CNA.42, a new monoclonal antibody directed against a fixative-resistant antigen of follicular dendritic reticulum cells.

A new monoclonal antibody (MAb), CNA.42, was generated using the CEM T-cell line. It recognizes a 120-kd formalin-resistant glycosylated antigen that is mainly expressed by follicular dendritic reticulum cells (FDRCs). This antigen is also expressed by a few mononuclear cells in the paracortical area of reactive lymph nodes and by some cortical thymocytes. Two hundred and eighty-nine cases of hematopoietic tumors of various types were tested with this antibody. They showed either intact FDRC networks or FDRC networks dispersed among malignant cells. In follicular lymphomas, the follicular pattern was highlighted by CNA.42 MAb. Expanded FDRC networks were found in angioimmunoblastic T-cell lymphomas. Neoplastic cells were positive in 43.6% (24/55) of T-cell and 4.6% (6/129) of B-cell lymphomas. The highest percentage of cases with positive neoplastic cells was found in anaplastic large-cell lymphomas (62.5%; 15/24). In Hodgkin's disease, FDRC networks, sometimes encasing Hodgkin and Reed-Sternberg (HRS) cells, were found. HRS cells were also stained by this antibody in 23 (21.9%) of the 105 cases examined. A variety of normal nonlymphoid tissues and nonhematopoietic tumors, such as some neurogenic tumors, carcinoma, and occasional sarcomas, were found to be positive. Analysis of the reactivity of CNA.42 antibody with FDRCs of lymphoid tissue from different animal species showed similar reactivity to that observed in humans, suggesting widespread evolutionary conservation of the antigen recognized by this antibody. In daily diagnostic practice, CNA.42 MAb seems to be a suitable FDRC marker and possibly has an auxiliary role in recognizing T-cell lymphomas.     

A monoclonal antibody against a new differentiation antigen of thymocytes

B14-2-14 is a monoclonal cytotoxic IgM antibody which reacts with thymocytes of all mouse strains tested. The fraction of positive cells (by visual immunofluorescence) varies between strains from about 25-45% in A.CA to 65-85% in C57BL/6, and high levels are dominant in F1 hybrids. In the periphery, the antigen is found on a few percent of lymph node and not on splenic T cells, and it is absent in nude mice. Among thymocytes, the distribution of the B14 determinant largely overlaps with that of the TL antigen and of molecules binding peanut agglutinin. The B14 antibody reacts only minimally with hydrocortisone-resistant thymus cells. Biochemical analysis shows that B14 antibody, anti-TL antibody and peanut agglutinin bind to separate molecules. The target of the B14 antibody may be either an immature, thymic form of Thy-1, or another molecule associated with it. Two polypeptides, of 40 and 35 kDa are precipitated by both B14 and anti-Thy-1 antibodies from biosynthetically labeled thymus cell lysates, and two others, of 27 and 17 kDa, from surface-iodinated thymus cell preparations. B14-2-14 offers an additional method for identification and selection of thymocytes at different stages of differentiation, and should also be useful for studies of the Thy-1 antigen.     

A monoclonal antibody directed against an organ-specific liver cell membrane antigen in rabbits

We generated monoclonal antibodies after immunization of mice with rabbit liver-specific protein (LSP) preparations. One of these antibodies (2D3) showed an organ-specific and species-specific binding pattern as determined by immunohistological and ELISA techniques. Immunoelectron microscopy studies demonstrated that this antibody is bound exclusively to the liver cell membrane except in the region of the bile canaliculi. We further describe a simple ELISA technique for the detection of anti-LSP antibodies. Our study clearly demonstrates the presence of at least 1 organ-specific liver cell membrane antigen in rabbit LSP and shows antigenic differences between areas of the plasma membrane of hepatocytes.     

A monoclonal antibody directed against a human cell membrane antigen prevents cell substrate adhesion and tumor invasion.

It was the aim of this study to design mouse monoclonal antibodies (MAbs) that can inhibit the invasion of breast cancer cells in the host tissue. Therefore, MAbs were raised against epitopes on the extracellular domain of SK-BR-3 human breast cancer cells, and biological assays were performed to test the capability of the MAbs to inhibit cell substrate adhesion. MAb 14C5 bound an extracellular plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells and inhibited the cell substrate adhesion of these cells in vitro. The MAb delayed the adhesion of MCF-7 and SK-BR-3 cells on precultured embryonic heart fragments (PHFS). It inhibited the destruction of the PHF by MCF-7 cells and the invasion of the PHF by SK-BR-3 cells. The MAb reacted with an epitope on the cell membrane of in situ and invasive ductal carcinomas of the breast in immunohistochemistry. Poorly differentiated, highly invasive ductal carcinomas show extensive staining of long plasma membrane extensions. Normal multilayered epithelia, normal connective tissue, and tumors derived from these tissues as well as normal breast tissue were negative. From both cell lines a protein complex consisting of two subunits with molecular weight of 50 and 90 kd, respectively, was immunoprecipitated. It is concluded that the 14C5 antigen plays a role in cell substrate adhesion and subsequently also in invasion of breast cancer cells. The 14C5 MAb was able to inhibit cell substrate adhesion and invasion in vitro of breast cancer cells.     

蓖麻毒蛋白A链与Ia抗原单抗偶联物对肿瘤细胞的作用

用ε—氨己酰半乳糖胺—琼脂糖和去唾液酰胎儿球蛋白—Sepharose 4B柱层析纯化蓖麻毒蛋白A链,以DE—52离子交换层析法纯化得Ia抗原MAb—HB_(55)5。将二者通过二硫键交联成免疫毒素RTA—HB_(55)。在体外条件下,RTA—HB_(55)对Raji细胞杀伤的IC_(50)为3×10~(-10)M,游离RTA的IC_(50)为10~(-8)M,RTA—HB_(55)对K_(562)细胞的IC_(50)0则为2×10~(-8)M。表明RTA—HB_(55)能选择性地杀伤靶细胞而不杀伤非靶细胞,可望在B淋巴细胞白血病和淋巴瘤的治疗中获得应用。     

Epitope mapping of 2E2-D3, a monoclonal antibody directed against human TDP-43

TDP-43 is now known to be a major component of ubiquitin-positive, tau-negative inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and sporadic amyotrophic lateral sclerosis. In this study, we mapped the epitope for the monoclonal anti-TDP-43 antibody 2E2-D3. Our mapping and peptide competition experiments showed that the antibody reacted with human TDP-43, but not mouse or rat TDP-43, and recognized amino acids 205-222 of human TDP-43, corresponding to a part of the second RNA recognition motif. These findings suggest that 2E2-D3 is a useful antibody for the characterization of mouse lines transgenic for human TDP-43.     

Diagnosis of rhabdomyosarcomas with HHF35, a monoclonal antibody directed against muscle actins.

The authors have recently developed a monoclonal antibody, HHF35, that recognizes the muscle-specific isoforms of actin. To determine its potential usefulness in the differential diagnosis of "small, round, blue cell" tumors of childhood, they immunolabeled formalinor B-5-fixed tissue sections from known cases of rhabdomyosarcoma or rhabdomyoma (30), neuroblastoma (9), retinoblastoma (2), and Ewing's sarcoma (9) with HHF35 and with antibodies to creatine kinase M, myoglobin, vimentin, and neuron-specific enolase. HHF35 reacted with 29 of 30 cases of rhabdomyosarcoma, whereas antibodies to creatine kinase M and myoglobin were positive on only 12 and 7 tumors, respectively. HHF35 did not react with any case of neuroblastoma, retinoblastoma, or Ewing's sarcoma when the antibody diluent contained 50 mM EDTA. These results indicate that HHF35 is a highly sensitive and specific marker for myogenic differentiation and that it will be useful in the differential diagnosis of rhabdomyosarcomas.     

An anti-Cryptococcus neoformans monoclonal antibody directed against galactoxylomannan

Six monoclonal antibodies (mAb) were produced by immunizing mice with a Cryptococcus neoformans serotype A spheroplast lysate (CNSL). Two mAb were of the IgG1 isotype; the others were IgM. The results obtained with one IgM mAb (CN6) are reported herein. This mAb recognized the four serotypes of C. neoformans and no cross-reactions were observed with extracts from Cryptococcus melibiosum, Candida albicans, Saccharomyces cerevisiae, Torulopsis glabrata or Trichosporon beigelii. Fractionation of the CNSL by gel filtration revealed that mAb CN6 recognized high molecular weight substances as well as a range of smaller molecules. Indirect ELISA inhibition studies showed that this mAb recognized substances in a cryptococcal culture filtrate. Inhibition studies and agglutination tests using latex beads sensitized with purified CN6 showed that CN6 strongly reacted with the C. neoformans serotype A cell envelope galactoxylomannan-mannoprotein complex (GAlXM-MP) and only weakly with the glucuronoxylomannan (GXM) component. These tests also showed that purified galactoxylomannan (GalXM) from C. neoformans serotype A was more reactive than purified mannoprotein (MP). An anti-GalXM mAb might be a useful tool for monitoring the clinical course of cryptococcal infections.     

Characterization of a human-human hybridoma antibody, C-OU1, directed against a colon tumor-associated antigen.

The human hybridoma cell line, B9165, was obtained after fusion of lymphocytes from lymph nodes draining the tumor region in a patient with adenocarcinoma of the colon with the human B-lymphoblastoid cell line WI-L2-729-HF2 (729-HF2). B9165 secretes the human monoclonal antibody, C-OU1 (IgM, kappa). Immunocytochemical and immunohistochemical analysis showed that the antibody bound to a differentiation antigen. Electron microscopy of colonic adenocarcinoma cells, intact tumor and colonic epithelium by the immunogold technique demonstrated that the C-OU1 antibody reacted with a molecule associated with areas of disruption of the intermediate filaments in the cytoplasm of the tumor cells. No reaction was seen with intermediate filaments in normal colonic epithelium. The molecular weight of the antigen was shown to be 43 Kda by SDS-PAGE and Western blotting of tumor extracts, and isoelectric focusing of sonicated extracts demonstrated reaction with molecular species of pI 5.4-6.2. These findings suggest that the C-OU1 antigen is a modified cytokeratin 18. The B9165 cell line has proved to be quite stable, and the antibody is of potential clinical value. Its usefulness for localizing tumors in patients is being investigated.     

Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-beta-adrenergic ligand antibody

A new method has been developed to raise monoclonal anti-idiotypic antibodies. Monoclonal anti-idiotypic antibodies were obtained by fusion of NS-1 myeloma cells with splenocytes of mice immunised by intravenous injections of fixed hybridoma cells bearing a monoclonal antibody specific for beta-adrenergic ligands. New screening tests were developed to analyse the resulting hybridoma supernatants for different anti-idiotypic properties. Among 23 hybridoma supernatants recognising the idiotype, 6 were found to inhibit hapten binding and 3 of these recognised beta-adrenergic receptors.     

Immunocytochemical characterization of a monoclonal antibody directed against mitochondria reactive in paraffin-embedded sections.

The monoclonal antibody mES 13 was previously produced against bacterially expressed BALB ras p21 and was reported to have both membrane and cytoplasmic reactivity in formalin-fixed, paraffin-embedded tissue sections. In the current study, the cytoplasmic reactivity of mES 13 is investigated and demonstrated to be mitochondrial. Immunoelectron microscopic studies showed specific labeling of mitochondria without labeling of other organelles. In normal tissues, the antibody strongly labeled tissues known to have large amounts of mitochondria such as renal tubules, hepatocytes, and myocardium. The pattern of reactivity of tumors generally mimicked that of normal tissues, with carcinomas and melanomas usually showing stronger staining than sarcomas and lymphomas. Two granular cell tumors were negative. Among renal neoplasms, mES 13 strongly labeled renal oncocytomas and granular cell renal cell carcinomas and showed weaker staining of clear cell and chromophobe cell tumors. The mES 13 antibody should be useful in the characterization and diagnosis of tumors in which oncocytoma is in the differential diagnosis, especially when only paraffin-embedded tissue is available for study.     

F4/80, a monoclonal antibody directed specifically against the mouse macrophage

A hybridoma clone which secretes a macrophage (MΦ)-specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate-induced mouse peritoneal MΦ with a mouse myeloma, NS1. Binding of antibody to primary cells and cell lines was detected by radioimmune indirect binding assay, autoradiography or fluorescence-activated cell sorter analysis. F4/80 binds to mouse MΦ from the peritoneal cavity or other sources, blood monocytes, MΦ derived from bone marrow precursors in culture and MΦ-like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to MΦ via Fc receptors, is not cytotoxic and is of the rat IgG_2b subclass. Since F4/80 binds to all MΦ defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the MΦ in the mouse. Large differences in expression of antigen F4/80 were found, depending on intraperitoneal stimulation, time in culture and stage of maturation. Immunoprecipitation experiments demonstrated that the antigen F4/80 is part of a component of Mr 160000 which is synthesized by the MΦ and, at least in part, exposed on the cell surface.     

Nuclear translocation of monoclonal antibody directed against cell-surface carbohydrate Y determinant.

Monoclonal antibody (MAb) Br 15-6A directed against the carbohydrate Y determinant expressed on tumor cells was found to be internalized and translocated to the nucleus of SK Br 5 breast carcinoma and SW 1116 and SW 707 colorectal carcinoma cells. Intracellular localization of MAb Br 15-6A was determined by cell fraction and by indirect immunofluorescence staining. Internalization of MAb Br 15-6A seems to be mediated by a specific cell surface protein of M(r) 108,000 in colorectal carcinoma cells and M(r) 92,000-96,000 in breast carcinoma cells. The MAb Br 15-6A precipitates an 88,000 M(r) chromatin protein and appears to be bound specifically to two EcoRI-digested and two HincII-chromatin fragments. Another MAb against the Y determinant (MAb Br 55.2) recognizes the same antigens as MAb Br 15-6A, but is not internalized.     

抗血小板糖蛋白Ⅵ单克隆抗体的制备及其功能研究

目的：制备抗血小板糖蛋白Ⅵ（GPⅥ）单克隆抗体，观察其在体外抗血小板黏附和聚集功能。方法：采用基因重组技术体外表达血小板糖蛋白Ⅵ胞外区重组蛋白（rGPⅥ）。以rGPⅥ免疫小鼠，经细胞融合及筛选后制备抗GPⅥ单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convulxin及ADP诱导的血小板聚集的影响；利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果：正确构建了rGPⅥ表达载体pET-20b（＋）-GPⅥ，rGPⅥ在原核细胞中有效表达。rGPⅥ能够被抗Penta-His单抗和抗GPⅥ多抗识别。制备的抗GPⅥ单克隆抗体SZ118能够识别rGPⅥ，并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convulxin诱导的血小板聚集，呈抗体剂量依赖性；对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明，SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论：成功制备抗GPⅥ单克隆抗体SZ118，该抗体与血小板有良好的结合能力，显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。     

抗血小板糖蛋白VI单克隆抗体的制备及其功能研究

目的:制备抗血小板糖蛋白VI(GPVI)单克隆抗体,观察其在体外抗血小板黏附和聚集功能。方法:采用基因重组技术体外表达血小板糖蛋白VI胞外区重组蛋白(rGPVI)。以rGPVI免疫小鼠,经细胞融合及筛选后制备抗GPVI单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convu lxin及ADP诱导的血小板聚集的影响;利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果:正确构建了rGPVI表达载体pET-20b(+)-GPVI,rGPVI在原核细胞中有效表达。rGPVI能够被抗Penta-H is单抗和抗GPVI多抗识别。制备的抗GPVI单克隆抗体SZ118能够识别rGPVI,并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convu lxin诱导的血小板聚集,呈抗体剂量依赖性;对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明,SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论:成功制备抗GPVI单克隆抗体SZ118,该抗体与血小板有良好的结合能力,显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。     

A monoclonal antibody directed against a serotype-specific, outer-membrane protein of Haemophilus influenzae type b

Monoclonal antibodies (Mabs) were produced against outer-membrane proteins (OMPs) of Haemophilus influenzae type b. The clones were screened by ELISA with outer-membrane preparations of H. influenzae type b and untypable strains as coating antigens. Antibodies directed against the proteins of mol. wt (10(3)) 43, 37 and 13 were identified by immunoblotting of SDS-PAGE patterns of OMPs. Proteolytic enzyme treatments of the OMPs resulted in reduction of Mab reactivity as measured by ELISA. Furthermore, the absence of reactivity of Mab Hb-2 with a preparation of lipopolysaccharide confirmed the protein nature of its corresponding epitope. Binding assays with live bacteria showed that Hb-2 reacted with a cell surface-exposed antigenic determinant. Mab Hb-2 was bactericidal in vitro in the presence of complement. The characterisation of Hb-2 (IgG2a) by Western immunoblotting analysis revealed that it was directed against the 37 X 10(3)-mol. wt OMP. In a dot-enzyme immunoassay, Hb-2 reacted specifically with 326 strains of H. influenzae type b. It did not cross-react with the other serotypes or untypable strains of H. influenzae or with other bacterial species. This is the first report of a monoclonal antibody identifying a serotype-specific surface-exposed OMP of H. influenzae type b.     

A new murine monoclonal antibody against human hepatoma.

A murine monoclonal antibody (MAb 336) reactive with human hepatocellular carcinoma has been raised after immunizing BALB/c mice with whole HepG2 cells. MAb 336 (IgG1) was reactive with HepG2 (whole cells and membrane fractions), but not normal liver or peripheral blood cells. Immunohistological studies indicated that 12/16 hepatocellular carcinoma and 6/11 cirrhotic livers expressed MAb 336-associated antigen, and most normal human tissues and tissues derived from other cancers were unstained. Direct and competitive binding assays ruled out the possibility that this MAb reacts with alpha-fetoprotein, carcinoembryonic antigen, or ferritin. Western blot analysis indicated that MAb 336 reacts with an antigen of approximately 30,000 daltons. This MAb may be potentially useful for studying antigenic expression in hepatocellular carcinoma and as a targeting agent for radioimmunodetection and immunoconjugate therapy.     

Supertypic HLA-Bw4 antigen detected by a new monoclonal antibody

A monoclonal antibody, HLAO1, was prepared by immunizing BALB/c mice with human lymphocytes of known HLA genotype. The monoclonal antibody is cytotoxic, of the IgG 2b isotype and binds to a protein of Mr 43,000 noncovalently associated with the beta 2-microglobulin. Genetic analysis proved complete concordance in the expression of the antigenic determinant defined by this monoclonal antibody and the presence of a supertypic (public) antigen, the HLA-Bw4. As expected, the monoclonal antibody also reacted with the antigens of HLA-A locus, A23, A24, and A32. The computer analysis of HLAO1 antibody binding to the Bw4/Bw6 heterozygous lymphocytes gave the approximate number of antigenic determinants, n = 2.2 X 10(4)/cell, and Ka = (5.9 +/- 0.8) X 10(9) M-1. Besides its potential immunochemical applications, the HLAO1 monoclonal antibody can become a useful tool in routine cytotoxicity typing of HLA antigens on peripheral lymphocytes.     

Detection of small cell lung cancer metastases in bone marrow aspirates using monoclonal antibody directed against neuroendocrine differentiation antigen.

To detect metastases in the bone marrow of patients with small cell lung cancer, immunofluorescence with a monoclonal antibody detecting a membrane antigen (MOC-1) associated with small cell lung cancer was performed on 53 bone marrow aspirates from 30 patients. In 19 (63%) patients MOC-1 reactive cells were detected. Simultaneous histopathological examination of the bone marrow biopsy specimens detected tumour cells in only six (20%). The method is more sensitive than conventional histochemical staining of bone marrow aspirate and may eventually be able to show additional subgroups, such as patients with limited disease who might benefit from adjuvant radiotherapy or surgery.     

Purification of woodchuck hepatitis surface antigen using a monoclonal antibody raised against the antigen

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.