Mammalian brain antigens defined by monoclonal antibodies

Seventy-two hybridoma lines that produce monoclonal antibodies to molecules of a rat synaptosomal plasma membrane fraction (SPM) were generated. The topographical distribution of the antigens in the cerebellum and other areas of the brain was studied by light microscopy immunocytochemistry. Some of the antibodies recognize exclusively neuronal antigens while others bind to specific glial molecules. Some of the antigens have a distribution limited to certain classes of neurons. There are antigens localized in both the cell bodies and processes while others are present only in the latter. Immunoblots of SPM proteins indicate that some antibodies react specifically with one or few of these proteins while other antibodies react with many. The latter antibodies also generally react with many brain cell types. Particularly interesting is the monoclonal antibody 8-6A2 which binds to many SPM proteins but only recognizes large neurons with long axons. A further characterization of the antigens was done by enzyme-linked immunosorbent assays and immunoblots of known purified proteins. The results indicate that antibody 8-2H5 binds specifically to clathrin, 8-7A5 to actin, 8-1E7 to the glial fibrillary acidic protein and both 8-3A5 and 7-2C12 to collagen. In contrast, the antibodies 4-4C3, 2-4H3, 4-4G7 and 6-6A8 bind to antigenic determinants present in many purified proteins.     

Inhibition of rat autoimmune T cell activation by monoclonal antibodies

The essential requirement for adoptive transfer of autoimmune diseases such as experimental allergic encephalomyelitis (EAE) by T lymphoblasts from established T cell lines, is a prior activation of these cells by autoantigen or mitogen. We have investigated the possibility of modulating this activation process by using monoclonal antibodies directed against rat leukocyte differentiation antigens. We report here that antigen-driven activation of autoimmune, encephalitogenic T cells from established myelin basic protein (MBP)-specific rat T cell lines can be inhibited by some, but not all, antibodies against RT1.B Class II restriction elements. In addition, monoclonal antibodies with specificity for rat leukocyte common antigen (OX-1) and T cell differentiation antigens W3/13 and W3/25 are inhibitory, while monoclonal antibody OX-8 with specificity for T cytotoxic/suppressor cells has no effect. We also observed that concanavalin A-induced activation of the T cells is more resistant to the inhibitory effect of monoclonal antibodies, and can be blocked effectively only by antibody OX-1. This demonstration that autoimmune T cell function can be inhibited by monoclonal antibodies points the way in suggesting cellular targets for immunotherapeutic purposes.     

Preferred use of primary radiolabeled anti-platelet monoclonal antibodies. Comparison of immunoblotting methods for the analysis of functional domains on human platelets.

Several methodologies used for the identification and characterization of platelet receptors for antiplatelet monoclonal antibodies are compared. Two antiplatelet monoclonal antibodies, are investigated due to their potent effects on human platelet function. A platelet-activating monoclonal antibody, called M.Ab.F11, is able to induce platelet aggregation and granular secretion. A platelet-inhibitory monoclonal antibody, named G10, strongly blocks the platelet aggregation and granular secretion induced by M.Ab.F11, as well as by physiological agonists. In order to identify the specific antigens recognized by these monoclonal antibodies, we tested a number of immunostaining methods. Comparison of various procedures revealed that a high degree of nonspecific interactions with platelet proteins occurred when the commonly used secondary reagents, protein A and radiolabeled or enzyme-conjugated secondary antibodies interacted with the platelet proteins either in the presence or absence of primary monoclonal antibodies. On the other hand, we observed a high degree of specificity and selectivity when only radiolabeled anti-platelet monoclonal antibodies were used as single reagents. It is established that M.Ab.F11 interacts with the platelet membrane proteins of 32 and 35 Kd, and M.Ab.G10 recognizes 100 Kd protein, which corresponds to GPIIIa molecule.     

Improved adhesion and proliferation of human endothelial cells on polyethylene precoated with monoclonal antibodies directed against cell membrane antigens and extracellular matrix proteins.

Endothelial cell seeding may improve the patency of synthetic vascular grafts provided that platelet reactivity of nonendothelialized sites is not increased. We have investigated if surface-adsorbed monoclonal antibodies directed against endothelial cell membrane proteins and against extracellular matrix proteins promote the adhesion and proliferation of cultured human endothelial cells, without causing platelet deposition at non-endothelialized sites. Adhesion of endothelial cells onto polyethylene coated with monoclonal antibodies directed against endothelial cell-specific membrane antigens, integrin receptors and glycoprotein CD31 was equal to or higher than adhesion onto fibronectin-coated polyethylene. Endothelial cells did not proliferate on these surface-adsorbed antibodies. However, pre-coating of polyethylene with mixtures of endothelial cell-specific monoclonal antibodies and monoclonal antibodies directed against fibronectin or von Willebrand factor, resulted in relatively high adhesion and optimal proliferation. Platelet reactivity of the polyethylene surface was found to significantly increase after adsorption of fibronectin, endothelial cell-specific monoclonal antibody or its Fc fragments. In contrast, adsorption of F(ab')2 fragments of endothelial cell-specific monoclonal antibody did not promote platelet deposition. Therefore, it is concluded that coating of vascular graft materials with mixtures of F(ab')2 fragments of monoclonal antibodies specifically directed against endothelial cells and against extracellular matrix proteins may be an effective way to both promote the growth of seeded endothelial cells and limit platelet-graft interaction.     

Identification and characterization of differentiation-dependent Schwann cell surface antigens by novel monoclonal antibodies: Introduction of a marker common to the non-myelin-forming phenotype

In an attempt to identify and characterize novel Schwann cell surface molecules with putative functions during development, maintenance, and regeneration of the peripheral nervous system (PNS), we have produced monoclonal antibodies against viable neonatal rat Schwann cells. Using a sensitive live cell ELISA protocol, three monoclonal antibodies reactive with cultured Schwann cells, designated 27B10, 26F2, and 27C7 were isolated. The 27B10 and 26F2 antibodies specifically labelled forskolin-stimulated secondary Schwann cells in vitro as determined by live cell ELISA implying that the expression of the antigens in situ is regulated by axonal contact. The observation that the antigens seemed to be associated with both Schwan cell phenotypes clearly discriminated them from the well characterized myelin proteins as well as from molecules known to be confined to the non-myelin-forming phenotype. Interestingly, both antigens were found to be concentrated at the nodes of Ranvier. Further studies therefore have to show whether the identified antigens share structural or functional homology with adhesion or channel molecules, which display a similar distribution. Following transection of the adult sciatic nerve, the 26F2 antigen was rapidly down-regulated in the distal nerve stump. The 27C7 antibody reacted with an 80 kDa cell surface molecule common to non-myelin-forming Schwann cells. No differences in expression of the antigen between forskolin-treated and untreated Schwann cells in vitro were found, suggesting that the antigen is expressed independently from axonal contract. Two weeks after nerve transection in the absence of myelinating Schwann cells, the antigen was associated with S-100-positive Schwann cells of the distal nerve stump. The antigen was found to be expressed also by non-neuronal tissues, the level fo the protein declined towards the adult stage. Comparison of the 27C7 antigen with previously described marker molecules suggests that we have identified a novel Schwann cell surface antigen of the non-myelin-forming phenotype. GLIA 19:213–226, 1997. © 1997 Wiley-Liss, Inc.     

Rat NGF receptor is recognized by the tumor-associated antigen monoclonal antibody 217c.

The expression of the epitope recognized by the tumor-associated antigen monoclonal antibody 217c increased on cultured rat Schwann cells with time. The same phenomenon has previously been reported for the rat nerve growth factor (NGF) receptor by using monoclonal antibody 192-IgG. The distribution of 217c antibody immunoreactivity closely paralleled that observed for NGF receptors on NGF-primed pheochromocytoma (PC12) cells in culture and a number of central neurons in vivo. Immunoprecipitation of affinity-labeled NGF receptors was used to examine whether these two antibodies shared common or unique antigens. Both the quantitative data and the electrophoretic mobility of the immunoprecipitated 125I-NGF/receptor complex indicate that the antigen recognized by the 217c mAb monoclonal antibody is, in fact, the NGF receptor. Furthermore, binding studies indicated that 192-IgG and 217c recognize different epitopes on the same molecule. The characterization of the 217c antibody should provide a valuable new tool in the study of NGF receptors.     

Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies

This study describes the identification and differentiation of neonatal rat retinal cells in monolayer cultures. A panel of monoclonal antibodies was used as a molecular probe of both cell type and developmental stage. Previously described cell-type specific monoclonal antibodies were used to label rod photoreceptors, horizontal cells, amacrine cells or ganglion cells. Two new antibodies that react with rat retina are described. The first, RET-G7, reacts with a cytoplasmic antigen of Muller glia, astrocytes and some horizontal cells. The second, RET-B2, reacts with bipolar cells and photoreceptor inner segments. Two main findings are presented. The first is that each of the major subclasses of retinal neurons have been unambiguously identified in these cultures. The morphology of some subclasses was very characteristic. All photoreceptors, as defined by reactivity with antibody RET-P1, were small spherical cells with one or fewer processes. Horizontal cells, as defined by reactivity with antibody B-1, were large with a characteristic multipolar network of processes. Bipolar and amacrine cells, on the other hand, were of similar size and could only be distinguished on the basis of immunocytochemical labeling. The second finding is that while RET-B2 antigen appeared on bipolar and photoreceptor cells after about 5 days in culture, several Muller cell and photoreceptor antigens were not expressed in monolayer cultures. The results suggest that the expression of some molecules in culture is the result of properties intrinsic to the cells whereas expression of others depends upon extrinsic factors or cell interactions that may not be present in monolayer cultures.     

Novel NGF-induced proteins in PC12 cells: immunological evidence for their presence in brain nerve endings using a single monoclonal antibody.

A monoclonal antibody, S-11D9, detected a group of novel polypeptides whose expression was induced 6 hr after incubation of PC12 cells with nerve growth factor. The antigens also were visualized by immune precipitation and Western blotting in synaptic vesicles, clathrin-coated vesicles, and synaptic plasma membrane prepared from bovine brain. A large polypeptide was detected on the synaptic plasma membrane; an intermediate size protein was visualized on the plasma membrane and on both synaptic and clathrin-coated vesicles, while a smaller molecule was found only on brain Golgi-enriched membrane preparations. Immunofluorescence labeling with S-11D9 on nerve growth factor-stimulated PC12 cells showed the antigens distributed in the cytoplasm and concentrated in discrete areas on the tip of most neurites. The particular distribution of these proteins in vesicles and synaptic plasma membrane and the finding that the different antigens share a common epitope recognized by a single monoclonal antibody open the possibility that these molecules are related markers for organelle transport pathways in nerve cells.     

A chick neural antigen identified by monoclonal antibodies

A monoclonal antibody technique has been used to locate a neural antigen which appears to be involved in developmental processes.

Hybridomas were prepared using chick embryo sympathetic neurons as an immunogen and one clone, H3, was found to secrete antibodies which bound to neurons of the peripheral and central nervous system. The antibodies bound to both membrane and cytoplasmic sites of neurons but only to cytoplasmic sites of glial cells. When added to newly prepared cultures of embryonic sympathetic neurons the H3 antibodies impaired both neurite outgrowth and long-term neuronal viability. No such effect was seen when the antibodies were added to established, differentiated neurons.     

A monoclonal antibody distinguishes anterior horn cells of human embryonic spinal cord during a transient period of development.

Monoclonal antibodies were prepared by using the anterior horn region of human embryonic spinal cord as immunogen. To increase the specificity of the immune response towards the anterior horn cells, mice were first injected with antigens from the posterior horn and then immunosuppressed with cyclophosphamide; subsequently antigens from the anterior horn were injected. One of the monoclonal antibodies recognizes a small population of anterior horn cells of human embryonic spinal cord during a transient period of development (9-10th embryonic week); these cells are probably motoneurons according to their location in the spinal cord, their positive staining for acetylcholinesterase and their large nuclei. The staining pattern has a special axial distribution as it is limited to the cervical and thoracic regions of the spinal cord. The antibody is species-specific and shows a high degree of tissue specificity. Since this antibody distinguishes a small group of anterior horn cells in the spinal cord during a specific developmental stage, it opens stimulating perspectives for further investigation on the nature of the antigen and its putative role during the development of the human embryonic spinal cord.     

Antibody response to different antigenic sites on measles virus surface polypeptides in patients with multiple sclerosis

Antibodies to different antigenic sites on measles virus (MV) surface proteins were examined in sera from 34 HLA-typed age- and sex-matched pairs of multiple sclerosis (MS) patients and control subjects by a previously developed competitive enzyme immunoassay in which human sera and monoclonal MV-specific antibodies compete for binding to the same antigen. Antibodies specific to 4 partly overlapping sites on the haemagglutinin protein and two sites on the fusion protein of MV were measured in addition to previously determined total antibody titres and antibody levels were compared between groups. MS patients had significantly higher levels of antibodies in six out of seven comparisons. Both the MS and the control group were further divided to subgroups with HLA-Dw2, -Dw1, -A2, -A3 and -B7 histocompatibility antigens and MV antibody levels in these groups were compared. The presence of the HLA-A2 or -A3 antigen did not have a significant effect on the antibody levels but both the HLA-Dw2 and -B7 antigens were associated with elevated antibody levels to antigenic sites defined by monoclonal antibodies. This was also supported by higher frequencies of these alleles in subgroups with high antibody titre both in MS patients and controls. Moreover, presence of Dw1 antigen was associated with lower mean titres especially against the fusion protein in control patients. The findings indicate that HLA antigens can control immune response to separate sites on a protein antigen either directly as immune response genes or through linkage to other genes.     

A monoclonal antibody against vimentin: characterization

A monoclonal antibody was developed using rat cerebral cortex astrocytes purified in vitro as the antigenic material. Screening was done by labeling antibodies bound to cerebellum slices with fluorescent tagged secondary antibodies. The monoclonal antibody (RBA1) bound to intracellular filaments in all cells examined in culture. The coloration of tissue sections by RBA1 was identical to that described for polyclonal antibodies against the intermediate filament protein vimentin. In the brain this included binding to meninges, blood vessels, ependymal cells, choroid plexus lining cells and a subpopulation of astrocytes. The latter included Bergman glial fibers, white matter astrocytes and tanycytes. Müller cells in the retina and fibroblast-like cells in the skin, tongue and intestine were RBA1-positive. In immunoblots in which purified vimentin and desmin were run on SDS and transferred to nitrocellulose paper, RBA1 bound to vimentin but not desmin. When cultured astrocyte proteins were blotted, the antibody bound to both GFAP and vimentin, but no GFAP staining was observed in any of the tissue section staining. Purified vimentin blocked tissue and cultured cell binding of the antibody. Therefore RBA1 is considered to be an antibody specific for the intermediate filament protein vimentin.     

Monoclonal antibodies specific for ganglion cells in the embryonic chicken neural retina

Monoclonal antibodies have been produced which recognize antigens on the surface of embryonic chicken neural retina cells. Two monoclonal antibodies, obtained from separate fusions, have been shown by fluorescence-activated cell-sorter analysis to bind to antigens on a subpopulation of neural retina cells. Immunohistochemical methods demonstrate that both monoclonal antibodies bind to the nerve fiber and inner plexiform layers of the embryonic neural retina. These monoclonal antibodies also bind to cell bodies and neurites of cultured retinal neurons and therefore selectively recognize antigens localized to the surface ganglion cells. One of the monoclonal antibodies binds to a 260 kdalton polypeptide, which has been shown to be developmentally regulated by immunoblotting and immunocytochemistry, as it is not detected in late embryonic neural retina. The second monoclonal antibody appears to bind to different antigens in neural and non-neural tissue. In liver extracts the monoclonal antibody recognizes a trypsin-sensitive antigen, but the antibody reacts with trypsin-resistant molecules in neural tissue. This monoclonal antibody may therefore bind a glycolipid component in the neural retina, although this remains to be determined. These antibodies can be used to obtain cell populations highly enriched in retinal ganglion cells.     

Cross-reactivities of monoclonal antibodies between select leech neuronal and epithelial tissues

Three monoclonal antibodies, originally studied because of their neuron-specific staining in the leech central nervous system, are characterized further here, both immunocytochemically and biochemically, with Western blot staining using the central nervous system and peripheral tissues. The three antibodies react both with neurons and select epithelial tissue in the central nervous system, gut, and penis. Antibody Lan3-8 reacts with neurons in the nerve cord and gut but with epithelial cells in the penis; it binds to a 65K molecule in all three tissues. Lan3-2 and Laz2-369 are considered as a related pair because in the central nervous system the former stains all (four) and the latter generally only half (two) of the neurons in a standard midbody ganglion responding to nociceptive stimulation. In the gut, both antibodies label patches of epithelial cells and Laz2-369 stains a previously unknown type of gut neuron. While a given antibody stains different bands in gut and central nervous system immunoblots, comparing the bands of both antibodies for the same tissue extract makes it apparent that there are similarities in the molecular species that both antibodies recognize. For each monoclonal antibody, the histologically identified tissue antigens need to be correlated with proteins identified on Western blots. Of particular interest are the broad 130K bands to which Lan3-2 and Laz2-369 bind. The question is raised whether the molecular species in these bands represent a family of proteins that serve a specific nociceptive cell function.     

Localization of phospholipase C beta 1 on the detergent-resistant membrane microdomain prepared from the synaptic plasma membrane fraction of rat brain

The membrane microdomain (MD), such as detergent-resistant low-density membrane microdomain fraction (DRM), has been paid much attention because many signal-transducing molecules are recovered in this fraction, although precise localization and interactions of these molecules are largely unclear. To identify neuronal MD-localized proteins, monoclonal antibodies (mAbs) against the DRM-components of synaptic plasma membrane fraction (SPM) were produced and the antigens were characterized. One of the antigens reacted with two closely positioned bands of about 140 kDa in SDS-PAGE and the antigen showed age-dependent localization on DRM. The antigen was immunoprecipitated with the mAb after partial solubilization with 0.6 M NaCl from SPM-derived DRM and identified as phospholipase C beta 1 through mass analysis. The identity was further confirmed with Western blotting using a specific polyclonal antibody. The enzyme purified from the DRM was activated by the alpha subunit of trimeric G protein, Gq, expressed in HEK293 cells. The lipid composition of the liposomes affected the enzymatic activity and the addition of NAP-22, a neuronal DRM-localized protein, inhibited the activity. These results suggest that there exists a signal-transducing MD that performs important roles in neuronal functions through PIP(2) signaling and Ca(2+) mobilization.     

Xenopus oocytes as immunological vectors to produce monoclonal antibodies to rat brain antigens

A novel approach was developed to raise a panel of monoclonal antibodies (mAb) against brain antigens using Xenopus oocytes as immunological vectors. Xenopus oocytes were injected to express proteins encoded by brain-derived mRNA extracted from rat cerebral cortex. A crude membrane preparation from mRNA-injected oocytes was then used to immunize mice previously rendered immunotolerant to native oocyte membranes. mAb reacting with cryostat cut sections from rat brain were selected and further characterized by immunohistological and immunobiochemical techniques. Several mAb recognized brain specific antigens, including some that were cell type specific and others that revealed a regional binding pattern. A particular group of antibodies recognized an epitope localized exclusively to the cerebellar pinceau terminals. Although some of the hybridomas found in this panel may be products of natural autoreactive lymphocytes, the presence of a specific immune response to mRNA expression products is discussed. These results indicate that mRNA injected oocytes are useful tools to raise mAb to study the molecular diversity of the nervous system.     

Partial purification and characterization of a murine glioma-associated antigen defined by syngeneic rat monoclonal antibodies

A glioma-associated antigen was previously identified on an avian sarcoma virus-induced F-344 rat astrocytoma cell line S69-c15 by four rat monoclonal antibodies (7G4, 9F1, 10E3 and 10E7) produced after syngeneic immunization. Earlier data suggested all four antibodies reacted with a polypeptide-associated epitope. We report here that the antigen activity was detected in the supernatant of tumor homogenates and could pass through a 1000 Da molecular weight cut-off dialysis membrane, as determined by antibody binding inhibition in a cell surface radioimmunoassay. When the dialysate was fractionated by Bio-Gel P-2 chromatography, antibody inhibiting activity eluted in the range of 300-600 Da. A highly purified material was further isolated by ion exchange high pressure liquid chromatography. Parallel purification product from an antigen-negative cell line failed to demonstrate antibody inhibiting activity. We conclude that greater than 400-fold purification enrichment of antigen can be achieved. We postulate that the partially purified antigenic determinant is a glioma-associated determinant of highly restricted expression and is presented in hapten-carrier form by the glioma cells.     

Immunohistochemical staining of the human forebrain with monoclonal antibody to human choline acetyltransferase

Monoclonal antibody to human choline acetyltransferase (ChAT) was successfully produced from a mouse hybridoma cell line. The antibody was found to be of the IgM molecular species. By using this monoclonal antibody, immunohistochemical staining for ChAT was obtained on human brain sections. Only large sized cells were stained in the putamen and the substantia innominata. The specificity of the staining was comparable to that with polyclonal rabbit antibody to human ChAT produced by standard immunization procedures. No staining was observed when mouse monoclonal antibodies prepared against other human or bacterial antigens, or when normal mouse IgM, was employed.     

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.     

An immunoradiometric assay for factor VIII related antigen (VIIIRAg) using two monoclonal antibodies-comparison with polyclonal rabbit antibodies for use in von Willebrand's disease diagnosis

Two monoclonal antibodies raised against FVIII/von Willebrand protein were used in an immunoradiometric assay (IRMA) to measure this antigen in normal plasma and plasma of patients with different forms of von Willebrand's disease. The first antibody, an IgG1, was used to coat polystyrene tubes, the second one, an IgG2a, iodinated and used in the second step. Both antibodies inhibit ristocetin induced platelet agglutination and react strongly with platelets, megakaryocytes and endothelial cells. The IRMA test using these antibodies showed greater sensitivity than that using rabbit polyclonal anti VIIIRAg antibodies. A good correlation between the two tests was nevertheless found when VIIIRAg was measured in the majority of patient's plasma. However 5 patients from 3 different families showed more antigenic material in the rabbit antibody IRMA than in the monoclonal antibody IRMA. It is suggested therefore that the monoclonal antibodies identify part of the VIIIR:Ag molecule showing structural abnormalities in these vWd patients, these structural changes remaining undetected by the polyclonal antibodies.