Production of Monoclonal Antibodies by the Use of pH-Dependent Vesicular Stomatitis Virus-Mediated Cell Fusion

Nagata, Satoshi; Yamamoto, Kiichi; Ueno, Yoshio; Kurata, Takeshi; Chiba, Joe

doi:10.1089/hyb.1991.10.317

Cited by 7 publications

(5 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Booster injections were given subcutaneously with the antigen mixed with incomplete Freund's adjuvant two times with an interval of two weeks. After the final booster injection, which was given intravenously, spleen cells of the mouse were taken and fused with the SP2/O cells by a polyethylene glycol method (21). Ten days after fusion, culture supernatant of the hybridomas were examined for the reactivity to purified GST-PIP 3 BP protein by enzyme-linked immunosorbent assay.…”

Section: Methodsmentioning

confidence: 99%

Evidence That a Phosphatidylinositol 3,4,5-Trisphosphate-binding Protein Can Function in Nucleus

Tanaka

Horiguchi

Yoshida³

et al. 1999

Journal of Biological Chemistry

Self Cite

140

View full text Add to dashboard Cite

PIP 3 BP is a phosphatidylinositol 3,4,5-trisphosphatebinding protein (PIP 3 BP) abundant in brain, containing a zinc finger motif and two pleckstrin homology (PH) domains. Staining of rat brain cells with anti-PIP 3 BP antibody and determination of localization of PIP 3 BP fused to the green fluorescent protein (GFP-PIP 3 BP) revealed that PIP 3 BP was targeted to the nucleus. Targeting was dependent on a putative nuclear localization signal in PIP 3 BP. Generation of PIP 3 in the nucleus was detected in H 2 O 2 -treated 293T cells, nerve growth factor (NGF)-treated PC12 cells, and platelet-derived growth factor (PDGF)-treated NIH 3T3 cells. Translocation of phosphatidylinositol 3-kinase (PI 3-kinase) to the nucleus and enhanced activity of PI 3-kinase in the nucleus fraction were observed after H 2 O 2 treatment of 293T cells, suggesting that PI 3-kinase can be activated in the nucleus as well as in the membrane after appropriate stimulation of the cells. Co-expression of the constitutively active PI 3-kinase with PIP 3 BP resulted in exportation of the protein from the nucleus to the cytoplasm, suggesting that PIP 3 BP can function as a PIP 3 -binding protein in the intact cells. These results imply that there may be an unknown function of PI 3-kinase in the nucleus.Phosphatidylinositol 3-kinase 1 is an enzyme that is activated immediately after growth factor or differentiation factor stimulation of the cells (1) and that generates second messengers, phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) (2-5). These 3Ј-phosphorylated phosphoinositides can activate serine, threonine kinases such as PKB/Akt, PKCs, and PDKs (6 -9). They are also suggested to be involved in other events such as rearrangement of cytoskeleton and vesicle transport because these phenomena are sensitive to the PI 3-kinase inhibitors and dominant negative mutants of PI 3-kinase (10). Recently, it was reported that the 3Ј-phosphorylated phosphoinositides can activate guanine nucleotide exchanging factors of Rac and Arf, small G proteins involved in actin rearrangement and vesicle transport, respectively (11,12). Therefore, G proteins as well as kinases are downstream of PI 3-kinase.We have identified PIP 3 BP as a PIP 3 -binding protein, using a PIP 3 analogue column (13). It is abundant in brain, implying that it may be involved in the function of nerve systems. PIP 3 BP binds to PIP 3 but not to PI 3,4-P 2 or phosphatidylinositol 4,5-bisphosphate (PI 4,5-P 2 ). It has a zinc finger motif homologous to that of Arf-GTPase activating protein (GAP) and two PH domains. Both PH domains are shown to be involved in binding to PIP 3 . Another PIP 3 -binding protein, centaurin ␣, is highly homologous to PIP 3 BP (14). No GAP activity to Arf has been detected in either protein. Although the binding of centaurin ␣ and PIP 3 BP to PIP 3 was specific, the role of the protein is unclear. To address this question, we determined the intracellular localization by immunological techniques, using mono...

show abstract

Section: Methodsmentioning

confidence: 99%

Evidence That a Phosphatidylinositol 3,4,5-Trisphosphate-binding Protein Can Function in Nucleus

Tanaka

Horiguchi

Yoshida³

et al. 1999

Journal of Biological Chemistry

Self Cite

140

View full text Add to dashboard Cite

show abstract

“…Kits containing inactivated Sendai virus are commercially available for the generation of murine hybridomas. The second virus that has been used for the fusion of cells to generate hybridomas for mAb secretion is vesicular stomatitis virus (33). In a study performed by Nagata et al, the efficiencies of hybridoma generation were compared using Sendai virus, vesicular stomatitis virus, and PEGmediated cell fusion (34).…”

Section: Fusion Methodsmentioning

confidence: 99%

Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies

Smith

Crowe

2015

Microbiol Spectr

View full text Add to dashboard Cite

The human hybridoma technique offers an important approach for isolation of human monoclonal antibodies. A diversity of approaches can be used with varying success. Recent technical advances in expanding the starting number of human antigen-specific B cells, improving fusion efficiency, and isolating new myeloma partners and new cell cloning methods have enabled the development of protocols that make the isolation of human monoclonal antibodies from blood samples feasible. Undoubtedly, additional innovations that could improve efficiency are possible. HISTORY OF HYBRIDOMASMonoclonal antibodies (mAbs) have revolutionized the conduct of science since their first description in 1975 (1). The use of these specific reagents also has made possible improved clinical diagnostics in the medical arena, and many antibodies have found their way to clinical use as prophylactic or therapeutic agents. Nevertheless, the potential of mAbs derived specifically from technology based on human hybridomas remains largely unfulfilled. The principal reason for the lack of a large number of hybridoma-derived mAb therapeutics has simply been the technical difficulty in generating stable hybridomas that secrete human mAbs of high affinity and functional activity. This chapter reviews recent efforts to develop and employ novel methods for the efficient generation of human hybridomas secreting human mAbs for clinical use.The principal advantage of the use of human hybridoma technology for mAb generation is that this approach preserves the authentic sequence and pairing of antibody DNA from a natural B cell for the expression of a naturally occurring full-length human mAb. There are significant theoretical advantages for expressing cDNAs encoding authentic heavy and light chains with chains that are paired using the coding sequence as it was generated naturally through B cell selection, class switch, and affinity maturation. No genetic modification of these sequences is required. Since antibody expression is typically very stable in hybridomas, sequence amplification of antibody variable genes is achieved easily if recombinant production or manipulation is desired. The resulting recombinant mAb retains most features of naturally occurring human antibodies, as the clone retains the native amino acid sequence and heavy/light chain pairing. Since the native constant region of the antibody in the original human B cell is retained in the mAb expressed by the resulting hybridoma, the functional properties of the particular Fc region can be studied for Fc-mediated activities, such as antibodydependent cellular cytotoxicity.

show abstract

“…The virus stock was prepared by infecting BHK-21 cells (Japanese Cancer Research Resources Bank) at a multiplicity of 0.1 PFU/cell. Virus harvested at 22 h postinfection was concentrated by ultrafiltration and ultracentrifugation [22,25], and purified by sucrose density gradient centrifugation [22]. This preparation containing 1.1 mg/ml of viral protein (2.3 x 1011PFU/ml) was stored at -80 °C.…”

Section: Virusmentioning

confidence: 99%

“…The spleen cells of the immunized mice and SP 2/O-Ag 14, BALB/c mouse non-secretory plasmacytoma cells, were fused with polyethylene glycol according to Oi and Herzenberg [28] or by the novel VSV-mediated cell fusion method described previously [25,26]. Media preparation and HAT selection of hybridomas were described previously [25]. In 2 weeks, the hybridomas were screened for production of anti-G protein antibody by enzyme-linked immunosorbent assay (ELISA) with purified G protein as the antigen (see below).…”

Section: Generation Of Monoclonal Antibody-producing Hybridomasmentioning

confidence: 99%

See 1 more Smart Citation

Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies

Nagata

Okamoto

Inoue

et al. 1992

Archives of Virology

View full text Add to dashboard Cite

Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1-6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein.

show abstract

Production of Monoclonal Antibodies by the Use of pH-Dependent Vesicular Stomatitis Virus-Mediated Cell Fusion

Cited by 7 publications

References 17 publications

Evidence That a Phosphatidylinositol 3,4,5-Trisphosphate-binding Protein Can Function in Nucleus

Evidence That a Phosphatidylinositol 3,4,5-Trisphosphate-binding Protein Can Function in Nucleus

Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies

Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies

Contact Info

Product

Resources

About