Identification and characterization of a novel antigen complex on mouse mammary tumor cells using a monoclonal antibody against platelet glycoprotein Ic

The rat monoclonal antibody GoH3 identifies a complex of glycoproteins Ic and IIa on human and mouse platelets. The GoH3 epitope is located on glycoprotein Ic. A novel glycoprotein complex is identified by GoH3 on the surface membranes of mouse mammary epithelial tumor cells. This antigen complex is composed of glycoprotein Ic noncovalently associated with a monomor or a disulfide-linked multimer of a high molecular weight glycoprotein (Ic-binding protein (IcBP]. Glycoprotein Ic is synthesized as a large precursor with asparagine N-linked high mannose oligosaccharides. Processing of this precursor involves a proteolytic cleavage of the large polypeptides into two smaller disulfide-linked polypeptide chains, Ic alpha (heavy) and Ic beta (light), and conversion of the majority of the high mannose oligosaccharides into complex-type glycans. Likewise, glycoprotein IcBP is initially glycosylated with high mannose asparagine N-linked oligosaccharides which are processed to complex units in the mature form. Association of glycoprotein Ic with IcBP occurs within the cell soon after their synthesis. The kinetics of labeling show non-coordinate processing consistent with the idea that the concentration of glycoprotein Ic limits complex formation and the subsequent processing of glycoprotein IcBP.     

A tubulin identified by a monoclonal antibody is not present in mitotic spindles

A monoclonal antibody, G8, which recognizes a form of tubulin (G8-tubulin) with a novel distribution in Rat-1 cells and Potorous tridactylis kidney (Ptk-2) cells was isolated. G8 labeled the interphase cytoskeleton of Rat-1 fibroblasts but not mitotic spindles or midbodies. G8 also stained a fiber network in some but not all Ptk-2 interphase cells but did not label mitotic spindles or midbodies in these cells. G8-tubulin is the only identified tubulin known to be absent from these structures. This distribution may indicate that G8-tubulin possesses functional specificity.     

Cell surface glycoproteins of hepatocytes and hepatoma cells identified by monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MABs) directed to cell surface components of rat hepatocytes were isolated. The antigens of seven MABs were identified as glycosylated plasma membrane proteins. The presence of these glycoproteins on normal hepatocytes and hepatocellular carcinoma cells was analyzed. A semi-quantitative enzyme-linked immunosorbent assay revealed that only two MABs (Be 8.7, Ne 11.3) recognized proteins which were expressed not only in normal liver but also in chemically induced transplantable Morris hepatomas and hepatoma-derived cell lines. The expression of six antigens was found to be sensitive to transformation. The domain specificity of the MABs was determined by indirect immunofluorescence on sections of liver tissue containing neoplastic nodules. Three MABs (Be 8.4, Ne 11.1, Ne 11.3) specifically bound to the sinusoidal domain and two MABs (Be 9.2, De 13.4) to the bile canalicular domain. These five antigens were transformation-sensitive except for the glycoprotein recognized by the MAB Ne 11.3. Three MABs (Be 8.7, Be 9.1, De 13.2) also showed intracellular immunofluorescence. Two of the antigens (Be 9.1, De 13.2) were not present in hepatomas. The relative molar masses (Mr) of the glycoproteins were determined after protein immunoblotting and immunoprecipitation. Four MABs (Be 8.7, Be 9.1, Be 9.2, De 13.4) recognized antigens with a Mr of 110 000 but did not mutually cross-react. The antigen recognized by MAB De 13.4 was identified as the ectoenzyme dipeptidyl peptidase IV (EC 3.4.14.-).     

Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody

We have produced a panel of monoclonal antibodies directed against nonlymphoid cells in central and peripheral lymphoid organs. In this paper we present the reactivity of one of these antibodies, ER-TR7. This antibody detects reticular fibroblasts, which constitute the cellular framework of lymphoid and nonlymphoid organs and their products. In frozen sections of the spleen incubated with this antibody, the red pulp and white pulp are clearly delineated. Furthermore, the major white pulp compartments--the follicles and periarteriolar lymphoid sheath as well as the marginal zone--are recognized by their characteristic labeling patterns. In lymph nodes, the capsule, sinuses, follicles, paracortex, and medullary cords are clearly delineated. In the thymus and bone marrow no such specialized compartments were demonstrated. ER-TR7 reacts with an intracellular component of fibroblasts. Since ER-TR7 does not react with purified laminin, collagen types I-V, fibronectin, heparan sulfate proteoglycan, entactin, or nidogen, it detects a hitherto uncharacterized antigen. The possible role of the ER-TR7 positive reticular fibroblasts in the cellular organization of peripheral lymphoid organs will be discussed.     

Characterization of a family of nuclear and chromosomal proteins identified by a monoclonal antibody

A monoclonal antibody (3C5) isolated from a mouse immunized with human chromatin stained the nuclei of all cultured cell types tested by indirect immunofluorescence. Experiments with HeLa and PtK1 cells demonstrated striking cell-cycle-related changes in the staining properties of the target antigen. A rapid increase in nuclear fluorescence was seen in prophase, with antigen located between the condensing chromosomes. In metaphase and anaphase cells antigen was present throughout the cytoplasm with the chromosomes apparently unstained. However, isolated metaphase chromosomes showed intense, peripheral staining. In telophase cells immunofluorescent staining was most intense among the decondensing chromosomes and by early G1 staining was predominantly nuclear. Nuclear fluorescence faded as cells progressed through interphase. By protein blotting and immunostaining, 3C5 recognized protein bands with subunit molecular weights of 130, 73, 50, 38, 32 and 22 to 25 kDa. These bands were present in all human and rodent cultured cell types tested. All bands were extracted by 6 M urea or 1% sodium dodecyl sulfate (SDS) but not by Triton X-100. Our results provide evidence against the involvement of a common carbohydrate moiety, in vitro proteolysis or non-specific cross reaction in this multi-banded pattern. The same family of proteins was detected in mitotic and interphase cells, suggesting that the changes in immunofluorescent staining through mitosis are due to changes in antigen accessibility. Subcellular fractionation experiments showed that all major bands were present in the nuclear fraction. Only two (50 and 32 kDa) were detected also in the post-nuclear membrane fraction and none were present in the soluble cytoplasmic fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human trophoblast glycoproteins defined by monoclonal antibody 1D2

A cell surface antigen has been defined by a monoclonal antibody 1D2, raised following immunisation with lectin-purified syncytiotrophoblast glycoproteins. 1D2 was nonreactive with any one of 8 common trophoblast proteins in immunodot. Analysis of nonreduced western blots of syncytiotrophoblast microvillous plasma membrane (StMPM) protein indicated that mAb 1D2 was reactive with a series of sialylated proteins with molecular weights of 16-22 kilodaltons. Immunoprecipitates of radiolabelled StMPM protein contained molecules that co-migrated with placental alkaline phosphatase in addition to those identified by western blotting. This set of human trophoblast molecules has not been previously identified by monoclonal antibodies; the antigenicity is widely distributed in human tissues.     

Novel conserved oligodendrocyte surface epitope identified by monoclonal antibody 4860

Oligodendrocytes are the myelinating cells of the central nervous system. They differentiate from oligodendrocyte precursor cells through several intermediate states that can be followed by characteristic morphological changes and the expression of marker molecules. However, most oligodendrocyte lineage markers demarcate either the precursor or the differentiated oligodendrocyte in restricted subcellular compartments. Here, we describe a novel marker of the oligodendrocyte lineage recognised by the monoclonal antibody clone 4860. It selectively labels the surfaces of differentiated oligodendrocytes in culture and clearly differs from other oligodendrocyte markers. Importantly, the 4860 epitope highlights developing white matter tracts in rodent and avian brains and thus represents a useful and conserved feature. The 4860 epitope is not associated with protein backbones as revealed by the related 487/L5 antibody. Furthermore, the epitope disappears upon lipid extraction from cryosections or inhibition of sphingolipid synthesis in cultured oligodendrocytes. Thus, we conclude that mAb 4860 represents a novel lipid-based oligodendrocyte marker.     

Basally located epithelial cell surface component identified by a novel monoclonal antibody technique

Tubular aggregates of glandular epithelial cells (gland fragments) were isolated from human endometrium by collagenase digestion of surrounding stroma, thus exposing the basal surfaces of the cells. Using these aggregates as immunogen, monoclonal antibodies could be derived that recognized basally located antigens. One such antibody, G71, is described, that binds to a basal epithelial cell antigen present in a variety of human epithelia. Epitope-bearing molecules in the range Mr 60 000-180 000 are present in two of the tissues studied, amnion and endometrium. The epitope is associated with areas of epithelial cell-extracellular matrix contact.     

Activation and immunorecognition by a monoclonal antibody of the tamoxifen-estrogen receptor complex

The interaction of tamoxifen with the estrogen receptor of fetal guinea pig uterus, the activation of the tamoxife-estrogen receptor complex and its immunorecognition by a monoclonal antibody raised against the human estrogen receptor is described in the present paper. The results show that: (1) the tamoxifen-receptor complex sediments at 8 S in low-salt and at 4.5 S in high-salt sucrose gradients, (2) this complex is partially recognized by the monoclonal antibody allowing the differentiation of two forms: the α form, which binds to the monoclonal antibody, and the β form, which does not react with it; (3) several factors such as time, temperature and high salt concentrations were capable of activating the tamoxifen-receptor complex, as determined by the increase of its binding to DNA-cellulose; (4) these factors also induced a partial transformation of the β form to the α form; (5) sodium molybdate inhibited both activation and transformation of the β into the α form. The correlation between activation and induction of the α form suggests that the monoclonal antibody recognizes selectively the activated form of the tamoxifen-receptor complex. These results indicate similar properties of the estrogen receptor when bound to either tamoxifen or estradiol; however, the differences observed in the behavior of the tamoxifen-receptor complex as compared with the estradiol-receptor complex. though quantitative rather than qualitative, suggest that the estrogen receptor is affected differently by tamoxifen and estradiol.     

Enterotoxic effects of Aeromonas sobria haemolysin in a rat jejunal perfusion system identified by specific neutralization with a monoclonal antibody.

Investigations into the pathogenesis of Aeromonas diarrhoea have demonstrated that several different cell-free products of motile aeromonads show enterotoxic activity in suckling mouse, rat, and rabbit assay systems. The relative contributions made by separate cytotoxic and cytotonic activities in the mixture produced by in vitro culture remains unresolved. Using a modified rat jejunal perfusion assay, we have studied the effects of A. sobria culture filtrates containing defined levels of haemolytic and cytotoxic activity and immunoreactivity for anti-cholera toxin. This material induced net water, potassium, and sodium loss with a rapid onset (less than 5 min) that was readily differentiated from the effects of purified cholera toxin (greater than 15 min). In filtrates containing up to 128 haemolytic and cytotoxic units of activity, the enterotoxic activity was neutralized by an anti-haemolysin/cytotoxin monoclonal antibody. No specific histological changes could be found in preparations perfused with enterotoxic material for up to 65 min. These findings indicate that the cytotoxic/haemolytic component of A. sobria culture filtrate is the dominant enterotoxic activity.     

Purification and chemical characterisation of membrane glycoproteins from rat thymocytes and brain, recognised by monoclonal antibody MRC OX 2

The MRC OX 2 monoclonal antibody recognises antigens present on rat thymocytes, brain, follicular dendritic cells in lymphoid organs, vascular endothelium, some smooth muscle and B-lymphocytes. The OX 2 antigens recognised by this antibody were purified from brain and thymus, by solubilisation with sodium deoxycholate, affinity chromatography with MRC OX 2 antibody and gel filtration. The purified brain and thymocyte OX 2 antigens were glycoproteins with apparent Mr 41000 and 47000 respectively as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Rabbit antisera raised against the purified antigens were analysed by radioimmunoassay and immunoperoxidase-staining of tissue sections. The brain and thymocyte OX 2 antigens were antigenically very similar to those on the other tissues. This indicates that the unusual pattern of distribution was not the result of fortuitous cross-reaction of the MRC OX 2 antibody, as the rabbit sera would be expected to recognise more determinants on the antigen than that recognised by the monoclonal antibody. The amino acid compositions of brain and thymus OX 2 antigens were very similar but with no distinguishing features. Carbohydrate compositions showed that the OX 2 antigens were highly glycosylated, with brain OX 2 antigen containing 24% and thymocyte OX 2 antigen 33% by weight of carbohydrate. Both OX 2 antigens contained carbohydrate residues typical of structures N-linked to asparagine but lacked galactosamine, indicating the absence of O-linked structures. Thymocyte OX 2 contained higher levels of galactose and sialic acid but less fucose than brain OX 2. Similar differences had been observed for brain and thymocyte Thy-1 antigens and were also observed in pooled glycoproteins purified by lentil affinity chromatography from these tissues, reflecting overall differences in the patterns of glycosylation in the two tissues. The OX 2 antigens showed many similarities to Thy-1 antigens in their odd patterns of distribution, characteristic migration on polyacrylamide gels in sodium dodecyl sulphate, and carbohydrate compositions. It is possible that OX 2 antigens, like Thy-1 antigens, have homologies with immunoglobulin domains. A possible role for OX 2 antigens in cell interactions necessary for tissue organisation is discussed.     

A monoclonal antibody identifies a glycoprotein complex involved in cell-substratum adhesion

The monoclonal antibody CSAT has been reported to perturb the adhesion of chick embryo cells to their substratum (Neff et al. [19]). Evidence is presented here that the antigen recognized by this monoclonal antibody is comprised of three membrane glycoproteins. The antigen is released from cells with non-ionic detergent and purified by monoclonal antibody affinity chromatography. When analysed by SDS-PAGE under non-reducing conditions, the antigen resolves into three components of apparent molecular weights 160 000 (band 1), 135000 (band 2), and 110 000 (band 3). Following reduction of each component, bands 1 and 2 migrate at slightly lower apparent molecular weights, while band 3 migrates at a higher apparent molecular weight, suggesting that band 3 has an internal disulfide bond. All three bands differ from one another as determined by peptide mapping and by immunologic cross-reactivity. It is postulated that the three glycoproteins function as a complex that plays a central role in cell-substratum adhesion.     

Development and distribution of a human B cell subpopulation identified by the HB-4 monoclonal antibody

Monoclonal antibodies have been successfully used to identify B cell differentiation antigens, few of which mark discrete B cell subpopulations. We have produced a monoclonal antibody, HB-4, against a cell surface antigen on the human B cell line, BJAB, which has an unusual distribution on normal lymphoid cells. HB-4, an IgM antibody, was found to react with an antigen that is expressed by a subpopulation of B cells, approximately 50% of natural killer cells, and not by other types of cells in bone marrow, blood, and lymphoid tissues. In two-color immunofluorescence assays, the HB-4-reactive antigen was found on less than 5% of immature IgM+ B cells in fetal liver and bone marrow and on 25% of B cells in fetal spleen. The HB-4 antibody reacted with 40% of IgM+ cells in newborn blood and 60% of B cells in adult blood. In contrast, only 2 to 26% of IgM+ B cells in the peripheral lymphoid tissues of adults were HB-4+. HB-4+ B cells could be induced to proliferate by cross-linkage of their surface immunoglobulins but not by T cell-derived growth factors. The subpopulation of activated B cells that is responsive to T cell-derived differentiation factors was HB-4-, as were plasma cells. The HB-4 antibody was reactive with some but not all B cell malignancies and cell lines, and not with malignancies or cell lines of other lineages. The HB-4 antigen may therefore serve as a useful nonimmunoglobulin marker for the identification of a subpopulation of mature resting B cells that are present in the highest frequency in the circulation.     

Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex

We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin.Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.     

Comparison of rat and human major platelet glycoproteins.

1. Using electrophoretic techniques combined with various detection methods we ascribed rat platelet glycoproteins (GPs) related to human GPIb, GPIIb and GPIIIa. 2. Rat GPIIb and GPIIIa crossreacted with rabbit polyclonal antibodies against human GPIIb and GPIIIa. 3. Species differences in glycosylation of GPs were shown using various lectins. 4. Molecular mass of rat major GPs was determined by SDS-PAGE (unreduced, reduced, kDa): GPIb (200, 166/26), GPIIb (140, 120/32) and GPIIIa (96, 106). 5. Isoelectric points of rat GPIIb and GPIIIa are shifted to the alkaline region as compared to human related GPs.     

A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-beta-lactam

We report the first example of a monoclonal antibody-catalysed hydrolysis of a beta-lactam where the antibodies were generated by a simple transition-state analogue. A rat monoclonal antibody (1/91c/4d/26) generated by using an acyclic 4-nitrophenylphosphate immunogen catalysed the hydrolysis of corresponding 4-nitrophenyl carbonates but, more importantly, also catalysed the hydrolysis of N-(4-nitrophenyl)-azetidinone at pH 8 with k(cat)=8.7 x 10(-6)s(-1) and K(M)=35 microM. This is the first example of a rat monoclonal catalytic antibody.     

Trichinella spiralis: a 76-kDa excretory/secretory larval antigen identified by a monoclonal antibody

Spleen cells from BALB/c mice were fused with myeloma cells following infection of the mice with Trichinella spiralis larvae and an ip booster injection with larval homogenate antigen. A monoclonal antibody (Mab), designated as TS 3G6 which did not react with sera or tissue extracts from noninfected mice, rats, and guinea pigs, was selected for further studies because of its high activity and specificity. When tested in ELISA TS 3G6 did not cross-react with Ascaris suum, A. lumbricoides, Toxocara canis, E. granulosus (larvae), Trichiuris suis, or T. ovis. Western blot analysis showed that Mab 3G6 recognized an antigen of 76 kDa located in the stichosome of the larvae as well as on the surface of the larval cuticle. Digestion of a larval extract with different enzymes suggests that the Mab TS 3G6 corresponding epitope is a polypeptide. The TS 3G6 antigen was detected in culture supernatants of Trichinella muscle larvae and in sera of experimentally infected animals using a sensitive ELISA assay. This secretory antigen also seemed to induce a specific immune response in the host since sera from infected animals could block the binding of Mab TS 3G6 to its target antigen when tested in a competitive ELISA.     

A rat oocyte differentiation antigen (OA-1) detected by a monoclonal antibody

Cell surface antigenic changes associated with differentiation of the rat oocyte and early embryo have been demonstrated with a monoclonal antibody (anti-OA-1). Antigen is first detectable coincident with initiation of oocyte growth, is a constant feature of all growing oocytes and displays a redistribution during meiotic maturation. Following fertilization, antigen is detectable on the surface of the embryo through the four-cell stage. This first monospecific marker for the rat oocyte and embryo should prove useful in probing structure/function relationships in oocyte growth, meiotic maturation fertilization, and/or early embryonic development.     

Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration

Kinesin is a microtubule-activated ATPase thought to transport membrane-bounded organelles along MTs. To illuminate the structural basis for this function, EM was used to locate submolecular domains on bovine brain kinesin. Rotary shadowed kinesin appeared rod-shaped and approximately 80 nm long. One end of each molecule contained a pair of approximately 10 x 9 nm globular domains, while the opposite end was fan-shaped. Monoclonal antibodies against the approximately 124 kd heavy chains of kinesin decorated the globular structures, while those specific for the approximately 64 kd light chains labeled the fan-shaped end. Quick-freeze, deep-etch EM was used to analyze MTs polymerized from tubulin and cross-linked to latex microspheres by kinesin. Microspheres frequently attached to MTs by arm-like structures, 25-30 nm long. The MT attachment sites often appeared as one or two approximately 10 nm globular bulges. Morphologically similar cross-links were observed by quick-freeze, deep-etch EM between organelles and MTs in the neuronal cytoskeleton in vivo. These collective observations suggest that bovine brain kinesin binds to MTs by globular domains that contain the heavy chains, and that the attachment sites for organelles are at the opposite, fan-shaped end of kinesin, where the light chains are located.