Two monoclonal antibodies recognizing determinants on human embryonal carcinoma cells react specifically with the liver isozyme of human alkaline phosphatase

From a series of hybridomas that produced monoclonal antibodies reactive with the surface of human embryonal carcinoma cells, two that specifically recognized determinants of the liver/bone/kidney isozyme of alkaline phosphatase were isolated. They did not cross-react with the intestinal or placental isozymes. Phylogenetic studies revealed that both antibodies cross-reacted strongly with liver alkaline phosphatase from higher primates, but exhibited marked differences in their respective cross-reactions with liver alkaline phosphatase from other mammalian species.     

The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum.

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of greater than 200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, kappa), the other by clone 12C11 (IgM, lambda). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.     

Recognition of a high molecular weight glycoprotein (HMW-GP) associated with rat colon cancer cells by rat monoclonal antibodies

In this report we describe an auto-immunogenic tumor associated high molecular weight glycoprotein (HMW-GP) present on most rat colon carcinomas as detected by syngeneic monoclonal IgM antibodies. The HMW-GP has an apparent molecular weight of more than 10(6) D and carries several epitopes for various lectins, carbohydrate specific mouse monoclonal antibodies and four rat syngeneic monoclonal antibodies. The epitope defined by the 10B 12 rat monoclonal antibody is present in colon carcinoma tissue, but only in very low amounts in normal gastro-intestinal-tissue. The epitope is present at multiple sites on the large molecule and is shown to be sterically related, but not identical to determinants for Dolichos biflorus lectin and a mouse mAB binding to blood group A. The 10B 12 mAB binding is insensitive to boiling, reduction with mercaptoethanol and treatment with trypsin, but abolished by pretreatment of the antigen with sodium periodate or neuraminidase. The epitopes defined by 1 G6 and 1F6 rat monoclonal antibodies are present both in tumor tissue and in normal colon mucosa and the antibody binding is increased after treatment with both neuraminidase and sodium periodate.     

Characterisation of renal antigens on distinct parts of the human nephron by monoclonal antibodies

Summary Ten monoclonal antibodies (TN 1-TN 10) directed against different renal antigens of distinct sites of the human nephron were derived from a fusion between P3-NS1/1-Ag4-1 mouse myeloma and spleen cells of a mouse hyperimmunized against isolated human kidney cells. Two of these reagents (TN 1, TN 10) were shown by immunoperoxidase labelling on frozen sections of five normal kidneys and of other selected human organs, as well as by immunofluorescence studies on normal peripheral blood cells and selected lymphohematopoietic cell lines, to detect antigens exclusively expressed on visceral glomerular or proximal tubular epithelial cells. The other eight antibodies were found to react with different determinants shared between renal structures, muscle cells, different epithelia, B-lymphocytes and granulocytes. In heterogeneous cultures of isolated kidney cells these monoclonal reagents could be used to identify distinct cell types of tubular origin. Thus such hybridoma-derived antibodies provide new tools to correlate structural characteristics of various renal epithelial cells with their functional properties and will contribute to the study of their influence on immunologically mediated kidney injuries in different forms of glomerulonephritis in man.This study was supported by two grants from the Deutsche Forschungsgemeinschaft, DFG, Mu 523/3-1 and SFB 120, Leukämieforschung und Immungenetik, Project A2 and A3     

Down-regulation of surface antigens recognized by systemic lupus erythematosus antibodies on embryonal cells following differentiation and exposure to corticosteroids

We have previously suggested that anti-DNA antibodies present in systemic lupus erythematosus patients can bind directly to tissues as a result of cross-reactivity with embryonal tissue-based antigens. Here we have analyzed the interaction between polyclonal and monoclonal mouse and human lupus autoantibodies and an embryonal cell line. We report that a murine embryonal stem cell line (ES) expresses a surface antigen which is recognized by mouse and human lupus autoantibodies. This surface antigen is down-regulated following maturation of the cells or incubation with corticosteroids. Adhesion molecules may serve as the target membrane antigen in ES cells since preincubation with these antibodies decreases the ability of ES cells to adhere to the plate.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRLHMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Five antigens on human T cells detected by mouse monoclonal antibodies

Five antigen systems were defined by the monoclonal antibodies (MoAb) produced against mature T cells. The antigens recognized were grouped into two categories based on the antigen distribution on T cells. (a) Tp 120 [mol. wt 120 kilodaltons (120kD)] and Tp40 (40kD), these are on most peripheral T cells, but not on any other cell lineages, i.e. pan-T antigen. (b) Ts32 (32kD), Ts145 (145kD) and TsA (not determined), these antigens are present only on certain populations of peripheral T cells, i.e., T subset antigen. Among these five, Ts145 and TsA are probably novel T cell antigens. Cell surface phenotypes of leukaemias and lymphomas were typed with these MoAb. Ia like antigen negative, null cell type acute lymphocytic leukaemia (Ia- null ALL) are Tp40+, suggesting that this type of ALL belongs to a T cell lineage. T cell ALL (T-ALL) and lymphoblastic lymphoma (LL) were both Tp40+, Ts32+, TsA+ and a half of the cases were Tp120+, but the expression of Tp40 was stronger on LL cells. Mature T cell (T2) lymphoma and adult T cell leukaemia (ATL) were Tp120+, TsA+, while Tp40 was weakly expressed on only one third of the cases. These MoAb were found to be useful to estimate the origin of various T cell malignancies.     

Human myeloid differentiation antigens identified by monoclonal antibodies: expression on leukemic cells

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.     

Localization of distinct surface antigens on Chlamydia trachomatis HAR-13 by immune electron microscopy with monoclonal antibodies.

Monoclonal antibodies were produced against the HAR-13 strain (A) of Chlamydia trachomatis. By an indirect immunofluorescence technique, three clones (1-7, 2-8, 9F) were found to produce antibody that reacted only with serotype A strains. Clone 3-5 produced antibody that cross-reacted with serotypes A, C, H, I, and J. Using an indirect immunoferritin technique, we examined the binding of these antibodies to strain HAR-13 by electron microscopy. Antibodies from all four clones were shown to bind to the outer membrane surface of both reticulate and elementary bodies. Two distinct binding patterns were demonstrated. Antibodies from clones 1-7 and 9F bound to the outer membrane surface in a homogeneous pattern, whereas antibodies 2-8 and 3-5 bound to the outer membrane surface in an irregular, patchy distribution. There was a direct correlation between the distribution of antigen on the outer membrane surface and neutralization of C. trachomatis HAR-13 infectivity in vitro. Neutralization of HAR-13 infectivity by antibodies 1-7 and 9F was complement dependent, whereas antibodies 2-8 and 3-5 did not neutralize under any conditions.     

A glycoprotein of molecular weight 85,000 on human cells of B-lineage: detection with a family of monoclonal antibodies

Immunization of BALB/c mice with glycoproteins purified from a detergent extract of human chronic lymphocytic leukemia (CLL) cells by affinity to Lens culinaris lectin led to the production of several monoclonal antibodies with similar reactivity. One of the antibodies, 50B4, was purified and the corresponding antigen was isolated from a B-lymphoblastoid cell line extract by affinity chromatography to the 50B4-IgG immunoadsorbent. Co-purification of the antigenic activities associated with five other monoclonal antibodies was achieved. Purified and radiolabelled 50B4 antigen could be specifically immunoprecipitated not only by 50B4 but also by the other five antibodies. SDS-PAGE analysis revealed that all antibodies precipitated the same component, a polypeptide chain of apparent mol. wt 85,000 under reducing conditions. Competitive-binding studies between the purified antibodies indicated the presence of two distinct epitopes on the antigen. The epitopes, each recognized by three different antibodies, were equally accessible on the cell surface of either a B-CLL (3 X 10(5) molecules/cell), a B-lymphoblastoid cell line (11 X 10(5) molecules/cell) or two acute lymphocytic leukemia (ALL) cell lines of pre-B phenotype (5 X 10(5) and 0.8 X 10(5) molecules/cell respectively). Although the antigens purified from the strongly positive ALL cell line gave a gel pattern identical to that of the B-lymphoblastoid cell line, the antigens purified from the B-CLL extract were resolved into two distinct glycosylated polypeptides of mol. wts 85,000 and 77,000 under reducing conditions. The distribution of the antigen(s) is not restricted to cells of the B-lineage as mature T-cells and a variety of non-hematopoietic cell types express both epitopes of the antigen(s).     

DR antigens on melanoma cells: analysis with monoclonal antibodies

Two monoclonal antibodies (691-13-17 and 37-7) can precipitate DR antigens from radioiodinated, detergent solubilized melanoma cells. However, after complete depletion of lysates with one antibody (691-13-17) alpha-like chains can be still be precipitated from some melanoma cells by the other antibody (37-7). Antibody 37-7 also precipitates an additional distinct antigen from SK-MEL 37 but not from any five other melanoma cell lines.     

Characterization of two distinct antigens expressed on either resting or activated human B cells as defined by monoclonal antibodies.

Two antigen systems (L29 & L30) expressed on two distinct human B cell subpopulations were identified by using BL1-4D6 and TB3-7D5 monoclonal antibodies, respectively. L29 was expressed on approximately one-third of B cells in human lymphoid tissues. These B cells associated with L29 were large activated B cells located in the germinal centres of lymphoid follicles. L30, on the other hand, existed on approximately two-thirds of B cells mainly located in the mantle zone of lymphoid follicles, most of which also expressed IgM and IgD on their cell membrane. In addition, L30 was shared on mature granulocytes. With the use of polyclonal activators such as pokeweek mitogen (PWM) and protein A-bearing staphylococci (SAC), L29 antigen was inducible on PWM- or SAC-stimulated B cells in correspondence with the emergence of Tac and T10 antigens of these B cells. In contrast, L30 antigen on the B cells stimulated by the polyclonal activators was decreased in its expression and was finally lost from these B cells. Although none of L29 and L30 was expressed on normal, non-activated human thymus and peripheral T cells, L29 but not L30 was expressed on concanavalin A-activated T cells. Immunochemical studies showed that L30 consist of a single polypeptide with mol. wt of 40,000. L29 antigen is presently under study.     

Internalization and processing of antibodies to surface antigens on human B cells. Monoclonal anti-IgM antibodies are processed differently than monoclonal antibodies towards non-Ig surface receptors

The internalization and intracellular processing of monoclonal antibody to immunoglobulin mu heavy chain (Mamu) have been investigated in two human Burkitt lymphoma cell lines (Ramos and Raji), in a human B cell lymphoma and in normal human peripheral blood B cells. In addition to the degradation of 125I-labeled Mamu to trichloroacetic acid (TCA) soluble material, a distinct pattern of larger 125I-Mamu fragments was detected in all sources of B cells tested. The particular fragmentation pattern, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, involved the cleavage of both peptide bonds and disulfide bridges. This type of antibody fragmentation appeared to be a selective mechanism associated with sIgM, as no other degradation than that leading to TCA-soluble material could be detected after the internalization and degradation of radiolabeled monoclonal antibodies towards a variety of non-Ig B cell surface receptors. Three fragments of 125I-Mamu degradation were also detected in the supernatant of Ramos cells, implying that the recycling and exocytosis of certain 125I-Mamu fragments also took place.     

Two distinct antigen systems in human B lymphocytes: identification of cell surface and intracellular antigens using monoclonal antibodies.

Two distinct antigen systems (L26 and L27) specifically expressed in human B lymphocytes were identified using TB2-2B3 (2B3) and T3-5B3 (5B3) monoclonal antibodies, respectively. Whereas L26 antigen defined by 2B3 were rarely expressed on the surface of B cells but abundant in the cytoplasm, 127 antigens detected by 5B3 was clearly expressed on the cell surface. These two antigens appeared to be restricted in their expression to B cells, as they were found in most B cells but not other cell types including thymocytes, T cells, monocytes and granulocytes. Functional studies demonstrated that L27 was more easily lost from B cells after activation with pokeweed mitogen than was L26. Likewise, plasma cell myeloma, as well as normal plasma cells, was devoid of both L26 and L27, whereas immunoblastic sarcoma of B cell type expressed L26 but not L27. These two antigens co-existed in the same B cell lines including Epstein-Barr virus transformed B cell lines, B cell type acute lymphatic leukaemia (B-ALL) cell line, Burkitt's lymphoma cell lines and myeloma cell lines, but pre-B and common ALL cell lines were entirely negative for both L26 and L27. Immunoprecipitation studies showed that L26 consisted of at least two polypeptide chains with molecular weights of 30K and 33K daltons, which were clearly distinct from HLA-DR antigens. The antigen L27 is presently under study.     

Human monoclonal antibodies reactive with cell surface antigens on human leukemia cell lines: many antibodies are (auto)antibodies

Primary Epstein Barr Virus (EBV) transformants from peripheral blood mononuclear cells (PBM) established in macrocultures were screened for the secretion of antibodies reactive with cell surface antigens on one or another of two indicator human leukemic cell lines and fused with the HMMA2.11TG/O human fusion partner. Human monoclonal antibodies (HuMAbs) were readily obtained. Relative oligoclonality of the primary EBV macrocultures was documented by the number of antibody secreting hybridomas (1-100%). The method permitted preselection for fusion of transformants producing antibodies of certain specificities and/or class. Fourteen HuMAbs, primarily of the IgM class, have been obtained. Those IgM HuMAbs obtained from patients with active diseases, e.g. Acute Lymphoblastic Leukemia (3 HuMAbs), and HIV infection (4 HuMAbs), were found to have a relatively broad spectrum of reactivity with cell lines of various hematopoietic lineages and normal cells, although several show selective reactivity with T cell lineage tumors or a selected population of cells. HuMAbs from normal donors of both the IgG and IgM class were obtained. The IgM HuMAbs from one volunteer reacted primarily with autologous and allogeneic macrophages (autologous PBM from the other patients were not available) as well as a diverse number of hematopoietic cell lines. From others, the IgG HuMAbs demonstrated a more restricted spectrum of reactivity, while the IgM HuMAbs reacted with both autologous and allogeneic normal cells. Thus, the B cell repertoire contains cells capable of secreting cell surface reactive antibodies and many of these antibodies express characteristics of autoantibodies. Those that did not react with autologous or allogeneic PBM may react with other autoantigens which have been expressed on the malignant cells used as screening targets or may represent true antitumor antibodies.     

Generation of monoclonal antibodies to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins.

We have expressed human CD40 and human B7 in insect cells using the baculovirus expression system and have used these insect cells to immunize mice for the generation of monoclonal antibodies. We demonstrate here that specific monoclonal antibodies to human CD40 and human B7 were obtained using this approach. One significant advantage of this method is that immunizing mice with insect cells did not evoke an immune response to human cells and, therefore, EBV-transformed human B cells could be used to screen for specific antibody production by the hybridoma clones.     

A rapid method for determining whether monoclonal antibodies react with the same or different antigens on the cell surface

A quick and simple method for determining whether monoclonal antibodies react with the same or different antigens is described which utilises an indirect radioactive binding assay against cells. Six antibodies were selected, BK19.45, BK20.20, GenOx4.17, GenOx4.21, W6-34 and W6-46, which detected antigens present either only on leukocytes (BK19.45 and BK20.20) or on a wider range of cell types including fibroblasts, liver cells and neuroblastoma cells. The saturation binding levels for each antibody, in terms of the amount of ¹²⁵I-anti-mouse immunoglobulin bound, were determined with respect to a fixed number of cells. The addition of two antibodies with different specities (BK19.45 or BK20.20 to either W6-34 or W6-46) resulted in a higher plateau value of ¹²⁵I-anti-mouse immunoglobulin bound per fixed number of cells than for either antibody singly. The increase in the amount of antibody bound is equivalent to the sum of the two individual saturation binding levels. In contrast, the addition of BK20.20 to BK19.45 or W6-45 produced no detectable rise in the saturation level. From these data it is concluded that BK20.20 and BK19.45, and W6-34 and W6-46 are bound to either identical epitopes which are in very close spatial relationship. On the other hand 19.45 and W6-34, as expected, detect unrelated antigens. Observations from autoradiograph binding studies on the semi-quantitative distribution on bone marrow cells of the antigens recognised by 19.45 and 20.20 supported the conclusion that the antibodies were recognising identical antigens.A study of the antibodies to HLA (2A1) and β₂ microglobulin (M3) showed an increase in the saturation level, when both antibodies were added together, which was less than the sum of the two individual saturation binding levels. The close association of β₂ microglobulin and HLA on the cell surface may to some extent prevent independent antibody binding. These data suggest that the above approach will differentiate between monoclonal antibodies which detect antigenic determinants that are located on closely associated surface molecules.