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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.

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86–91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158–163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3–11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

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.

Recently we described the generation of the prostate tissue-specific monoclonal antibody (MAb) 107-1A4, its expression pattern and preliminary targeting of human prostate cancer xenografts. In this report we demonstrate that the target antigen for MAb 107-1A4 is prostate-specific membrane antigen (PSMA) using immunoaffinity absorption followed by SDS-PAGE and mass spectrometric analysis of peptides produced by in-gel tryptic digestion. Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in men in the United States. In spite of increasing attention and accumulating knowledge, advances in treatment that improve survival remain elusive. Prostate specific antigen (PSA) is the most widely used marker of prostate cancer. Immunoassays for PSA using monoclonal (MAb) or polyclonal antibodies have clinical applications, such as monitoring and early detection of prostate cancer. However, PSA is not a perfect tumor marker because serum levels often are elevated in men with benign prostatic hyperplasia, prostatitis and other nonmalignant disorders and also because levels are not always elevated in individuals with early prostate cancer. 1 In an effort to identify additional proteins that could be of value for the diagnosis and treatment of prostate cancer, we have recently described the generation of a new prostate cancer-reactive MAb, designated 107-1A4, using a two-phase immunization protocol involving the prostate cancer cell line, LNCaP. 2 This MAb recognizes an antigen, which appeared to be distinct from those previously described and shows specific tumor targeting in preliminary in vivo studies. In part because MAb 107-1A4 recognized a conformational epitope and, thus, could not be used in Western blots, we were unable to characterize its antigen using conventional approaches. 2 In this report we describe the identification of the target antigen of MAb 107-1A4 using ProteinChip array, surface-enhanced laser desorption/ionization (SELDI) technology from Ciphergen Biosystems (Fremont, CA). SELDI, a concept introduced by Hutchens and Yip 3 combines ProteinChip technology with timeof-flight mass spectrometry and offers the advantages of speed, simplicity, sensitivity and accuracy. Briefly, each ProteinChip array has a number of spots that contain functional groups, chemical or biological "docking sites," for the selective binding and washing of proteins/peptides from complex mixtures. After the sample is applied to the surface, unbound proteins and interfering substances are washed away. A solution containing laser energyabsorbing molecules, often referred to as a matrix, is then added and allowed to dry. In this fashion, the laser energy absorbing matrix molecules co-crystallize with the adsorbed proteins. The captured proteins are then detected using laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS). A more comprehensive description of the overall history and recent advances in SELDI technology and comparisons with other laserbased mass spectrometry...

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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.

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 α1‐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. © 1996 Wiley‐Liss, Inc.