A monoclonal antibody detecting a novel antigen expressed in the HTLV-I- infected cells

A monoclonal antibody, FTF 148, was prepared by hybridizing murine myelomal cells (NS-1) and spleen cells of BALB/c mice immunized with cultured cells derived from an adult T cell leukemia (ATL) patient (KUT- 2 cells). This monoclonal antibody reacted with all of the human T cell leukemia virus I (HTLV-I)-infected cell lines tested but did not react with other T cell lines derived from acute lymphocytic leukemia, Epstein-Barr virus-transformed B cell lines, or an erythroleukemic cell line. This monoclonal antibody was not directed to viral antigens because it reacted equally well with almost all KUT-2 and MT-1 cells, only 1% to 3% of which were ATL-associated antigen-positive. In contrast to interleukin 2 receptors expressed on both ATL cells and normal phytohemagglutinin-stimulated blasts, this antigen was not expressed on the latter cells. The antigen, mainly expressed on the cell membrane, was analyzed by metabolic labeling with 3H-leucine and surface labeling with 125I followed by cell lysis and immunoprecipitation with the FTF 148 antibody. The findings obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that p50 and p74 proteins were specifically precipitated and the antigen was also different from the product of the Xs gene of HTLV-I.     

A novel apoptosis-inducing monoclonal antibody (anti-LHK) against a cell surface antigen on colon cancer cells

Background Apoptosis is a crucial element in the behavior of mammalian cells in many different situations. We here report the establishment of a novel monoclonal antibody (anti-LHK mAb) that has apoptosis-inducing activity against colon cancer Colo205 cells. Methods The mechanism of anti-LHK mAb-induced cell death was assessed by microscopic morphology, Annexin V/Hoechst 33528 staining, and detection of DNA fragmentation. The molecular weight of LHK antigen was determined by Western blotting. Growth inhibition of Colo205 cells induced by anti-LHK mAb was determined by in vitro and in vivo studies. Results Anti-LHK reacted with a 70-kDa antigen and completely blocked the proliferation of Colo205 cells bearing LHK in vitro in a manner characteristic of apoptosis. Strikingly, anti-LHK mAb suppressed tumor growth in a murine peritoneal dissemination model. Conclusions LHK antigen, which is restricted to epithelial cells, may be a novel death receptor that plays a critical role in controlling the growth, invasion, and metastasis of human colon cancer cells.     

A novel membrane antigen selectively expressed on terminally differentiated human B cells

A monoclonal antibody (MoAb) that defines a novel terminal B-cell- restricted antigen, termed HM1.24, was developed against a human plasma cell line. The MoAb, designated anti-HM1.24, reacted with five different human myeloma cell lines, as well as with monoclonal neoplastic plasma cells obtained from the bone marrow or peripheral blood of patients with multiple myeloma or Waldenstrom's macroglobulinemia. The HM1.24 antigen was also expressed by mature Ig- secreting B cells (plasma cells and lymphoplasmacytoid cells) but not by other cells contained in the peripheral blood, bone marrow, liver, spleen, kidney, or heart of normal individuals or patients with non- plasma-cell-related malignancies. The anti-HM1.24 MoAb bound to human myeloma RPMI 8226 cells with an affinity constant of 9.2 x 10(8) M-1, indicating approximately 84,000 sites/cell. By immunoprecipitation assay under reducing conditions, this MoAb identified a membrane glycoprotein that had a molecular weight of 29 to 33 kD. Our studies indicate that the HM1.24-related protein represents a specific marker of late-stage B-cell maturation and potentially serves as a target antigen for the immunotherapy of multiple myeloma and related plasma cell dyscrasias.     

The monoclonal antibody 97A6 defines a novel surface antigen expressed on human basophils and their multipotent and unipotent progenitors.

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However, little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb), 97A6, that specifically detects human Ba, MC (lung, skin), and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils, eosinophils, monocytes, lymphocytes, platelets) did not react with MoAb 97A6, and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (>98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40), Ba-eosinophil colonies (7 of 40), Ba-macrophage colonies (3 of 40), and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast, no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells, starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (>30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors, MC progenitors, Ba progenitors, as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors.     

Identification and characterization of a new surface membrane antigen found predominantly on malignant B lymphocytes.

A monoclonal antibody, 1D10, was derived that identifies a new antigenic epitope on the surface of malignant B lymphocytes. Normal resting and stimulated lymphocytes do not express the antigen. The majority of individuals with acute Epstein-Barr virus infection express the antigen on their lymphocytes, and in these patients, the T lymphocyte may also be antigen positive. The antigen was found on B-lymphoid neoplasia from the early pre-B cell stage through terminally differentiated plasma cells, a characteristic not reported for other B cell-associated antigens. Studies on homozygous typing cells and cells from individuals with known HLA phenotypes indicate that the antigen does not segregate in a pattern characteristic for major histocompatibility antigens. The molecule is a heterodimeric polypeptide with the molecular weight and isoelectric points of the alpha and beta chains being 32,000 d/4 and 28,000 d/6, respectively. Evidence is presented that the 1D10 molecule is not HLA-DR, -DP, or -DQ. By extrapolation, we suggest that this novel molecule may represent HLA D-region gene expression of a gene(s) not normally expressed. Potential candidates are D-region pseudogenes. We conclude that the antigenic epitope identified by the 1D10 monoclonal antibody is unique among previously described B-lymphocyte antigens. Further studies of the factors controlling the expression of this molecule, as well as studies designed to look at the possible cellular function, may provide insights for understanding crucial events in the malignant transformation of lymphocytes.     

Membrane antigen on Epstein--Barr virus-infected human B cells recognized by a monoclonal antibody.

This paper describes a monoclonal antibody (B532) that detects a membrane antigen present on greater than or equal to 95% of the B cells from lines carrying the Epstein-Barr virus (EBV) genome. Evidence suggesting that B532 is EBV-related was originally obtained by using a cell-binding radioassay with different cell line substrates. Immunofluorescence and cell-sorter analysis confirmed that the antigen was present in high density on all EBV-infected lymphoblastoid B-cell lines, but not on EBV-negative B-, T-, myeloid, or null cell lines. Isolated normal peripheral blood B and T lymphocytes and monocytes failed to bind B532. The monoclonal antibody did not inhibit in vitro EVB infection nor did it block the killing of EBV-infected targets by cytotoxic T lymphocytes. The cell surface antigen recognized by B532 was shown by immunoprecipitation to have a molecular weight of approximately 45,000.     

Identification of a human erythroid cell surface antigen by monoclonal antibody HAE9

A noncytotoxic monoclonal antibody (IgM) HAE9 that selectively binds to 36% CFU-E and more than 90% nucleated erythroid cells in human bone marrow is described. This antibody recognizes a 70-kDa-membrane protein. It is suggested that HAE9 is directed to a human epitope of Ag-Eb, an interspecies mammalian erythroid-specific cell surface marker.     

A monoclonal antibody which identifies an antigen in endothelial cell and epithelial basement membrane.

We isolated a monoclonal antibody which decorates the endothelial cells of normal and lymphatic vessels in formalin-fixed, paraffin-embedded tissue. In addition, the antibody recognizes a previously undescribed substance found in the basement membrane zone and subbasement membrane zone of a variety of epithelial. By ELISA assay, the antigen recognized by this monoclonal antibody is not laminin, type IV collagen or fibronectin. This antibody may be used as a diagnostic tool because it identifies an epitope in neoplasms differentiating towards endothelial cell such as angiosarcoma and Kaposi's sarcoma.     

The murine monoclonal antibody, 14A2.H1, identifies a novel platelet surface antigen

A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (10(3)/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on Fc gamma RII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.     

Identification of neurophysin immunoreactivity in hypothalamus by a monoclonal antibody to a carcinoma cell surface antigen

Mouse monoclonal antibody (mAb) L6 identifies an antigen expressed on the cell surface of many different human carcinomas. While studying the binding activity of mAb L6 to intracerebral tumor xenografts of human lung carcinoma LX-1 cells in nude rats using immunohistological techniques, we observed that L6 can also bind to a cytoplasmic antigen expressed in the magnocellular component of the hypothalamo-neurohypophysial system. Double-labeling experiments with antisera to vasopressin and oxytocin confirmed the localization of L6 immunoreactivity within both peptide-containing cell groups. L6 immunoreactivity in Brattleboro rats (with genetic deletion in the vasopressin gene) was exclusively localized within oxytocin neurons. Oxytocin and vasopressin failed to block L6 staining which suggested that its target epitope resides within the neurophysin sequence, and this explanation was supported by the finding that adsorption of L6 with porcine neurophysin completely eliminated hypothalamic immunoreactivity. Western blot analysis of bovine neurophysin and human pituitary extracts identified L6-immunoreactive bands which corresponded to the position of neurophysin and pro-pressophysin, confirming that L6 immunoreactivity in hypothalamus is related to neurophysin. Thus, monoclonal antibody L6, which is highly reactive with a membrane antigen of human lung cancer cell line LX-1, recognizes a cytoplasmic epitope in hypothalamic neurons identified as neurophysin by immunohistochemistry and Western analysis.     

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.     

An analysis of islet cell surface antigen defined by monoclonal islet cell surface antibody 5C12

Monoclonal antibody 3A4 to islet cell surface antigen has been previously established in our laboratory, using hybridization of spleen lymphocytes from non-obese diabetic (NOD) mice transferred into immunologically incompetent recipient mice. In the present study, monoclonal islet cell surface antibody 5C12 could be newly obtained in the 10:1 ratio of NOD mice spleen cells and mouse myeloma cells (SP2/0) without any modifications. Protein A radioligand assay and indirect immunofluorescence on living cells showed that 5C12 antibody reacted to normal rat islet cells and cultured rat insulinoma cells (RIN-r), but not to cultured lymphocytes (Bri-7, IM-9) and Chang-liver cells. Analysis of 125I-labeled antibody binding revealed that unlabeled 5C12 effectively inhibited subsequent 125I-5C12 binding to RIN-r cells, whereas unlabeled 3A4 did not. The scatchard plot from these data showed the curvilinearity, and about 150,000 binding sites to antibody per RIN-r cell were counted. The treatment of RIN-r cells with papain and neuraminidase reduced the binding of 5C12 to RIN-r cells, whereas the effect of trypsin was not observed. Immunoprecipitation of 125I-labeled insulinoma cell lysates followed by SDS-PAGE and autoradiography indicated that 5C12 recognized 105K dalton cell surface protein in RIN-r cells. Immunoblotting also showed that 5C12 antibody recognized 105K dalton cell surface protein in RIN-r cells. These results demonstrated that 5C12 was an important tool for clarifying the immunoresponse against certain antigenic determinants on pancreatic B cells. Furthermore, 5C12 has not only qualitatively and quantitatively improved diagnostic methodology, but it may also provide new reagents useful to the treatment and prevention of type 1 diabetes.     

A monoclonal antibody recognizes a 39 kDa protein expressed in atretic granulosa cells

A major problem in ovarian physiology is the lack of conveniently quantifiable markers of atresia. Towards this end, we identified a monoclonal antibody (anti-OA-2) that selectively recognizes granulosa cells in atretic follicles. When cryostat sections of rat ovaries were incubated with anti-OA-2, granulosa cells in atretic follicles showed intense immunofluorescent labeling. In contrast, no anti-OA-2 immunoreactivity was observed in the granulosa of the healthy follicles. The amount of anti-OA-2 binding was significantly enhanced when atresia was stimulated by treatment with human chorionic gonadotropin, testosterone, or estrogen withdrawal. The results of immunoprecipitation and Western blot analyses indicated that the OA-2 antigen is a 39 kDa protein which is actively synthesized by the granulosa during atresia. The 39 kDa protein is localized at or near the inner surface of the plasma membrane. We conclude that the anti-OA-2 monoclonal will prove useful as a convenient analytical tool to study the regulation of granulosa atresia.     

Apo2.7抗体标记法原位检测细胞凋亡

探讨利用免疫细胞化学方法代替流式细胞仪原位检测细胞凋亡的可行性。 1.材料:(1)细胞株:HepG2细胞系高度分化的肝癌细胞株,生物学特性与正常肝细胞相近,用于建立体外肝细胞凋亡的模型。(2)试剂:鼠抗人Apo2.7抗体:购自美国Coulter公司。异硫氰酸荧光素(FITC)标记的羊抗鼠IgG:购自北京中山生物技术有限公司。免疫组织化学试剂盒:购自福州迈新生物公司。 2.方法:(1)乙醇诱导HepG2细胞凋亡模型的建立:HepG2     

Identification and characterization of a monoclonal antibody to an antigen expressed on activated macrophages.

A hybridoma clone secreting a monoclonal antibody, designated MA158.2, that reacts with an antigen expressed on lymphokine-treated macrophages was produced by fusion of mouse myeloma cells with rat spleen cells immunized against C57BL/6 peritoneal macrophages rendered tumoricidal in vitro by incubation with the lymphokine macrophage-activating factor. The specificity of the antibody for activated macrophages and lack of reactivity with histologically diverse cell types was determined by radioimmune indirect binding and flow cytometry. MA158.2 antibody binds to mouse peritoneal macrophages elicited by nonspecific inflammatory agents and to tumoricidal macrophages elicited with Corynebacterium parvum. Resident peritoneal, splenic, and alveolar macrophages were only weakly positive. Several macrophage cell lines (P388D1, WEH1-231, J774, RAW 264.7), murine fibroblasts, and neutrophils did not bind detectable amounts of MA158.2. Radioimmune indirect binding analysis demonstrated that cell suspensions prepared from C57BL/6 mouse spleen, thymus, and lymph node as well as polymorphonuclear leukocytes, lymphocytes, and T- and B-cell murine lymphomas were MA158.2 negative. Expression of the reactive antigen on the macrophage cell surface was enhanced 3-fold following in vitro activation of elicited macrophages with macrophage-activating factor and the kinetics of activation to the tumoricidal state paralleled the increased expression of the antigen recognized by MA158.2. MA158.2 is a rat IgG2a antibody containing a single specific heavy and light chain that does not detect a polymorphic determinant. This monoclonal antibody will be a useful tool for monitoring the efficacy of agents in activating murine macrophages to the tumoricidal state and in analyzing the sequence of biochemical events that culminate in macrophage activation.     

Identification of a surface antigen on Theileria parva sporozoites by monoclonal antibody.

A mouse monoclonal antibody (mAbD1) that neutralizes sporozoites of different stocks of the protozoan parasite Theileria parva has been used to localize and identify a sporozoite antigen. Protein A-colloidal gold was used to localize bound mAbD1 in immunoelectron microscopic studies. mAbD1 bound to sporozoite antigen, which was evenly spread over the surface of all sporozoites. Immune complexes were obtained by incubation of sporozoite suspensions with mAbD1 followed by Zwittergent 3-14 extraction and precipitation with protein A-Sepharose. One- and two-dimensional NaDodSO4/polyacrylamide gel electrophoretic analyses were performed on these complexes, and a major protein with a molecular size of 68 kDa was identified. Other related components of 52 kDa, 47 kDa, and 28 kDa were also detected. Since antibody to this antigen(s) neutralizes T. parva sporozoites from different stocks, the results could be of relevance to the development of a broad spectrum vaccine against the cattle disease East Coast fever, which is caused by T. parva.     

Platelet activation induced by a murine monoclonal antibody directed against a novel tetra-span antigen

MAb 14A2.H1 identifies a novel low-abundance platelet surface antigen, PETA-3, which is a member of the tetra-span (TM4) family. This MAb brings about platelet aggregation and mediator release, which is completely inhibitable by prostaglandin E₁, and partially inhibitable by aspirin and ketanserin. Platelet activation by MAb 14A2.H1 is dependent on interaction with both the platelet Fc receptor, FcγRII, and the specific antigen as it was prevented by either a blocking MAb to FcγRII (IV.3) or F(ab')₂ fragments of 14A2.H1. The extent of platelet activation by the antibody varied considerably between donors, and is believed to reflect the polymorphism of FcγRII. Subaggregating concentrations of 14A2.H1 synergized with other platelet agonists, ADP, adrenaline, collagen and serotonin, indicating signalling via a pathway distinct from these activators. Synergy was also blocked by MAb rv.3, or F(ab')₂ fragments of 14A2.H1. The similar low copy number of PETA-3 and FcγRII in the platelet membrane (approximately 1000/platelet). together with the dependence on FcγRII for activation by MAb 14A2.H1, suggests that PETA-3 may be a component of the FcγRII signal transducing complex in platelets.