A monoclonal antibody against a novel intracellular neural antigen expressed differentially in neural cell types

A monoclonal antibody, GTE52, was isolated from a fusion of myeloma cells with the lymphocytes of a mouse immunised with enzymatically dissociated guinea-pig trigeminal ganglion cells. GTE52 was found to stain the nuclei of satellite cells and Schwann cells, but not neurones, in the peripheral nervous system of guinea-pig and mouse. In the central nervous system GTE52 labelled glia and some neurones. Double-labelling experiments on primary cultures of optic nerve using antibodies to glial fibrillary acidic protein, galactocerebroside and fibronectin showed that GTE52 labelled a sub-population of astrocyte glia, possibly corresponding to the type 2 astrocytes, oligodendrocytes and not fibroblasts. Adult non-neural tissue was not stained by GTE52 with the exception of the smooth muscle of the gut. However, during development of the guinea-pig the antigen recognised by GTE52 was expressed in all cells of 16-day embryos but was lost from the tissues studied, which were not stained in the adult, from about embryonic day 60 onwards.     

Presence of a novel epithelial antigen on rat cerebellar cell lines as detected by a monoclonal antibody

We have derived a monoclonal antibody, MCAb 51, following immunization of BALB/c mice with a Rous sarcoma virus-transformed rat cerebellar cell line. When assayed by immunofluorescence on primary rat cerebellar cultures MCAb 51 recognizes only islands of cells with an epitheloid morphology. Double-label immunofluorescence experiments with MCAb 51 and antisera to tetanus toxin, glial fibrillary acidic protein, galactocerebroside and fibronectin reveal that these cells do not appear to be neurons, astrocytes, oligodendrocytes, or fibroblasts, respectively. In contrast, cells from kidney, liver, tongue and choroid plexus epithelium are positive for the antigen. Of 12 Rous sarcoma virus-transformed cell lines, in contrast to 2 out of 9 chemically transformed lines, 11 exhibit the MCAb 51 antigen. These findings demonstrate that MCAb 51 recognizes an epithelial cell surface marker. Possible explanations for the difference in the expression of the antigen on Rous sarcoma virus and chemically transformed neural lines are discussed.     

Oligodendrocyte cell surface recognized by a novel monoclonal antibody specific to sulfatide.

A rat monoclonal antibody (OL-1) was obtained by in-vitro immunization of rat spleenocytes with paraformaldehyde-fixed primary cultured glial cells derived from newborn rat brain and subsequent fusion with a rat myeloma cell line. The antibody secreted by the hybridoma immunostains live rat and mouse oligodendrocytes in primary and secondary cultures. The antibody binds specifically to oligodendrocytes and myelin structures in-situ. Radioimmunolabelling assays with a number of purified glycolipids offer thin layer chromatography separation show that OL-1 antibody binds strongly to sulfatide and to a lesser extent to galactosyl diglyceride.     

抗人脑胶质瘤单克隆抗体的制备及识别抗原的提取

目的：制备新的抗人脑胶质瘤单克隆抗体（monoclonal antibody,McAb)，并提取其识别抗原。方法：以人脑恶性胶质瘤细胞系SHG－44为抗原免疫Balb/c小鼠，通过杂交瘤技术获得稳定分泌抗胶质瘤McAb的杂交瘤细胞株。以酶联免疫吸附测定及免疫组化等方法研究McAb的特性。通过亲和层析法提取该McAb识别的抗原。结果：得到1株稳定分泌抗体、效价高的杂交瘤细胞株，命名为SU－2000。鉴定表明该McAb属IgGl亚类，效价高，特异性高。识别的抗原位于胞膜，可能属于神经外胚层抗原；通过亲和层析法成功地提取出该抗原，十二脂硫酸3钠－聚丙烯酰胺凝胶电泳证实提取的抗原纯度高，分子量约为75kDa。结论：SU－2000 McAb及其识别抗原可进一步用于相关实验研究。     

A monoclonal antibody to the endothelium of rat brain microvessels

A rat brain fraction enriched with microvessels was used as the immunogen to produce mouse hybridoma cell lines secreting monoclonal antibodies. One of these antibodies, selected from 156 supernatants by enzyme-linked immunosorbent and immunofluorescent assays, reacted only with the endothelium of microvessels in the brain. The endothelium-specific antibody labelled the cytoplasm of microvascular endothelial cells, their luminal membranes, and an extracellular layer, the endocapillary coat, which covered the luminal surface of these cells. In the kidney, the antibody specifically stained the brush border of the proximal tubuli, and in the liver, the antibody specifically stained bile canaliculi. This demonstrates that 3 morphological structures with important transport functions, cerebral microvascular endothelium, brush border of kidney proximal tubuli, and liver bile canaliculi, express the same epitope.     

A novel monoclonal antibody against carbohydrates of L1 cell adhesion molecule causes an influx of calcium in cultured cortical neurons

We have studied the function of carbohydrates of the L1 molecule, a member of the immunoglobulin superfamily of adhesion molecules, using a novel monoclonal antibody, mAb-L1(2E12), against L1 molecule. This antibody was specific for the 200 kDa component of mouse L1 molecule and its epitope was N-linked for complex-type oligosaccharides. The mAb-L1(2E12) was found to induce a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured mouse embryonic cortical neurons. The rise in [Ca²⁺]_i was dependent on the concentrations of mAb-L1(2E12). The rise seemed to be due to an influx of extracellular Ca²⁺ as EGTA treatment abolished it. Both cadmium and nifedipine blocked the effect of mAb-L1(2E12), suggesting the Ca²⁺ influx was through voltage-operated Ca²⁺ channels, particularly L-type Ca²⁺ channels. These results provide an important insight for understanding the mechanisms by which oligosaccharides of the L1 molecule influence various functions of neural cells.     

Establishment of a monoclonal antibody against senile plaques and its application for immunohistological and immunoelectron microscopical studies in the brain of the elderly

Summary A monoclonal antibody (Am-3) was produced against senile plaques in the brain of a patient with Alzheimer's disease. Am-3 was reactive with senile plaques of typical, primitive and diffuse type not only in the brain used as immunogen, but also those in the brain of 15 out of 25 autopsy cases of the aged people. Moreover, Am-3 was also reactive with granular materials of various sizes scattered in the 1st, 3rd and 4th layers of the cerebral cortices of the cases with severe dementia. Am-3 was also reactive with vessel wall of the congophilic angiopathy. By immunoelectron microscopic examination, Am-3 was positive with amyloid fibril in the core and crown of senile plaques, and in the congophilic angiopathy.     

The monoclonal antibody 23E9 defines a novel developmentally-regulated Schwann cell surface antigen

The present study describes the identification and partial characterization of a novel Schwann cell surface molecule by means of a monoclonal antibody (23E9). The 23E9 antigen was found in association with Schwann cells of the peripheral nerve but not with sensory neurons and satellite cells of the dorsal root ganglion. The expression of the antigen in the sciatic nerve starts after birth, is high around postnatal day 8 and becomes down-regulated towards the adult stage. This suggests that it may be involved in the induction of myelin formation. On Western blots, the antibody identified two major bands of approximately 27 and 42 kDa. Treatment of cultured Schwann cells with forskolin, an agent known to mimic neuronal contact in vitro, stimulated the up-regulation of the antigen. This implies that the expression of 23E9 is induced and maintained by axon-derived signals in vivo. Comparison of the presented data with the literature suggests that we have identified a novel cell surface molecule not previously characterized in the context of Schwann cell biology. To clarify the molecular identity of the antigen and define its physiological relevance, the antibody will be used in future studies for immunoprecipitation and functional in vitro assays.     

Heterogeneity in migrating neural crest cells revealed by a monoclonal antibody

A monoclonal antibody, GlN1, obtained by immunization with extracts of the 14 d embryonic quail nodose ganglion, is described. GlN1 recognizes an antigenic determinant present in virtually all the satellite cells of the peripheral ganglia, all Schwann cells of the peripheral nerves, and in subpopulations of sensory and autonomic neurons of embryonic and adult quails and chickens. The molecular weight of the antigen(s) revealed by GlN1 in embryonic day 12 quail dorsal root ganglion (DRG) cultures is around 80 kDa. In the neural crest, GlN1 determinant is found as soon as the crest cells leave the neural primordium. Only a proportion (25%) of the migrating neural crest cells carry the antigen. This demonstrates that the neural crest is composed of a heterogeneous population of cells from its early migratory stages. Being selectively distributed on neural crest cells and its derivatives, the GlN1 determinant may be considered as a "differentiation antigen" that will be useful in further studies on cell-line segregation during the ontogeny of the PNS.     

Endogenous benzodiazepine-like molecules in the human, rat and bovine brains studied with a monoclonal antibody to benzodiazepines

The anti-benzodiazepine monoclonal antibody 21-7F9 has been used for the identification and study of endogenous benzodiazepine-like molecules in the human, rat and bovine brains. A sandwich radioimmunoassay has been designed for the quantification of the membrane-bound endogenous benzodiazepine-like molecules. The localization of these molecules is not restricted to the brain tissue. They are also present in kidney, liver and spleen as well as in the neuroblastoma × glioma NG108-15 hybrid cells line. Immunoblots show benzodiazepine-like immunoreactivity in the membrane proteins of all these tissues. The membrane-bound benzodiazepine-like molecules are resistant to limited proteolysis of the membranes. Moreover, this treatment increases the binding of the monoclonal antibody 21-7F9 to the membranes, probably by exposing sites that normally are not accessible to the antibody. Immunocyto experiments show that benzodiazepine-like molecules are also present in samples of human cerebella that have been stored in paraffin since 1940, 15 years before the first chemical synthesis of benzodiazepines. The results indicate that the cerebellar benzodiazepine-like molecules recognized by the antibody are the product of biological (not chemical) synthesis. Benzodiazepine-like immunoreactivity has also been detected in NG108-15 cells that have been cultured for 3 months ib serum-free medium. These results suggest that the cells could biosynthesize benzodiazepine-like molecules.     

Ultrastructural localization of monoclonal antiphospholipid antibody binding to rat brain

Antiphospholipid antibodies (aPL), in the presence or absence of systemic lupus erythematosus, are associated with a number of neurologic complications. However, the role aPL play in pathology is unclear. A thrombotic etiology seems likely for many associated disorders, but not for others. Here we describe aPL-reactive sites in the central nervous system (CNS). Previously, using light microscopy, we showed direct binding of two monoclonal phosphatidylserine-reactive antibodies (aPS) to ependyma and myelin of fixed cat brain. In this study we determined the ultrastructural localization of their binding sites in rat CNS using immunogold electron microscopy techniques. Both monoclonal antibodies reacted strongly with myelin, preferentially with the major dense line formed by the cytoplasmic apposition of the oligodendrocyte plasma membrane. Both monoclonal antibodies also reacted with an antigen that appears associated with the axoneme in cilia of ependymal and choroid plexus epithelium. One monoclonal aPS also showed some reactivity with brain vascular endothelium and reacted slightly with mitochondria, while the other aPS did not react with these structures. While the etiology of aPL-associated neurologic disorders remains unclear, our data suggest possible target sites within the CNS with which aPL can react.     

Differentiation potential of a monoclonal antibody-defined neural progenitor cell population isolated from prenatal rat brain by fluorescence-activated cell sorting

We have studied the differentiation potential in vitro of a subpopulation of neural progenitor cells from BDIX-rat brain. These cells transiently express a cell surface determinant (CSD) specified by monoclonal antibody (Mab) RB13-2 (Kindler-R?hrborn, A. et al., Differentiation 30 (1985) 53-60), and recently identified as a set of O-acetylated gangliosides (Reinhardt-Maelicke, S. et al., submitted) also recognized by Mab D1.1 (Levine, J.M. et al., J. Neurosci., 4 (1984) 826-831) and partly by Mab JONES (Schlosshauer, B. et al., J. Neurosci., 8 (1988) 580-592), respectively. As analyzed by immunofluorescence, Mab RB13-2 binding brain cells (prenatal days 11-22; postnatal days 7 and 89) were localized in different areas of the proliferative ventricular layer of the prenatal cerebrum and in the external granular layer of the early postnatal cerebellum. No Mab RB13-2 positive brain cells were found in adult brain. Following their isolation by fluorescence activated cell sorting on prenatal day 18, the differentiation potential of Mab RB13-2 binding brain cells was studied by double-immunofluorescence analysis under different conditions of monolayer culture. In the presence of 10% fetal calf serum (FCS), these cells differentiated into glial fibrillary acidic protein (GFAP) positive flat astrocytes, whereas neurons (neurofilament-positive) and a smaller number of stellate astrocytes (GFAP-positive) developed in a chemically defined medium containing 0.5% FCS. Neural progenitor cells binding Mab RB13-2 may thus either retain more than one option for differentiation into specific cell types, or the expression of the CSD specified by Mab RB13-2 may be common to more than one subset of neural progenitor cells (with or without predetermined unidirectional differentiation pathways) whose survival and/or proliferative behavior could be differentially affected by microenvironmental conditions.     

Preparation of a monoclonal antibody to citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain

Using hybridoma technology, an IgM monoclonal antibody (mAb), designated as F95, was developed against a deca-citrullinated peptide (DCP) consisting of 10 citrulline residues and a carboxyl Gly-Gly-Cys through which DCP was covalently linked to an activated carrier protein, keyhole limpet hemocyanin (KLH). Clones were selected on the basis of not reacting with human unmodified and noncitrullinated myelin basic protein (MBP), MBP-C1, but reacting well with human citrullinated MBP (MBP-C8). When tested by ELISA, this mAb demonstrated minimal reactivity with human MBP-C1, varying reactivity with the C2-C5 isomers of human MBP, moderate binding with guinea pig MBP-C8, and strong reactivity with human MBP-C8. By ELISA, mAb F95 was directed predominantly against citrulline, not MBP, as revealed by its binding to DCP linked with activated KLH, bovine serum albumin (BSA), or ovalbumin (OA), but not with KLH, BSA, or OA alone. Immunohistochemistry of normal human brain demonstrated that F95 stained central nervous system myelin and a subset of astrocytes. Given the citrulline-directed features of mAb F95, this immunohistochemical pattern suggests that certain astroglial filaments expressing glial fibrillary acidic protein also contain citrulline-bearing components. These potentially implicate citrullinated proteins, notably in astroglial filaments, in a variety of normal and pathological neurobiological processes.     

Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 26, and D-37 MG, with 2.9×10⁴, 1.5×10⁵, 8.6×10⁵ and 1.59×10⁶ EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 26 by 39%. U-343 MGa Cl 26 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MGH, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.Supported by Grants CA-11898, NS-20023, CA-43722, and the Association for Brain Tumor Research (MHW, PAH)     

Characterization of a monoclonal antibody, Namu mAb, which reacts to the subventricular zone in mouse brain

The lateral ventricle in adult mammalian brain is widely acknowledged as one of the areas that undifferentiated neural cells such as neural stem cells and neural progenitor cells inhabit. However, immunological aspects of neural stem cells in the lateral ventricle are still under debate. Here, we report the generation and characterization of a novel monoclonal antibody (mAb), called Namu mAb, which stains the subventricular zone in the lateral ventricle of adult mouse brain. Namu mAb reacted to the cells in the subventricular zone and never reacted to differentiated neural cells such as neurons and glial cells such as astrocytes and oligodendrocytes. Its reaction pattern for the subventricular zone and the neurospheres was similar to that of Nestin and glial fibrillary acidic protein mAbs. Namu mAb recognition molecule, Namu antigen, was a 50 kDa protein present in the cytoplasmic fraction of mouse brain, and its expression was clearly observed in neurospheres cultured in the presence of epidermal growth factor, but it was never or only weakly induced in the presence of basic fibroblast growth factor or leukemia inhibitory factor. Collectively, it is concluded that Namu mAb specifically reacts to undifferentiated neural cells in mouse brain.     

Monoclonal antibody directed against glutaraldehyde conjugated glutamate and immunocytochemical applications in the rat brain

Like other small-sized neurotransmitter molecules, glutamate (Glu) was conjugated to carrier proteins via glutaraldehyde (G). Human serum albumin (HSA) and thyroglobulin (TH) conjugates were alternately injected into mice. When a relevant immune response was obtained for antibody affinity and specificity, hybridization of spleen activated lymphocytes with SP2/O/Ag myeloma cells was performed. Supernatant culture media of hybridomas were tested for the presence of anti-conjugated Glu antibodies with our ELISA method. Selected hybridomas giving good antibody affinity and specificity were then cloned by the limiting dilution technique. Using DEAE-chromatographed ascites fluid, Glu reactivity was observed on the cortex and the hippocampus. Staining obtained with this monoclonal antibody was in agreement with that observed with previous polyclonal antisera directed against conjugated Glu or monoclonal anti-γ-glutamyl-Glu antibody.     

Oligodendroglia development in cell culture as monitored with a monoclonal antibody

A new marker for young oligodendrocytes has been identified by a monoclonal antibody (mOg-1, IgM isotype) prepared from cerebellar plasma membrane stimulated mouse lymphocytes. mOg-1 reactive cells in the mouse cerebellum first appear at day 19 of gestation. Future white matter layers of fixed sections of neonatal rat cerebellum were labeled with mOg-1. Although EM analysis has shown cell-surface binding by presumptive oligodendroglia in neonatal cerebellum, the antibody does not bind to compact myelin. In cell cultures prepared from 6-d-old mice, 1.1% of the cells bound mOg-1 after 3 d in culture, but up to 5% of the cells bound mOg-1 after 2 weeks in culture. Of these same Og-1-positive cells, 69% bound anti-galactocerebroside and 65% bound anti-myelin basic protein. After a week in culture Og-1-positive cells often produced lamellar sheets extending a millimeter over the polylysine substratum in the absence of normal myelin formation. mOg-1 recognizes a cell-surface determinant distinct from well-characterized oligodendroglial molecules (galactocerebroside, sulfatide and myelin basic protein) that is expressed early in oligodendrocyte development. The antibody has been used to follow the maturation of oligodendrocytes in cultures of both normal and jimpy mouse cerebellum.     

A novel monoclonal antibody recognizes a previously unknown subdivision of the habenulo-interpeduncular system in zebrafish

The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of almost all vertebrates. We prepared a monoclonal antibody (6G11) which very specifically recognizes only a part of this system. 6G11 is a monoclonal antibody prepared from a neuronal membrane protein in adult zebrafish brain. In western blot analysis of the adult zebrafish brain, the antibody recognized a 95 kDa protein, and the class of the antibody was determined to be IgM. The 6G11 antigen was not detected in zebrafish muscle, intestine, testis or ovary. A group of neurons stained by the 6G11 antibody was located in the caudomedial part of the zebrafish habenula. The 6G11-immunopositive neurons extended their axons into the fasciculus retroflexus (FR). One group of immunopositive neurons projected toward the interpeduncular nucleus (IPN), especially to the intermediate and the central subnucleus (type 1 neuron). The other group projected to the ventral midline at the level of the raphe nucleus; these axons passed ipsilaterally beside the IPN and converged in the ventral midline under the raphe nucleus (type 2 neuron). Both type 1 and type 2 fibers are relatively minor components of the FR. Little has previously been known about this topological pattern in any species. The 6G11 monoclonal antibody could be a useful tool for expanding knowledge of the habenulo-interpeduncular system.