Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).     

Mouse monoclonal antibodies specific for endothelial cells

Summary Balb/c mice were immunized with a human endothelial cell pool. Spleen cells were then fused with a NS-0 hybridoma cell line. A number of hybridomas secreted antibodies that reacted with the immunizing endothelial cell pool as well as with every other tested umbilical cord vein~derived human endothelial cell. These monoclonal antibodies also stained pig, rabbit and ox aortic endothelial cells indicating their specificity for this cell type. Five of 16 monoclonal antibodies additionally reacted with human fibroblasts (HFIB). The produced monoclonal antibodies did not recognize FVIIIRAG or MHC determinants. They can therefore be regarded as additional and reliable markers for endothelial cells in vitro.     

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.     

Application of chemically desialylated and degalactosylated human glycophorin for induction and characterization of anti-Tn monoclonal antibodies

Human erythrocyte glycophorin was desialylated by mild acid hydrolysis and degalactosylated by Smith degradation. Two monoclonal antibodies (Tn5 and Tn56) obtained by immunization of mice with this artificial Tn antigen were characterized and compared in some experiments with two antibodies (BRIC111 and LM225) obtained in other laboratories by immunization with Tn erythrocytes. The specific binding of the antibodies to glycophorins desialylated and degalactosylated on the nitrocellulose blot and to asialo-agalactoglycophorin-coated ELISA plates, and reactions with authentic Tn antigen served for identification of their anti-Tn specificity. The antibodies were further characterized in inhibition assay with various glycoproteins. The antibody Tn5 (similar to BRIC111) was shown to be specific for human erythrocyte Tn antigen, whereas Tn56 reacted strongly with different glycoproteins carrying O-linked GalNAc- residues, and was strongly bound to the murine adenocarcinoma cell line Ta3-Ha. The antibodies Tn5, Tn56 and BRIC111 were similarly inhibited by ovine submaxillary mucin (OSM) and asialoOSM, but the antibody LM225 showed a distinct preference in reaction with OSM (sialosyl-Tn antigen). The results show that Tn antigen, obtained by chemical modifications of human glycophorin, enables the preparation and characterization of anti-Tn monoclonal antibodies, without using rare Tn erythrocytes.Abbreviations HuGph human erythrocyte glycophorin - HoGph horse erythrocyte glycophorin - OSM ovine submaxillary mucin - mAb monoclonal antibody - ELISA enzyme-linked immunosorbent assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - PBS phosphate buffered saline (0.01m Na₂HPO₄/0.15m NaCl, pH 7.2) - BSA bovine serum albumin - TBS 0.05m Tris-HCl/0.15m NaCl, pH 7.2 - TGr transformation grade     

Preparation and characterization of monoclonal antibodies specific for GoCodGdCGoCo duplex with high-anti conformation

Monoclonal antibodies were prepared using a self-complementary oligonucleotide duplex containing cyclonucleosides with the high-anti conformation as an antigen. A competition ELISA assay showed that the monoclonal antibodies specifically recognized high-anti left-handed oligonucleotides but not oligonucleotides with B- or Z-conformation.     

Development and characterization of monoclonal antibodies specific for the genus Listeria

Abstract Monoclonal antibodies were obtained by the classic hybridoma technique with lymphocytes of BALB/c mice immunized with formalin killed Listeria monocytogenes cells. Among 1000 hybridomas issued from the fusion, four monoclonal antibodies (mAbs A6 A E4, C10 A F7, G4 A D6, G7 A D5) gave interesting results. By Western-blot analysis with various soluble extracts of different Listeria species, the four mAbs reacted with two major antigens of 38 and 41 kDa, with all Listeria species tested. The mAb A6 A E4 is an IgG2b with κ light chains and reacted only with Listeria antigens without any cross reaction with other organisms tested by ELISA, dot-blotting and Western-blotting. With the same conditions, the three other mAbs reacted with Listeria and with other genus extracts, particularly with Streptococcus and Enterococcus . mAb A6 A E4-reactive antigens are proteins, and glycoprotein immunoassay indicated that the epitope is devoid of carbohydrate moiety. This mAb A6 A E4-reactive protein was neither expressed on cell surface nor released outside the bacteria; immunogold electron microscopy showed that these antigens were localized in the cytoplasma area.     

Production and characterization of two monoclonal antibodies specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies

[This corrects the article on p. 1498 in vol. 3.].     

Production and characterization of monoclonal antibodies specific for Shewanella colwelliana exopolysaccharide.

Six monoclonal antibodies were produced to whole cells of Shewanella colwelliana (Aco1 to Aco6) and two (Aco22 to Aco23) to purified exopolysaccharide (EPS). Aco1, -4 to -6, -22, and -23 bound to both the cell surface and the purified EPS, while Aco2 and -3 bound to cells only. The EPS of S. colwelliana was antigenically unique from those of nine other species of marine bacteria that were tested. Mapping studies revealed that all of the EPS-specific monoclonal antibodies bound to the same epitope. This EPS epitope was sensitive to cleavage of ester bonds, but neither pyruvate, acetate, nor terminal nonreducing sugars were required for antigenicity. When S. colwelliana was grown on rich media, most of its EPS was loosely associated with the cell surface.     

Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies

Monoclonal antibodies raised against the rat liver glucocorticoid receptor were used to investigate receptors of wild-type and glucocorticoid-resistant variants of mouse lymphoma cells. Two of the variant types contained receptors of 'nuclear transfer deficient' (nt-) and 'increased nuclear transfer' (nti) phenotypes, respectively, while the third was of the 'receptorless' (r-) phenotype with negligible hormone binding activity. Three monoclonal antibodies of the IgM class and one of the IgG class reacted with both wild-type and nt- receptors but not with the steroid binding form of nti receptors. Some of the antibodies bound the wild-type and nt- receptors more efficiently after activation at 20 degrees C. By use of an immuno-competition assay we were able to detect cross-reacting material in considerable amounts in extracts of nti and r- cell variants. This material was further characterized by gel filtration and immunoblotting. The immunoreactive material of wild-type, nti and r- cells gave a major band of mol. wt. 94 000 upon SDS-gel electrophoresis while the steroid-binding polypeptides of wild-type and nti receptors have mol. wts. of 94 000 and 40 000, respectively. The data show that in S49.1 mouse lymphoma cells the products of two receptor alleles can be distinguished.     

Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.     

抗重金属汞离子抗体的制备及鉴定

汞、镉、铅等重金属引起的环境污染已在世界范围内造成危害。快速、廉价地监测生境中重金属是减小其对人类及动物危害的先决条件。传统检测方法无法满足高通量的现场检测,建立更快速、更经济的免疫分析法检测汞离子是生产及经济发展的需要。本研究中,报道了汞特异性单克隆抗体的制备与筛选方法和结果。因Hg2+太小以至于不能引起免疫反应,所以用螯合剂(二乙烯三胺五乙酸,DTPA)将金属离子与载体蛋白(匙孔血蓝蛋白,KLH)连接起来。成功合成、鉴定汞复合物抗原后,免疫BALB/c小鼠,通过细胞融合获得了稳定分泌抗体的杂交瘤细胞。用极限稀释法亚克隆,通过ELISA筛选,获得了2株稳定分泌抗汞离子抗体的细胞株(H2H5,H1H8)。小鼠腹腔注射1×107H2H5、H1H8细胞株制备腹水,腹水抗体效价都在1∶51200以上。经鉴定两株杂交瘤均为IgG1亚类,轻链为kappa型且分泌抗体稳定性较好。实验结果为汞离子残留免疫学检测方法的建立提供了技术基础,对提高风险评估工作的效率和质量,保障食品安全有重要现实意义。     

Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDTTPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.     

Generation and characterization of monoclonal antibodies specific for the human IFN-gamma receptor

Purified preparations of the human IFN-gamma R derived from placental membranes were used to produce receptor-specific murine mAb. Supernatants from growth-positive wells were screened for their ability to block binding of 125I-IFN-gamma to human placental membranes. Ten inhibitory cultures were identified. Two of these (GIR-208 and GIR-301) abrogated all binding of radioligand to either intact placental membranes or soluble, purified IFN-gamma R. Three others (GIR-72, 76 and 94) showed moderate blocking activity (65, 59, and 49%, respectively) whereas the remaining five (GIR-57, 67, 83, 109, and 153) blocked binding to a low but significant extent (20 to 40%). Specificity experiments demonstrated that the antibodies reacted with the receptor and not the ligand (IFN-gamma). None of the antibodies reacted with IFN-gamma by ELISA. Moreover, GIR-208 and GIR-301, but not isotype-matched controls, identified the receptor by Western blot analysis. GIR-208 and GIR-301 also completely abrogated binding of 125I-IFN-gamma to either mononuclear phagocytes (U937) or human fibroblasts (WISH). Competition experiments revealed that GIR-208 and GIR-301 recognized similar epitopes on the IFN-gamma R and that these (or this) epitopes were identical to or linked to the ligand binding site of the receptor. In addition, both antibodies inhibited development of IFN-gamma-dependent anti-viral activity in WISH cells in a dose-dependent fashion. These data thus indicate that the IFN-gamma R expressed on human placental cells, mononuclear phagocytes, and fibroblasts are similar.     

空肠弯曲菌FlaA单克隆抗体的制备与鉴定

【目的】原核表达空肠弯曲菌鞭毛蛋白FlaA,并制备其单克隆抗体。【方法】克隆目的基因并将其构建到pET30a(+)和pGEX-6p-1表达载体,分别以变复性纯化后的rHis-FlaA、rGST-FlaA蛋白为免疫原和检测原进行杂交瘤细胞的筛选。采用间接ELISA法测定细胞上清和单抗腹水效价,Dot-ELISA、Western blot分析单抗特异性。【结果】成功构建pET30a(+)-flaA和pGEX-6p-1-flaA重组原核表达质粒,并融合表达rHis-FlaA和rGST-FlaA蛋白,Western blot试验显示天然蛋白多抗血清能与体外表达的蛋白呈现特异性反应,表明表达蛋白具有免疫原性。筛选获得3株稳定分泌抗FlaA的单克隆杂交瘤细胞株,分别命名为2D12、5E12、6A9,其Ig亚类分别为IgG2a、IgG1、IgG1,腹水效价分别为1∶102400,1∶102400和1∶51200;Western blot试验显示,3株单抗均能与表达rHis-FlaA重组蛋白的细菌发生特异性反应;Dot-ELISA试验表明,3株单抗均能与不同来源的空肠弯曲菌分离株发生特异性反应。【结论】本研究制备的单克隆抗体有较高特异性,具有良好的应用价值。为进一步研究空肠弯曲菌鞭毛蛋白的生物学特性、致病机理,以及建立快速检测技术奠定基础。     

Isolation and characterization of monoclonal antibodies specific for the cardiac muscarinic acetylcholine receptor

A number of monoclonal antibodies were raised against the purified porcine atrial muscarinic acetylcholine receptor. The antibodies were shown to exhibit a high degree of specificity for the receptor by their ability to recognize the purified receptor but not other porcine atrial glycoproteins in enzyme-linked solid-phase immunosorptive assays and by immunoblot analyses. Several of the antibodies were able to quantitatively precipitate the muscarinic receptor in both pig and rat heart and a portion of the receptor from rat cerebellum but little if any receptor from rat cerebral cortex. Thus, these monoclonal antibodies not only exhibit specificity for the muscarinic receptor but also are specific for the cardiac receptor subtype.     

1组曲霉单克隆抗体的制备与鉴定

目的制备并鉴定一组抗曲霉不同抗原的单克隆抗体。方法采用烟曲霉细胞壁抗原成分、分泌抗原和灭活分生孢子,分别免疫BALB／c小鼠,制备单克隆抗体,免疫荧光法鉴定单克隆抗体与曲霉属和念珠菌属抗原的交叉反应。结果获得29株稳定分泌抗曲霉单抗的杂交瘤细胞株,其中用烟曲霉细胞壁抗原成分免疫获得11株,用分泌抗原免疫获得13株,用孢子免疫获得5株;Ig亚类鉴定,11个克隆株为IgG1亚类,3个克隆株为IgG3,15个克隆株为IgM。免疫荧光法鉴定29株单抗特异性识别烟曲霉细胞壁抗原,与其他曲霉抗原有交叉反应。结论29株单克隆抗体,对于建立侵袭性曲霉感染早期诊断方法、筛选曲霉保护性抗体以及研究抗体保护机制奠定了实验基础。