Identification of cytosolic antigens from GW-39 adenocarcinoma cells by crossed immunoelectrophoresis and immunofluorescence

Rabbits were immunized with a cytosolic fraction prepared from human adenocarcinoma cells of the colon (GW-39). The antibodies obtained were analyzed using crossed immunoelectrophoresis and were found to precipitate 23 distinct antigens in the cytosolic fraction from colon tumor cells. After preabsorption with human serum and plasma as well as acetone powders prepared from 2 normal human tissues and 4 normal hamster tissues, 1 major immunodominant cytosol antigen (CA-3) and two less intense immunoprecipitin peaks (CA-1 and CA-5) remained detectable by the crossed immunoelectrophoretic method. The preabsorptions with normal tissues were sufficiently complete to remove anti-carcinoembryonic antigen antibodies and showed that antigens CA-1, CA-3 and CA-5 are immunologically distinct from carcinoembryonic antigen. Indirect immunofluorescence localization studies with preabsorbed anti-cytosol antibodies and FITC-conjugated second antibody showed that these antigens were expressed in the cytoplasm and at the cell surface in several human colon tumor cell lines, their subclones and in primary colon tumor specimens.     

Human-human hybridoma producing monoclonal antibodies against colorectal cancer-associated antigens

Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B-lymphoblastoid cell lines, LICR-LON-HMy-2 (HMy-2) and WI-L2-729-HF2 (729-HF2), to generate hybridomas synthesizing antibodies reacting with tumor-associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been further analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9165 and D4213) showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (F11348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G4146 to two components from colon cancer cells with Mr of 59 K and 61 K, while B9165 bound to a 43 K component and F11348 to several components with Mr from 30 to 200K. D4213 showed no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens.     

A New Approach for the Immunogenic Presentation of Membrane-Bound Human Colon Tumor Antigens

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.     

A new approach for the immunogenic presentation of membrane-bound human colon tumor antigens

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.     

Production and characterization of monoclonal antibody to a 60-kD glycoprotein in ovarian carcinoma

Monoclonal IgG1 antibodies 2C8 and 2F7, derived by immunization of mice with a glycoprotein-enriched fraction of human ovarian adenocarcinoma, recognized a 60 kD glycoprotein in the ovarian tumor but not in normal ovary. Survey of other normal adult tissues by an indirect solid-phase radioimmunoassay (RIA) revealed the presence of the antigen in trace amounts in various normal organs such as small intestine, liver colon and urinary bladder, except in lung where its concentration was as high as in tumors. Among fetal tissues tested, intestine and placenta had the highest activities. By RIA, about 50% of ovarian and colonic tumors had elevated levels of the antigen. All ovarian cyst fluids, both benign as well as malignant, also contained a high level of the antigen. Immunodepletion studies indicated that the antigen was distinct from carcinoembryonic antigen and the ovarian cancer antigens described in our laboratory with other monoclonal antibodies. The antigen bound to Con A-Sepharose and was eluted with 2% alpha-D-mannoside, was soluble in 0.6 M perchloric acid and stable at 100 degrees C for 30 min. The antigenic activity in isolated plasma membrane enriched fractions of ovarian adenocarcinomas was sensitive to trypsin, chymotrypsin or protease treatment but unaffected by neuraminidase, beta-galactosidase, periodate or methanol treatment. By immunoperoxidase staining, the antigen was localized in a variety of human tumors showing widespread distribution.     

抗人肝癌单克隆抗体HL2的制备及鉴定

用肝癌细胞株QGY-7703、BEL-7402、SMMC-7721以贯序法免疫BALB/c小鼠,获得一株分泌肝癌单抗的杂交窟株HL_2,其染色体数目为97—105。单抗HL_2系IgG_2b亚类。吸收实验及阻断实验证明HL_2抗原与CEA、AFP、HBsAg、HBcAg及HBeAg无免疫同源性。ABC法和ELISA法说明单抗HL_2与四株肝癌细胞、六例肝癌组织均呈明确阳性反应,除与SGC-7901、H_(128)、K_(562)、Hela细胞株及部分胚胎肝脏,胚胎结肠有较弱性反应外,与肝癌旁组织、正常肝脏、其它肿瘤组织和细胞株、二种正常人细胞及其它胚胎组织无反应。显示了单抗HL_2的特异性较好、阳性率较高,是一个有应用前景的单抗。     

Mast cell and neuroendocrine cytoplasmic autoantigen(s) detected by monoclonal pANCA antibodies

pANCA is a marker antibody expressed in most patients with ulcerative colitis, and its cognate antigen is potentially an immunologic target in this disease. This study evaluates whether pANCA detects an autoantigen that is expressed in the colonic mucosa. Immunohistochemistry of colon specimens with human pANCA monoclonal antibodies (Fab 5-2 and 5-3) revealed a minor population of immunoreactive mucosal cells bearing a cytoplasmic vesicle antigen. By distribution, morphology, and tryptase expression, these were identified as mast cells. Immunofluorescent analysis revealed similar immunoreactivity of mouse mast cell lines and human KU812. Western analysis of mouse mast cell lines revealed immunoreactive proteins, and these were distinct from previously proposed pANCA antigens (histone H1, HMG 1 and 2, and neutrophil vesicle antigens). Cognate antigen for Fab 5-2 and 5-3 was also expressed in other tissue mast cells, cerebellar neurons, and pancreatic islet cells. These findings identify a novel cytoplasmic autoantigen(s) associated with UC by its presence in colonic mucosa and recognition by a disease-associated marker antibody.     

The i antigen complex: A new specificity unique to dividing human cells

Evidence is presented that erythroagglutinin, anti-i, sera recognize a membrane antigen characteristic of dividing human cells. The antigen was present on fetal tissues, cultured cell lines and mitogen-stimulated lymphocytes and absent from normal adult tissues. Cell synchrony studies with cultured lymphoblasts indicate that antigen expression is maximal during the S and G₂ phases of the cell cycle. Absorption studies have shown that antibodies to this determinant are distinct from the erythroagglutinin activity of sera. The significance of “division antigens” in relation to oncofetal antigens is discussed.     

A murine monoclonal antibody raised against human non-small cell carcinoma of the lung

Murine monoclonal antibodies (MAbs) reactive with human non-small cell carcinoma of the lung (NSCCL) were produced following immunization with a membrane preparation of Calu-3, a human adenocarcinoma of the lung cell line grown in nude mice. Positive hybrids unreactive with normal liver membranes and human peripheral white blood cells were selected for further testing. One MAb (RS5-4H6) recognized an antigen expressed in a variety of lung and other carcinoma cell lines as detected by flow cytometry. Immunohistology showed a selectivity for normal and neoplastic lung epithelium, as well as other cancers. The antigen was detected by immunohistology in 87% of tumor specimens derived from the lung, breast, colon, kidney, and ovary. Most other normal human tissues were not stained but occasional cells of the stomach, salivary and other glands, as well as kidney tubules were reactive. This MAb is an IgG1. Western blot analysis indicated that the antibody reacts selectively with an antigen greater than 300 kD. The antigen is resistant to neuraminidase treatment and periodate oxidation, but sensitive to pronase treatment, suggesting that the epitope is peptide in nature. This antibody may be potentially useful as a targeting agent for radioimmunodetection and immunoconjugate therapy.     

Mouse monoclonal antibodies with restricted specificity for human renal cell carcinoma and ability to modulate the tumor cell growth in vitro

Three mouse monoclonal antibodies (MAbs), KRC-1, -2 and -3(IgG1) were produced against cultured cells of human renal cell carcinoma (RCC) cell lines. All of them had restricted specificity for subset of RCC, but did not react with other tumor cell lines, tumor and normal tissues including kidney. The effect of these MAbs on the tumor cell growth in vitro was examined both in soft agar and in plate culture systems. All of them showed modulator effect on the tumor cell growth: KRC-1 showed stimulation effect and others, KRC-2 and -3, suppression one in the both systems. The specificity was observed in the modulator effects. KRC-1 detected a 135 kd glycoprotein in immunoblotting. KRC-2 detected a high molecular weight antigen (ca. 1200 kd) in gel filtration. All the antigens showed affinity for Con A lectin. Molecular properties of the antigens appeared to be different from those of previously reported RCC-associated antigens. The three MAbs seem to be useful to study mechanism of growth regulation in transformed cells.     

Monoclonal antibody localization of Lewis antigens in fixed tissue

Monoclonal antibodies that bind specifically with Lewisa (Lea) and Lewisb (Leb) antigens were used in an immunoperoxidase assay to characterize Lewis (Le) antigenic profile of a variety of fixed tissues. Representative sections of normal and malignant tissue from the stomach, colon, pancreas, and kidney were examined and compared. Lea antigen was expressed more often in gastric adenocarcinomas (80%) than normal gastric mucosa (40%). In gastric tumors with concomitant expression of Le antigens, larger areas within an individual tumor expressed Lea antigen rather than Leb antigen. Expression of Leb antigen in normal colonic tissue was seen only in the proximal colon and not distal colon; colon carcinomas, on the other hand, expressed Leb antigen (64%) regardless of where the primary arose. Leb antigen was expressed in large pancreatic ducts (three of three) more often than Lea antigen (one of three); exclusive expression of Lea antigen was demonstrated in the proximal convoluted tubules of normal kidney (three of three) and in renal cell carcinomas (six of eight).     

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method

This report describes a method for the production of murine monoclonal antibodies (MAbs) against cellular antigens preserved during formol fixation and paraffin embedding of human tissues in an attempt to select markers that would be useful in immunopathology. Hybridomas were prepared using spleen cells from mice immunized with cell suspensions obtained from formalin-fixed paraffin block sections of a human breast carcinoma. A monoclonal antibody 83 D4 was selected, which was reactive with paraffin embedded breast carcinoma tissues, but not with normal breast. The reactive antigen has a high molecular weight (400-1000 kD) and was detected on the cell surface of live human breast cancer cell lines and on frozen tissues sections. These results demonstrate that the MAb 83 D4 identifies a native breast tumor associated epitope conserved during tissue fixation and embedding and could be used as an immunohistochemical marker.     

Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells

Two monoclonal antibodies (TRA-1-60 and TRA-1-81) recognizing distinct cell surface antigens on human embryonal carcinoma (EC) cells were produced and characterized. These antibodies reacted strongly with undifferentiated human EC cells in indirect radioimmunoassays (RIA) and immunofluorescence (IF) assays, but only weakly or not at all with cells derived from pluripotent EC cells differentiating in vitro or in xenograft tumors, nor with other germ cell tumor cell lines that did not also express the typical features of human EC cells. They did not react with murine teratocarcinoma cell lines. A survey of other human tumor cell lines and normal human tissues disclosed that molecules recognized by these antibodies are not confined to human EC cells but that cross-reacting epitopes appear on several neoplastic and normal tissues, although in a different anatomical pattern for each antibody. Both antibodies immunoprecipitated a major polypeptide (apparent molecular weight approximately 240,000) and a minor polypeptide (apparent molecular weight approximately 415,000) from lysates of 125I surface-labeled human EC cells, in this respect resembling another monoclonal antibody, 8-7D, previously described by Blaineau et al. (1,2) However, sequential immunoprecipitation revealed that each of the three antibodies reacted with different molecules of slightly different molecular weights. The epitopes defined by the present antibodies differ from those recognized by the other human EC cell-specific monoclonal antibodies that have been described and provide new markers for studying the differentiation of pluripotent human EC cells.     

Antiseea to Carcinoembryonic Antigen: Removal of Persistant Impurities (1)

Antisera to carcinoembryonic antigen preparations frequently contained antibodies to a distinct normal serum protein(s). The latter antibodies persisted following absorptions with cross-linked human plasma proteins, normal colon, and Group 0 red cells. A simple method for removal of the contaminating antibodies involved absorptions with the perchloric acid-soluble fraction of normal serum. Excesses of the absorbant and its soluble immune complexes were then removed on anion exchange columns.     

Clonal expression of differentiation and Ia-like antigens on alloreactive human T lymphocytes

A series of human T lymphocyte clones showing specific responses for major histocompatibility complex-encoded alloantigens were isolated from a single culture. They were classified into distinct functional groups based on measurement of alloantigen-specific proliferation and cytotoxicity. Surface antigen expression on these clones was analyzed using a panel of monoclonal antibodies specific for T cell differentiation antigens and for Ia-like antigens. Four distinct groups were identified based on the Leu series of differentiation antigens: (a) Leu1+,2+,3+,4+; (b) Leu1+,3+,4+; (c) Leu1+,2+,4+; and (d) Leu2+,4+. Functionally distinct clones showed not only differential expression of Leu2 and Leu3 , antigens which had previously been shown to distinguish helper/inducer and cytotoxic/suppressor cells, but also of Leu1, an antigen classified as a common T cell marker. All of the clones were found to express Ia antigens as detected by three framework monoclonal antibodies, but none were found to express a determinant associated with the DR3 allospecificity , although it was found on normal B lymphocytes and a B cell line established from the HLA-DR3 individual from whom these clones were derived.     

Immunologic marker analysis of normal and malignant histiocytes. A comparative study of monoclonal antibodies for diagnostic purposes

The authors investigated the phenotype of "monocyte-derived histiocytes/macrophages" on frozen sections of various human tissues, in 12 histiocytic tumors, and in 15 large cell non-Hodgkin's lymphomas. The monoclonal antibodies (MAbs) considered specifically directed against antigens associated with monocytes/histiocytes included the following: Leu-M1, Leu-M3, Leu-M5, My4, My7, My8, My9, anti-Monocyte 1, anti-Monocyte 2, RFD-7, RFD-9, OKM1, and FMC17. The histiocytes in normal tissues and the tumor cells of the histiocytic malignancies expressed these antigens in various degrees. They were not reactive with MAbs specific for lymphocytes, myeloid cells, or Reed-Sternberg cells (Ki-1 antigen). Out of these 13 MAbs, only the labeling by MAb Leu-M3, Leu-M5, anti-Monocyte 1, and RFD-7 was restricted to normal and malignant monocytes/histiocytes. In combination with their broad labeling of different types of monocytes/macrophages, these MAbs are of value in differential diagnostic purposes to distinguish histiocytic malignancies from large cell lymphomas. However, none of the 13 MAbs can be considered as pan-histiocytic reagents because they did not recognize all cell types belonging to the mononuclear/phagocytic system.     

Production of monoclonal antibody SP-21 to colon-ovarian tumor antigen, COTA

An IgG monoclonal antibody, SP-21, directed against colon-ovarian tumor antigen, COTA, is reported. The antibody had no reactivity with CEA, normal colonic mucin, CSAp, ABO blood group antigens, or with normal human lung, liver, spleen, kidney, plasma and saliva in studies using the enzyme-linked immunoassay method (ELISA). Immunoperoxidase staining of colon, lung, kidney, and prostate cancer tissues and benign and inflammatory colon disease tissues revealed a specificity identical to that of the polyclonal (goat) anti-COTA antibodies.     

Antiseea to Carcinoembryonic Antigen: Removal of Persistant Impurities (1)

Antisera to carcinoembryonic antigen preparations frequently contained antibodies to a distinct normal serum protein(s). The latter antibodies persisted following absorptions with cross-linked human plasma proteins, normal colon, and Group 0 red cells. A simple method for removal of the contaminating antibodies involved absorptions with the perchloric acid-soluble fraction of normal serum. Excesses of the absorbant and its soluble immune complexes were then removed on anion exchange columns.     

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.     

Renal cell adenocarcinoma and retrovirus p30-related antigen excreted to urine

The authors have previously demonstrated in syncytiotrophoblastic cells of human placenta, hydatidiform and destructive moles, and choriocarcinoma antigens reacting with antibodies to the endogenous feline retrovirus RD114 p30 and human T cell leukemia-lymphoma virus p19 proteins. The authors now report that a monoclonal IgG1 antibody (HPS-1), recognizing both syncytiotrophoblasts and RD114 p30, also reacts with an antigen in the tumor cell cytoplasm of all 27 renal cell adenocarcinomas studied. Positively stained antigenic material was also seen in the lumen of normal tubuli of tumor kidneys, suggesting its excretion into urine. None of 10 normal kidneys, 17 Wilms' tumors, 5 transitional cell carcinomas of the renal pelvis, 5 similar tumors of the urinary bladder, 20 adenocarcinomas of the cervix uteri, 20 adenocarcinomas of the corpus uteri, or 20 adenocarcinomas of the colon showed any positive staining. All 3 renal oncocytomas studied gave a positive staining reaction. In RD114 p30 radioimmunoassay antigenic activity was detected in the urine of renal cell adenocarcinoma patients in amounts up to 1.93 ng/mg protein, but not in the serum. After nephrectomy, a decline of the excreted antigen was observed, the preoperative values being 0.16 to 1.93 ng/mg protein and the postoperative ones ranging from immeasurable to 0.58 ng/mg protein. The patients with measurable postoperative urine values had clinically detectable distant metastases. The p30-related antigen may provide a marker for renal cell adenocarcinoma.