Anti-CD13 Anticorpo [WM15] (A86405)

Urinary expressed prostatic secretion or "EPS-urine" is proximal tissue fluid that is collected after a digital rectal exam (DRE). EPS-urine is a rich source of prostate-derived proteins that can be used for biomarker discovery for prostate cancer (PCa) and other prostatic diseases. We previously conducted a comprehensive proteome analysis of direct expressed prostatic secretions (EPS). In the current study, we defined the proteome of EPS-urine employing Multidimensional Protein Identification Technology (MudPIT) and providing a comprehensive catalogue of this body fluid for future biomarker studies. We identified 1022 unique proteins in a heterogeneous cohort of 11 EPS-urines derived from biopsy negative noncancer diagnoses with some benign prostatic diseases (BPH) and low-grade PCa, representative of secreted prostate and immune system-derived proteins in a urine background. We further applied MudPIT-based proteomics to generate and compare the differential proteome from a subset of pooled urines (pre-DRE) and EPS-urines (post-DRE) from noncancer and PCa patients. The direct proteomic comparison of these highly controlled patient sample pools enabled us to define a list of prostate-enriched proteins detectable in EPS-urine and distinguishable from a complex urine protein background. A combinatorial analysis of both proteomics data sets and systematic integration with publicly available proteomics data of related body fluids, human tissue transcriptomic data, and immunohistochemistry images from the Human Protein Atlas database allowed us to demarcate a robust panel of 49 prostate-derived proteins in EPS-urine. Finally, we validated the expression of seven of these proteins using Western blotting, supporting the likelihood that they originate from the prostate. The definition of these prostatic proteins in EPS-urine samples provides a reference for future investigations for prostatic-disease biomarker studies.

In clinical chemistry, determination of serum alanine aminopeptidase (AAP) levels has been found to be useful for detecting or confirming biliary obstructions from either intra- or extra-hepatic disorders. In haematology, 'gp150' is a surface-expressed protein molecule, recognised by monoclonal antibodies belonging to the so-called 'Cluster of Differentiation' (CD-) 13, which has independently been found to be a useful marker of myeloid leukaemia in addition to providing potential prognostic value. The current report links these two independent research streams and provides evidence that the hepatobiliary disease marker, serum AAP, predominantly comprises a circulating isoform of 'gp150'. Thus, a (CD-13) monoclonal antibody raised to, and specifically reactive with, cell surface myeloid 'gp150' is able to specifically and almost completely block serum (or plasma) AAP activity otherwise observed in its absence. This holds true for serum (or plasma) derived both from normal individuals or from patients suffering hepatic dysfunction, with or without associated biliary obstruction. In the case of patients with obstructive jaundice, raised levels of AAP are observed, which fall to near normal levels following preincubation with this monoclonal antibody. In addition, data are presented to support the view of varying isoforms of AAP within flowing blood. Finally, preliminary data is provided on AAP activity in cases of leukaemia. These studies should thus prove of use to clinical laboratories investigating the involvement of AAP activity in various pathological processes.

This study is the first to report the presence of CD13/glycoprotein 150 (GP150)/aminopeptidase-N activity in cell-free plasma. We have determined that aminopeptidase-N in plasma provides, quantitatively, aminopeptidase-N's predominant functional activity within flowing blood. Thus, while aminopeptidase-N activity observed in whole blood can be partly, but significantly, blocked by the CD13 monoclonal antibody (MAB) WM15, the magnitude of such inhibition is low (< 25%) and similar to that observed using washed cell fractions selectively enriched for neutrophils (30.6% inhibition) or monocytes (21.8% inhibition). Plasma, free of cell components, possesses substantial aminopeptidase-N activity that is largely inhibitable (> 70%) by WM15. Blood collected into heparin or citrate yields similar data, while blood collected into EDTA gives rise to reduced CD13/aminopeptidase-N activity, consistent with inhibition of the known heavy-metal ion association necessary for proper functioning of this molecule. Although monocyte- and granulocyte-enriched cell fractions possess aminopeptidase-N activity significantly inhibitable by CD13 antibodies, lymphocyte-enriched cell fractions also possess aminopeptidase-N-like activity; however, in the latter case, this activity is not inhibitable by CD13 antibodies. Immunoaffinity isolation of plasma aminopeptidase-N has also been carried out; further characterization using functional studies and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis indicates that CD13 MABs can completely clear plasma of aminopeptidase-N activity and that the purified protein has similar electrophoretic characteristics to cell-derived material. These data, therefore, provide evidence for the presence within blood both of a soluble (that is, non-cell-associated) form of CD13/GP150/aminopeptidase-N localizable to plasma and of cell-associated, aminopeptidase-N-like proteins other than CD13/GP150. These findings have significant implications for our understanding of the many functions of this molecule in blood.

A mouse monoclonal antibody, WM-15, has been developed which reacts with a human myeloid lineage-restricted cell surface antigen. WM-15, an IgG1 antibody, reacts with a mean of 3.8% of normal bone marrow mononuclear cells, identifying predominantly promyelocytes and myeloblasts, and in fluorescence-activated cell sorting experiments produces enrichment of bone marrow granulocyte-macrophage progenitor cells. Normal mature monocytes and granulocytes are weakly labelled by WM-15, but other haemopoietic cells including erythroblasts and all lymphoid cells are unreactive. The myeloid specificity of this antibody is highlighted by its reactivity with myeloid leukaemia cell lines and 65% of cases of acute myeloid leukaemia, while lymphoid cell lines and leukaemias are WM-15 negative. WM-15 appears to be a useful reagent for further investigation of normal and abnormal myelopoiesis.

Six murine monoclonal antibodies reactive with human myeloid lineage differentiation antigens are described. These antibodies, designated WM-12, WM-14, WM-15, WM-16, WM-19, and WM-20, react with normal peripheral blood neutrophils and monocytes, as well as a proportion of myeloid precursor cells in bone marrow, but fail to react with the majority of normal lymphoid cells. Immunoprecipitation studies have demonstrated binding of WM-12, -14, -16, -19, and -20, to elements of a protein multimer with sub-units of 50, 105, and 170 kilodalton molecular weight under reducing conditions. WM-15 antibody, however, reacts with a separate protein of 165 kilodaltons. These 6 antibodies reacted with 89% of cases of acute myeloid leukemia, but showed significant binding with only occasional cases of acute lymphoblastic leukemia. Cases of AML (FAB M1 and M2) were more frequently positive with WM-15 (60% of cases positive) than with the other 5 antibodies, whereas the converse was true for leukemias with monocytic differentiation (FAB M4 and M5; 82-100% of cases positive with WM-12, -14, -16, -19, -20). These antibodies appear useful for diagnosis and classification of acute leukemia.

We have investigated the binding of over 30 different monoclonal antibodies (MAB) belonging to three distinct clusters of differentiation (CD-11b; CD-13; CD-33; as defined by the Third International Workshop on Leucocyte Differentiation Antigens (ILWS), 1986), and which are reactive with three distinct myeloid restricted surface antigens ('gp160,95'; 'gp150'; 'gp67'). By investigating reactivity with non-haemopoietic cells, we have confirmed that CD-11b and CD-33 MAB reactivity is largely restricted to haemopoietic cells, whilst CD-13 MAB showed additional binding to a wide range of non-haemopoietic cells. Epitopic heterogeneity was also investigated within each cluster of differentiation. Tested anti-CR3 (CD-11b) MAB varied in their ability to block the binding of complement coated sheep red blood cells and zymosan particles. A more detailed analysis of MAB binding heterogeneity was performed by competitive inhibition assays. It was demonstrated that MAB from both CD-11b and CD-13 bind to several distinct epitopes (at least six and five respectively) on their respective antigen molecules. In contrast, CD-33 MAB appear to bind to only a single site on 'gp67'. These data may allow for a clearer appreciation of the disparate functional effects obtained using different MAB reagents to individual myeloid antigens, as reported by a number of workers.

This report details experimental results which show the presence of enzymic aminopeptidase-N-like activity on endothelial cells, concomitant with cell surface expression previously detected by both ELISA and indirect immunofluorescence. This activity, detected using selected chromogenic substrates in 'functional' assays, is shown to be due (at least in part) to a molecule previously termed 'gp 150' and recognized by MoAb belonging to CD-13, since such antibodies can be shown to inhibit this activity. Activity, as detected on endothelial cells, is similar to that observed on various haemopoietic cells. These assays not only provide a functional basis to cell surface gp150 (CD-13) co-expression on haemopoietic and endothelial cells but have also been used to help define structural epitopes present on aminopeptidase-N/gp150, previously analysed using radiolabelled antibodies in competitive binding assays. Appraisal of these new data suggests that the number of distinct antibody binding sites on this molecule is greater than that previously demonstrated. This study is therefore an important first step in investigating the potential involvement of this selective peptidase molecule in the control of haemopoietic cell growth and differentiation and in haemostatic mechanisms.