Generation and characterization of mouse monoclonal antibodies to the myelin-associated glycoprotein (MAG)

Dobersen, Michael J.; Hammer, Jeffrey A.; Noronha, Antonio; MacIntosh, Tracy D.; Trapp, Bruce D.; Brady, Roscoe O.; Quarles, Richard H.

doi:10.1007/bf00964654

Cited by 69 publications

(32 citation statements)

References 34 publications

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“…The lysates were incubated at 95°C for 5 min, after which they were subjected to SDS-PAGE (10%). The proteins were transferred to polyvinylidene difluoride membranes and immunostained with MAG monoclonal antibody (Millipore, Temecula, CA) (2 g/ml), which recognizes MAG Ig domains 1-3, or B11F7 (Dobersen et al, 1985) (2.6 g/ml), which recognizes MAG Ig domain 4. For MAG-Sn chimeric expressing cells, a combination of two monoclonal antibodies, 3D6 and SER4 (each at 1 g/ml; gifts from Dr. Paul R. Crocker, University of Dundee, Dundee, UK), which recognize sialoadhesin Ig domains 1 and 2/3, respectively, were used.…”

Section: Methodsmentioning

confidence: 99%

The Inhibition Site on Myelin-Associated Glycoprotein Is within Ig-Domain 5 and Is Distinct from the Sialic Acid Binding Site

Cao¹,

Qiu²,

Domeniconi³

et al. 2007

J. Neurosci.

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Section: Methodsmentioning

confidence: 99%

The Inhibition Site on Myelin-Associated Glycoprotein Is within Ig-Domain 5 and Is Distinct from the Sialic Acid Binding Site

Cao¹,

Qiu²,

Domeniconi³

et al. 2007

J. Neurosci.

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“…Murine monoclonal antibodies to MAG were produced as described (11). Briefly, P3X63-Ag8.653 myeloma cells were fused with spleen cells from a female NZB/N mouse immunized with purified human MAG isolated from the central nervous system.…”

Section: Methodsmentioning

confidence: 99%

Murine monoclonal antibodies to the myelin-associated glycoprotein react with large granular lymphocytes of human blood.

Dobersen¹,

Gascón²,

Trost³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Monoclonal antibodies prepared to human myelin-associated glycoprotein were shown to react with a population of human peripheral blood mononuclear cells. The population is similar to the large granular lymphocytes or natural killer cells defined by antibody Leu 7 (also called HNK-1). The population also includes cells exhibiting the Leu 2 marker for suppressor/cytotoxic T cells. The results indicate a shared antigenicity between the nervous system and the immune system and may be relevant to the pathogenesis of demyelinating diseases.Evidence has been accumulating for shared antigenic determinants between the nervous system and the immune system (1-5). This crossreactivity may be involved in the etiopathology of such neurological diseases as multiple sclerosis (5) and myasthenia gravis (3, 4). It recently has been demonstrated that Leu 7 (or HNK-1)-a mouse monoclonal antibody, raised to a human lymphoblastoma, that recognizes human natural killer cells-reacts with components of nervous tissue (6, 7). The myelin-associated glycoprotein (MAG) was identified as a molecule in nervous tissue containing the epitope recognized by this monoclonal antibody (7,8). Furthermore, the epitope in MAG is in its carbohydrate moieties (9) and also is in other glycoconjugates of peripheral nerve (9, 10).In this paper, we describe the reaction of mouse monoclonal antibodies prepared to MAG (11) with a subpopulation of human peripheral blood mononuclear cells. Further experiments show that this population is similar to the large granular lymphocytes (LGL) recognized by Leu 7. The results indicate that MAG and a population of peripheral blood lymphocytes share a common antigenic determinant. This finding may have relevance in the pathogenesis of demyelinating diseases.MATERIALS AND METHODS Monoclonal Antibodies. Murine monoclonal antibodies to MAG were produced as described (11). Briefly, P3X63-Ag8.653 myeloma cells were fused with spleen cells from a female NZB/N mouse immunized with purified human MAG isolated from the central nervous system. Hybrids and subsequent clones were screened for antibody production to MAG by using a solid-phase enzyme immunoassay system. The following monoclonal antibodies were used in this study: G7C8, E8B8, and C6D9 of the IgM subclass with K light chains, which react with determinants in the carbohydrate moieties of human MAG; F7F7, an IgG2b subclass antibody with K light chains, which also reacts with a carbohydrate determinant of human MAG; and B11F7 (IgG2b) and D7E10 (IgGl) with K light chains, which react with polypeptide determinants of human and rat MAG. The hybridomas were grown as ascites tumors in pristane-primed NIH/Swiss nude mice. Monoclonal antibodies were purified from ascites fluid by Sepharose-protein A affinity chromatography (12) for antibodies F7F7 and B11F7 and by euglobulin fractionation followed by Sephadex G-200 chromatography (13) for antibody G7C8. Hybridoma HNK-1 (14) was purchased from the American Type Culture Collection. The hybridoma was grown and the monoclonal a...

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“…Blood was drawn from rabbits prior to immunization. The serum was titered for immunoreactivity against bovine milk GT by using an ELISA (23 (Fig. 1), the longest cDNA clone, that contained an additional 165 base pairs (bp) at the 5' end compared to pLbGT-1 but that still lacked the translation start codon.…”

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confidence: 99%

Expression of bovine beta-1,4-galactosyltransferase cDNA in COS-7 cells.

Masibay

Qasba

1989

Proc. Natl. Acad. Sci. U.S.A.

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A bovine (-1,4-galactosyltransferase (GT; EC 2.4.1.90) cDNA in an Okayama-Berg vector, pLsGT, was constructed from a partial cDNA clone and a genomic fragment. We report that the cDNA sequence of pLsGT, in a transient expression assay in COS-7 cells, codes for an enzymatically active GT protein. There is an approximately 12-fold increase in the GT activity in pLsGT-transfected cells compared to cells transfected with the antisense bovine GT construct, pLasGT, or pSV2Neo or mock-transfected cells. The increased activity is correlated with the increase in bovine GT mRNA, which is distinguishable from COS GT mRNA with a 3'-end-specific probe of pLsGT. The expressed GT activity is modulated by a-lactalbumin, which changes the acceptor specificity to glucose to synthesize lactose. Polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and a monoclonal antibody (mAb 4-10) directed against a synthetic peptide corresponding to the amino-terminal region of the protein encoded by pLsGT bind the expressed protein, and the resulting immunoprecipitates exhibit GT enzymatic activity.N-Acetylglucosamine 8-1,4-galactosyltransferase (GT; EC 2.4.1.90) is but one member of the glycosyltransferase family of enzymes. Many of these enzymes are involved in the synthesis and extension of oligosaccharide chains on glycoproteins and glycolipids, generating a series of disaccharide linkages (1-3). Molecular approaches have been utilized to study the various transferases and cDNAs for some transferases have been cloned (4-9).GT transfers galactose from UDP-galactose to N-acetylglucosamine (GlcNAc) to produce N-acetyllactosamine with a f8-1,4-glycosidic linkage. The cDNA for GT has been independently cloned by several laboratories from lactating bovine mammary gland (4), the bovine kidney cell line MDBK (5), human liver (6), F9 embryonal carcinoma cells (7), and the mouse mammary cell line c127 (8). All these clones show strong homology to each other. On the other hand the 5' sequence of human liver GT cDNA clone isolated by Humphreys-Beher et al. (10) does not exhibit homology to any of the reported GT cDNAs (4-8), in spite of the fact that the deduced partial amino acid sequence of the cDNA clone showed partial homology to the amino-terminal sequence of the bovine GT protein they isolated. This raises the question whether the described clones (4-8) do indeed code for active GT.To address this problem, we constructed a full-length bovine GT cDNA in an Okayama-Berg expression vector (pLsGT)*, transfected it into COS-7 cells, and obtained a 12-fold increase in GT enzymatic activity in the transfected COS-7 cells compared to mock-transfected cells. This GT activity is modulated by a-lactalbumin, which changes the acceptor specificity from GlcNAc to glucose. In addition, this GT activity is immunoprecipitable by a polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and by a monoclonal antibody (mAb) directed against a synthetic peptide whose sequence was derived from the cDNA sequence that spans a portion o...

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Generation and characterization of mouse monoclonal antibodies to the myelin-associated glycoprotein (MAG)

Cited by 69 publications

References 34 publications

The Inhibition Site on Myelin-Associated Glycoprotein Is within Ig-Domain 5 and Is Distinct from the Sialic Acid Binding Site

The Inhibition Site on Myelin-Associated Glycoprotein Is within Ig-Domain 5 and Is Distinct from the Sialic Acid Binding Site

Murine monoclonal antibodies to the myelin-associated glycoprotein react with large granular lymphocytes of human blood.

Expression of bovine beta-1,4-galactosyltransferase cDNA in COS-7 cells.

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