Biochemical and histological characterization of antigens preferentially expressed on the surface and cytoplasm of breast carcinoma cells identified by monoclonal antibodies against the human milk fat globule

The preparation of monoclonal antibodies (MAbs) against the human milk fat globule membrane with preferential binding to breast carcinoma cells is described. Using BALB/c mouse myeloma cells; inter-specific, intra-strain, and inter-strain hybridomas were isolated that identified three different components of the human milk fat globule of approximately 46,000, and 70,000 daltons and a mucin-like glycoprotein complex (NPGP) ranging from 400,000 to over a million daltons, respectively. Three MAbs (BrE1, BrE2, BrE3) identified the latter component which consists of at least three different size molecules for which the aforementioned MAb's have different binding specificities. MAbs, BrE2 and BrE3, bound to normal breast epithelial cells but to a lesser extent than to tumors and only at the apical surface facing the lumen, while they bound breast carcinomas strongly, and often in the cytoplasm as well as on the surface. Higher concentrations of BrE3 were required to stain normal breast compared to breast tumors. BrE1 also stained breast carcinomas both on the surface and cytoplasmically but did not stain normal breast tissue. The MAb, Mc13, as well as the previously reported MAb McR2, both against the 70,000 dalton component, did not significantly stain either normal or cancerous breast tissue in histological sections but did bind significantly to cultured breast epithelial cells and to the milk fat globule membrane. The MAbs, Mc8 and Mc3, reported previously to be against the 46,000 dalton component, stained histologically only malignant breast tissue but only weakly; however, they bound strongly to intact breast carcinoma cells and breast cell membrane preparations with a radioimmunobinding assay. These MAbs should be useful in characterizing the surface of breast epithelial cells, studying surface alterations in malignancy, and possibly in breast cancer diagnosis and therapy.     

An immunohistochemical study using monoclonal antibodies of the antigen of fat globule membranes in human milk

Monoclonal antibodies (Mab) IKO-21 and IKO-25 obtained to the membrane antigen of the woman milk lipid globules were tested immuno- and cytochemically on the samples of human normal definitive and embryonal tissues and tumours. Mab IKO-21 are shown to react, apart from epithelial tissues, with a vascular endothelium and blood cells. Mab IKO-25 are specific to the epithelial tissue and malignant epithelial tumours. Their intensive reaction with the cells of malignant tumours of the mammary gland, lung, ovary and the type of their distribution in the organs and tissues enables their use in the differential diagnosis of malignant epithelial and non-epithelial tumours as for revealing metastases both in vitro and in vivo.     

Characterization of several monoclonal antibodies generated against ferret tracheal epithelial cells

Monoclonal antibodies have been generated to ferret tracheal epithelial (FTE) cells. Four of the antibodies exhibiting positive reactivity in a fluorescence-activated cell sorter were further characterized. In tracheal sections, three of the antibodies reacted with submucous gland cells and material intermeshed in the cilia while the fourth antibody failed to react with epithelial cells. The three antibodies which reacted in tracheal sections also reacted with void volume fractions following Sepharose CL-6B chromatography of a solubilized FTE cell preparation. Reactivity of the void volume fractions to these antibodies was sensitive to periodate oxidation, and unaffected by heating (100 degrees C X 30 min) or methanol extraction suggesting a carbohydrate epitope. The fourth antibody recognized periodate-insensitive antigen in the void volume and included column fractions. Binding of one of the antibodies to periodate-sensitive antigen was completely blocked by 10 mM N-acetyl galactosamine as demonstrated in an ELISA with fixed FTE cells. In a western blot analysis, two of the antibodies which recognized periodate-sensitive antigen also reacted with a large heterogenous macromolecule.     

Ultrastructural localization of milk fat globule membrane antigens in human breast carcinomas

The localization of milk fat globule membrane components has been assessed using post-fixation immunoelectron microscopy with three different antibodies for a group of breast carcinomas of different type and histological differentiation. For well differentiated carcinomas localization was in relation to the cell membrane, with polarization being evident in a proportion of cases. Moderately differentiated carcinomas showed a combined picture of cell membrane, vesicular, and intracytoplasmic luminal localization. The latter is a feature of infiltrating lobular carcinomas. Poorly differentiated carcinomas exhibited vesicular labelling throughout the cytoplasm, with no cell membrane localization. No labelling was seen over endoplasmic reticulum. It is proposed that carcinomas exhibit defects in intracellular transport of milk fat globule membrane components resulting in failure of expression at the cell surface and accumulation of vesicles within the cytoplasm, the extent of change relating to tumour differentiation.     

Lectin binding affinities of human milk fat globule (HMFG) membrane antigens

Six biotinylated lectins with differing specificities and two monoclonal antibodies (III D 5 and III H 2) were used to characterize the sugar-residues in human milk fat globule (HMFG) membrane antigens. Immunoblotting analysis revealed that most of the antigens contain several sugars. However, the molecules exclusively reacting with anti-HMFG III D 5, a monoclonal antibody previously shown to detect antigen(s) positively correlating with the expression of estrogen receptors in mammary and gynaecological carcinomas, could only be stained with peanut agglutinin and Ricinus communis-lectins. One of these antigens, a 42-57 kDa molecule, was shown to have a complexed quaternary structure with galactose determining the antigenic specificity. It is suggested that the production of this glycoprotein in estrogen sensitive tissues results from activation of galactosyl-transferase-enzyme at the same time as the expression of estrogen receptors.     

Characterization of monoclonal antibodies against human lactoferrin

The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties.     

Demonstration of HLA-DR-like antigens on milk fat globule membranes.

Milk fat globules (MFG), which are formed by exocytosis of lipid from epithelial cells of the mammary gland, are enveloped by plasma membrane from the epithelial cells. Highly purified, detergent-solubilized MFG membranes have been shown to contain molecules reactive with a rabbit antiserum against HLA-DR antigens. Indirect immunoprecipitation combined with polyacrylamide gel electrophoresis in sodium dodecyl sulfate demonstrated that the MFG membrane material reactive with the antiserum comprised molecules which under denaturing conditions displayed molecular weights of 28,000 and 35,000. The two types of polypeptide chains, which were both glycosylated, were held together by noncovalent forces under nondenaturing conditions. Various types of chemical and physicochemical analyses failed to reveal any significant differences between the HLA-DR-like antigens from MFG and from spleen cells. Since the HLA-DR-like antigens bound detergent in micellar form and were expressed on the outside of intact MFG, as revealed by indirect immunofluorescence, it is concluded that these antigens are embedded in the hydrocarbon matrix of the MFG and not merely passively adsorbed onto the MFG.     

Differentiation antigens defined by mouse monoclonal antibodies against human germ cell tumors

We have developed two mouse monoclonal antibodies, M912-2A2 and M912-2G10, against cell surface antigens of a human infantile embryonal carcinoma cell line, MTE. The distribution of these antigens (designated as 2A2 and 2G10) was almost identical in human germ cell tumors in which they hallmarked yolk sac components and some tubular endodermal structures. Immunoelectron-microscopically, the antigens were located on the microvilli of MTE tumor cells. These antigens were not found on other common childhood tumors. In normal and fetal tissues they exhibited quite different distributions. In the kidney, 2A2 and 2G10 were present on the collecting tubules and proximal/distal tubules, respectively. Expression of both antigens was already observed in fetal kidneys of 10 weeks gestational age. In hematopoietic cells 2G10 was present only on granulocytes and on erythrocytes regardless of ABO blood group, whereas 2A2 was not present on any peripheral blood cells. Both antigens were equally expressed in testis and epididymis. Biochemically, reactivity of both antibodies was abolished with periodate treatment, suggesting their carbohydrate nature. Further biochemical characterization revealed that antibody to 2G10 reacts with the nonreducing terminal structure of type 2 carbohydrate chain, Ga1 beta 1-4G1cNAc, common to nLc4 (paragloboside), nLc6 (neolactohexaose), and Y4 neutral glycolipids of O-type erythrocytes. These data illustrate the complexity of carbohydrate antigens on yolk sac components of human germ cell tumors and provide a basis for the study of primitive endodermal and yolk sac differentiation in these tumors.     

Characterization of Candida albicans cell wall antigens with monoclonal antibodies.

The antigenic composition of Candida albicans is very complex. In order to study the antigenic relationship between blastoconidia and germ tubes of C. albicans, we produced several monoclonal antibodies and analyzed their reactivity against cell wall antigens either in intact cells or in cells treated with dithiothreitol. Overall, four types of reactivity were found. Monoclonal antibodies 3D9 and 15C9 stained the germ tubes only when tested by indirect immunofluorescence. However, they showed a different reactivity by immunoblotting. Monoclonal antibody 3D9 reacted with antigens with molecular masses of > 200 and 180 kDa specifically expressed in the germ tube. Monoclonal antibody 15C9 reacted with antigens of 87, 50, and 34 kDa present in the germ tube extract and with antigens of 92, 50, 34, and 32 kDa present in the blastoconidium extract. The reactivity of blastoconidia treated for different times with dithiothreitol with these monoclonal antibodies was also studied by enzyme-linked immunosorbent assay. The reactivity of monoclonal antibody 3D9 did not significantly change during the cell wall extraction. However, the reactivity of monoclonal antibody 15C9 was increased for blastoconidia extracted for 60 min and decreased markedly for blastocondia extracted for 120 min. Monoclonal antibody G3B was nonreactive by indirect immunofluoresence but reacted with antigens of 47 and 38 kDa present in the germ tube extract and with an antigen of 47 kDa present in the blastoconidium extract. Monoclonal antibody B9E stained both morphological phases by indirect immunofluorescence. By immunoblotting, it reacted with antigens of > 70 kDa present in the germ tube extract and with antigens of > 63, 56, 47, and 38 kDa present in the blastoconidium extract. Based on the results presented in this study, four types of antigens are described. Type I antigens are expressed on the outermost layers of the germ tube cell wall only. Type II antigens are expressed both on the germ tube cell wall surface and within the blastoconidium cell wall. Type III antigens are found within the cell wall of both blastoconidia and germ tubes. Type IV antigens are expressed on both the blastoconidium and germ tube surface. Two types more can be hypothesized for antigens expressed on the blastoconidium cell surface and within the germ tube cell wall (type V) and for those expressed on the blastoconidium surface only (type VI).     

抗人肿瘤相关核仁抗原单克隆抗体的建立

本研究以A549细胞系和人肺腺癌组织细胞核仁为抗原,建立了7株McAbs,并用ELISA技术对其反应性进行了初步分析。结果表明:各株McAb均能与人癌细胞核仁起反应,但各自的抗原却不尽相同。MA1、MA2、MA3和MA6株的抗原可能是HMNA类物质;MA4、MAS和ML1株的抗原可能是属于核仁的正常成份,但优势表达于癌细胞中。本组抗体的建立,对于研究肿瘤细胞核仁的分子组成和生物学功能,并进而利用HMNA为临床肿瘤病理服务可能有重要意义。     

Characterization of human thymic epithelial cell surface antigens: Phenotypic similarity of thymic epithelial cells to epidermal keratinocytes

Cellular interactions between developing thymocytes and cells of the thymic microenvironment are necessary for maturation of thymocytes into mature T cells. While much is known about the molecules on developing T cells that mediate these interactions, little is known about the surface molecules of human thymic epithelial (TE) cells. In this study, using a panel of 276 MAb including 255 MAb from the 5th International Workshop on Human Leukocyte Differentiation Antigens (HLDA-V), we have determined the expression of CD1 through CDw130 and other surface molecules on resting and IFN--activated cultured human TE cells and on resting epidermal keratinocytes (EK). We demonstrate the surface expression of 50 of the 161 molecules assayed for on TE cells, including a number of adhesion molecules, cytokine receptors,Apo-1, and MHC-encoded molecules. While activation of TE cells with IFN- for 48 hr induced a greater than fivefold increase in the expression of four surface molecules (CD38, CD54, MHC class I, and MHC class II), it also induced a greater than 50% increase in the expression of 14 other surface molecules (CD12, CD29, CD40, CD44, CD47, CD49b, CD49c, CD49e, CD55, CD66, CD87, CD104, TE4, and STE3) and a decrease in the expression of three molecules (CDw65, CDw109, and STE2). In comparing the phenotype of TE cells to 83 other cell lines studied in HLDA-V, we found that TE cells were strikingly more similar to EK than to any of the other cell types tested.     

Detection of monoclonal antibodies against cell surface antigens: the use of antiglobulins coupled to red blood cells

An antiglobulin-coupled red cell assay is described for screening monoclonal antibodies against cell surface antigens. A monoclonal antibody specific for rat immunoglobulin kappa chains was coupled to red blood cells and used to detect binding of rat monoclonal antibodies to cells attached to the wells of microtitre plates. The method was found to be simpler and more rapid than the alternative enzyme-linked binding assay and useful for rapid screening and selection of antibodies for use as differentiation markers of human and mouse haemopoietic cells.     

Characterization of monoclonal antibodies against human rotavirus hemagglutinin

Three monoclonal antibodies capable of specifically inhibiting hemagglutination of human rotavirus were produced. Their hemagglutination inhibition (HI) activity was specific to the homologous strain (KUN) used for immunization. The monoclonal antibodies with HI activity were highly effective in neutralizing the infectivity of the KUN strain. These antibodies reacted with Vp80, and 80,000 molecular weight (MW) protein present in the viral outer shell. It was confirmed by immunoblotting assay with the monoclonal antibodies that the antigenic site of human rotavirus hemagglutinin (HA) resides on Vp80 and on its smaller trypsin cleavage products Vp30 (MW 30,000) and Vp24 (MW 24,000). Immunofluorescence studies using the antibodies revealed that the HA antigen of the KUN strain developed at the final stage of virus maturation.     

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.     

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 against surface antigens of Moraxella bovis.

Six hybridoma lines producing monoclonal antibodies (MAbs) against Moraxella bovis were established from fusions between the SP2/0 myeloma cells and BALB/c mice splenocytes. Three antibodies were of the IgG1 isotype, two were IgG2a, and one was IgG2b. The specificity of the antibodies was determined by indirect enzyme-linked immunosorbent assay (ELISA) using whole cells of M. bovis and of other Gram-negative bacteria, and lipopolysaccharide (LPS) from M. bovis JUR2 and E. coli as antigens. Ascitic fluid produced by the six hybridoma lines inhibited hemagglutination by M. bovis GF9. One MAb (35F) reacted specifically with purified M. bovis LPS in the ELISA test. The MAb panel detected heterogeneity among the isolates recovered from different geographical regions.     

Production of monoclonal antibodies against gastric parietal cell antigens.

Two mice DBA/1 were each immunized with a single injection of one million enriched parietal cells in the hind foot pads. Monoclonal antibodies to be used as research tools in studies on regulatory mechanisms in gastric parietal cells were obtained after fusion of mouse myeloma cells (SP2) with cells from the popliteal lymph nodes of the mice. Twelve hybridomas produced antibodies reactive with structures only present in parietal cells as assessed by immunohistochemistry of oxyntic mucosa sections. Three hybridomas were subcloned and the antibodies produced by them, designated as PC4, PC8, and PC117, were characterized. In an enzyme-linked immunosorbent assay, all antibodies reacted with H,K-ATPase-containing vesicles. The antibody PC8 recognized a 94 kDa protein after immunoblotting of H,K-ATPase-containing vesicles and all antibodies precipitated a 94 kDa protein from [125I]H,K-ATPase-containing vesicles. The antibodies PC4 and PC117 recognized extracellular structures with a polarized distribution in viable, purified parietal cells. The results suggest that the structure recognized by all three antibodies is the alpha-subunit of the H,K-ATPase. The antibodies produced by another hybridoma, PC43, recognized a structure present in parietal and surface epithelial cells of the oxyntic mucosa. In an enzyme-linked immunosorbent assay, they reacted with a high-activity carbonic anhydrase which had been affinity-purified from pig oxyntic mucosa and they recognized a 30 kDa protein after immunoblotting. Thus, monoclonal antibodies against both intracellular and extracellular parietal cell structures were obtained after immunization with a small number of parietal cells.     

Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigens. Quantitation of binding and staining of individual cells

An immunoperoxidase method has been developed which allows accurate and sensitive quantitation of the binding of monoclonal antibodies to cell surface antigens. Monolayers of fixed cells were prepared in wells of Terasaki micro-test plates and monoclonal antibodies bound to cell surface antigens were identified by the unlabeled antibody-enzyme method of Sternberger (1974). The cell-bound peroxidase could either be quantified per well or visualized on individual cells by the use of appropriate substrates for peroxidase. Experimental procedures are described in detail and results obtained with several monoclonal antibodies with specificity for different target cells are shown. Limitations and applications of the technique are discussed.     

In vitro modulation of cellular localization of milk fat globule membrane antigens in human breast carcinomas

Alterations in the cellular localization of cell surface components such as the milk fat globule membrane are a common feature of breast carcinomas and relate to the differentiation of a tumour. This study has examined the potential modulation of such components. A group of carcinomas were cultured with and without insulin and/or hydrocortisone and the site of staining for milk fat globule membrane, as detected by the antibodies HMFG 1, HMFG 2, and NCRC 11, was assessed using light microscopic and electron microscopic immunohistochemistry. Modulation of localization, with a shift from cytoplasmic vesicle labelling to submembraneous vesicles/cell surface labelling and intracytoplasmic luminal labelling, was observed in 5 of 14 moderately differentiated and 8 of 11 poorly differentiated carcinomas. Two well differentiated carcinomas continued to show peripheral labelling; three poorly differentiated carcinomas showed no change from cytoplasmic labelling only; and the other carcinomas exhibited heterogeneous localization, making any change difficult to assess. Insulin was required for any change to be observed and it is suggested that this has an effect on the mechanisms for intracellular transport of membrane and secretory proteins.     

Characterization of Plasmodium yoelii monoclonal antibodies directed against stage-specific sporozoite antigens.

A battery of monoclonal antibodies against Plasmodium yoelii sporozoites was produced. Five of these (NYS1 through NYS5) were selected for characterization. All five were positive in the indirect immunofluorescent antibody test with P. yoelii sporozoites; however, each showed a different immunofluorescence pattern. Although NYS1 (immunoglobulin G3 [IgG3]), NYS2 (IgG3), and NYS3 (IgM) were positive in the circumsporozoite precipitation test, only NYS1 and NYS2 were able to neutralize sporozoite infectivity in mice. NYS4 (IgM) and NYS5 (IgG1) were not positive in the precipitation test and did not protect mice from sporozoite infection. All except NYS4 were species as well as stage specific. NYS4 cross-reacted with sporozoites of P. berghei. Electrophoretic immunoblotting analysis showed that these monoclonal antibodies detected sporozoite antigens of various molecular weights. Inhibition enzyme-linked immunosorbent assays indicated that each recognized a different antigenic epitope. The differences in their immunochemical and biological reactivity make them useful for screening a variety of P. yoelii antigens in recombinant DNA libraries. These antigens will be used in an animal model system for vaccine development.