[15] High efficiency fusion procedure for producing monoclonal antibodies against weak immunogens

The recently established culture medium, GIT, is applicable to many kinds of cells including mouse and human myeloma cells, and most adhesive cell lines. We applied this GIT medium to mouse B cell hybridoma production. When the medium was used to propagate myeloma cells before cell fusion and also for HAT selective medium, the fusion efficiency was more than twice as high as when the regular medium (RPMI-1640 supplemented with FBS) was used. Constantly more than 80% wells were hybridoma positive irrespective of the antigens used. To determine the optimal cell concentration at the hybridoma selection, a graded number of myeloma and spleen cells was distributed to each well ; the best result was obtained when 3 X 104 myeloma and 3 X 105 spleen cells were distributed to each well. In addition, the GIT medium shows very little lot-to-lot variation. These results indicate that fusion efficiency in mouse B cell hybridoma production was greatly improved by using GIT medium. hybridoma production ; GIT medium ; monoclonal antibody Since Kohler and Milstein (1975) introduced a new technology to obtain a monoclonal antibody directed against a single epitope on an antigen, monoclonal antibodies have been used extensively. The production of monoclonal antibodies to various antigens by the fusion of antigen-sensitized spleen cells with myeloma cells has been expanded to every biological field. Though basic methods have been well established, there is still a need for the more effective, stable fusion efficiency. Good and constant fusion efficiency makes it possible to effectively establish desired hybridoma clones and contributes markedly to the progress of

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“…This efficiency is much better than those reported previously (Cianfriglia et al 1986;Lane et al 1986;Long et al 1986). …”

Section: Discussioncontrasting

confidence: 52%

A great improvement of fusion efficiency in mouse B cell hybridoma production by use of the new culture medium, GIT.

Kudo

Morishita

Suzuki³

et al. 1987

Tohoku J. Exp. Med.

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“…The synthetic peptide (Rockland Immunochemicals, Gilbertville, PA) was conjugated to keyhole limpet hemocyanin to impart immunogenicity to the small peptide and then mixed with adjuvant (Titermax Gold for the first injection and Freund's incomplete adjuvant for subsequent booster doses) and used to immunize Balb/C mice. The fusion of mouse spleen with mouse myeloma cells, Sp2/0, was done using standard hybridoma techniques (17). The selection of the antibody was done by screening the supernatant by ELISA and using the synthetic peptide as an antigen.…”

Section: Methodsmentioning

confidence: 99%

Adenylyl cyclase type 5 protein expression during cardiac development and stress

Hu¹,

Chandra²,

Ge³

et al. 2009

American Journal of Physiology-Heart and Circulatory Physiology

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Adenylyl cyclase (AC) types 5 and 6 (AC5 and AC6) are the two major AC isoforms expressed in the mammalian heart that mediate signals from ␤-adrenergic receptor stimulation. Because of the unavailability of isoform-specific antibodies, it is difficult to ascertain the expression levels of AC5 protein in the heart. Here we demonstrated the successful generation of an AC5 isoform-specific mouse monoclonal antibody and studied the expression of AC5 protein during cardiac development in different mammalian species. The specificity of the antibody was confirmed using heart and brain tissues from AC5 knockout mice and from transgenic mice overexpressing AC5. In mice, the AC5 protein was highest in the brain but was also detectable in all organs studied, including the heart, brain, lung, liver, stomach, kidney, skeletal muscle, and vascular tissues. Western blot analysis showed that AC5 was most abundant in the neonatal heart and declined to basal levels in the adult heart. AC5 protein increased in the heart with pressure-overload left ventricular hypertrophy. Thus this new AC5 antibody demonstrated that this AC isoform behaves similarly to fetal type genes, such as atrial natriuretic peptide; i.e., it declines with development and increases with pressureoverload hypertrophy. adenylyl cyclase isoforms; monoclonal antibody; pressure overload; hypertrophy ADENYLYL CYCLASE (AC) is an enzyme that catalyzes the conversion of ATP to cAMP. The complexity in understanding the signaling pathway of cAMP can be attributed, in part, to 10 isoforms of AC: nine membrane bound and one soluble, which have been cloned and characterized in mammals (4,6,9,10,29). Each of these membrane-bound isoforms consists of two hydrophobic domains (with 6 transmembrane spans) and two cytoplasmic domains. The cytoplasmic domains constitute the catalytic site, which is subject to intracellular regulation (6). The AC isoforms have high amino acid homology in their cytoplasmic domains but differ in the sequence of the transmembrane region. The specificity of tissue distribution, the functional properties, and the chromosomal location of the corresponding genes also differentiate these isoforms (4, 10, 12). The major AC isoforms expressed in the heart are type 5 (AC5) and type 6 (AC6) (7,11,30). Tissue distribution and developmental expression of AC5 and AC6 mRNA have been previously studied in rats, chicks, and humans (7,30,31). However, in the absence of an AC5-specific antibody, it has not been possible to determine the levels of protein expression. It is very important to study the differential regulation of AC5 and AC6 in the heart since AC5 and AC6 behave differently in the pathogenesis of heart failure. We demonstrated that the AC5 knockout (KO) mouse model lives longer and is resistant to stress (32), including chronic pressure overload (21) and chronic catecholamine (22) stress. However, studies by others have suggested that the overexpression of AC6 is beneficial and might be considered for heart failure therapy (23). In addition, based on mRNA...

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“…Monoclonal antibodies against syntaxin 6 were obtained from the corresponding hybridoma cell lines generated by fusion of NS-1 mouse myeloma cells with spleen cells from a BALB/c mouse immunized with a glutathione S-transferase (GST)-syntaxin 6 fusion protein (Lane et al, 1986). This mouse's serum recognized a band of 29 kDa in rat brain postnuclear supematant.…”

Section: Materials and Methods Monoclonal Antibodiesmentioning

confidence: 99%

Syntaxin 6 functions in trans-Golgi network vesicle trafficking.

Bock¹,

Klumperman²,

Davanger³

et al. 1997

MBoC

282

237

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The specific transfer of vesicles between organelles is critical in generating and maintaining the organization of membrane compartments within cells. Syntaxin 6 is a recently discovered member of the syntaxin family, whose constituents are required components of several vesicle trafficking pathways. To better understand the function of syntaxin 6, we generated a panel of monoclonal antibodies that specifically recognize different epitopes of the protein. Immunoelectron microscopy shows syntaxin 6 primarily on the trans-Golgi network (TGN), where is partially colocalizes with the TGN adapter protein AP-1 on clathrin-coated membranes. Additional label is present on small vesicles in the vicinity of endosome-like structures. Immunoprecipitation of syntaxin 6 revealed that it is present in a complex or complexes with alpha-soluble NSF attachment protein, vesicle-associated membrane protein 2, or cellubrevin and a mammalian homologue of VPS45, which is a member of the sec1 family implicated in Golgi to prevacuolar compartment trafficking in yeast. We show that mammalian VPS45 is found in multiple tissues, is partially membrane associated, and is enriched in the Golgi region. Converging lines of evidence suggest that syntaxin 6 mediates a TGN trafficking event, perhaps targeting to endosomes in mammalian cells.

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[15] High efficiency fusion procedure for producing monoclonal antibodies against weak immunogens

Cited by 72 publications

References 7 publications

A great improvement of fusion efficiency in mouse B cell hybridoma production by use of the new culture medium, GIT.

A great improvement of fusion efficiency in mouse B cell hybridoma production by use of the new culture medium, GIT.

Adenylyl cyclase type 5 protein expression during cardiac development and stress

Syntaxin 6 functions in trans-Golgi network vesicle trafficking.

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