromaffin granules) from bovine adrenal medulla and human pheochromocytoma. The numerous soluble proteins in the granules: (a) have a spectrum of sizes; (b) have a spectrum of charges; and (c) display considerable interspecies qualitative homology. Dopamine-beta-hydroxylase (DBH) was isolated from human and bovine vesicles by Concanaval in A affinity chromotography. DBH is a tetrameric glycoprotein consisting of 2 non-covalently joined dimeric subunits, each of which is 2 disulfide linked monomers; interspecies molecular weight differences were noted. Ongoing studies concern chromogranin A, the quantitatively major vesicle protein, in bovine and human vesicles.

Radioimmunoassay of urinary free cortisol.

The radioimmunoassay for urinary free cortisol described in this paper is simple, rapid, and reproducible. The method uses a commercially available antibody preparation and is performed in two steps. The first step includes an extraction and a column purification to remove materials antigenically similar to cortisol from the urine. The second step is the radioimmunoassay using dextran-coated charcoal to separate bound and unbound cortisol. 3H-cortisol is added prior to any mechanical manipulation to allow calculation of analytical recovery for the purification procedure. The coefficient of variation for interassay determinations was a maximum of 10.3% and for intraassay determinations a maximum of 5.7%. Analytical recovery averaged 97.7%. One technician can analyze 100 samples per week.