Rapid isolation of monoclonal hybridoma cultures by a ‘fusion-cloning’ method: the requirement for aminopterin

Butcher, R.N.; Obi, Takeshi; McCullough, Kenneth C.

doi:10.1016/1045-1056(91)90031-e

Cited by 15 publications

(5 citation statements)

References 10 publications

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“…B. Charley and C. La Bonnardière (INRA, France) kindly contributed the interferon reagents. A MAb against GM-CSF (64-10) was generated in our laboratory by established methods (Butcher et al , 1991 ). Hybridoma supernatant from MAb 64-10 was used at 25% (v/v), a concentration found to block GM-CSF-induced BMHC proliferation (A. Summerfield, H. Gerber and K. C. McCullough, unpublished data).…”

Section: Methodsmentioning

confidence: 99%

Induction of apoptosis in bone marrow neutrophil-lineage cells by classical swine fever virus

Summerfield

Zingle

Inumaru

et al. 2001

Journal of General Virology

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The pathogenesis of bone marrow atrophy during classical swine fever (CSF) was investigated in vitro by using CSF virus (CSFV) infection of bone marrow haematopoietic cells (BMHC). The monocytic lineage had the highest susceptibility to CSFV infection, whereas the more mature SWC8 M granulocytic cells were not directly susceptible to infection. However, myelomonocytic precursors were targets for CSFV infection and continued to differentiate into SWC8 M granulocytic cells, which remained infected. This explains the occurrence of infected peripheral blood granulocytes during CSF. The infection of BMHC resulted in increased apoptosis and necrosis, mainly within the granulocytic lineage. Caspases 3 and 9 were particularly activated, relating to the mitochondrial pathway of apoptosis. Interestingly, the majority of infected cells were nonapoptotic, the apoptotic cells being primarily non-infected. This indicated an indirect mechanism for induction of apoptosis, but no role could be identified for bone marrow stroma cells such as macrophages or fibroblastoid cells. Furthermore, soluble factors including cytokines and reactive oxygen species were not primarily responsible. In contrast, contact between infected and noninfected BMHC was critical for increasing apoptosis in the latter. Taken together, these results in vitro relate to and help to explain further the apoptosis of BMHC that occurs in vivo during CSF. This experimental system will also be particularly useful for the study of CSFV gene products involved in leukocyte apoptosis.

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Section: Methodsmentioning

confidence: 99%

Induction of apoptosis in bone marrow neutrophil-lineage cells by classical swine fever virus

Summerfield

Zingle

Inumaru

et al. 2001

Journal of General Virology

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“…MAR mutants produced using any of the C29 group of MAbs (MAbs C29, C70 and C74) all had the same amino acid substitution at the same site. The method of production (Butcher et al, 1991) meant that it is possible that these three MAbs are all subclones of a single hybridoma. Finding the same substitution in three individually raised MAR mutants indicates the importance of residue 261 of VP1 in the binding of these MAbs.…”

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confidence: 99%

Identification of neutralizing epitopes on a European strain of swine vesicular disease virus.

Nijhar

Mackay

Brocchi

et al. 1999

Journal of General Virology

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Six neutralizing monoclonal antibodies (MAbs) were used to isolate MAb neutralization-resistant (MAR) mutants from a recent European strain of swine vesicular disease virus (SVDV), ITL/9/93. Sequencing of MAR mutants identified two epitopes located at positions analogous to sites 2A (VP2) and 3B (VP3) on poliovirus (PV) which have been previously identified on a Japanese strain of SVDV. A third epitope near to the C terminus of VP1, not previously recognized on SVDV, was tentatively identified in a region analogous to site 1 of PV. A fourth epitope, located in the C-terminal region of VP3, has never before been recognized as a site of neutralization on picornaviruses. All four epitopes were predicted to be surface-exposed.

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“…The highest hybridoma production rate found in the literature is 2% using a laser-PEG-HAT protocol. (39) It seems that this novel result is attributable to the avoidance of the PEG (33,(35)(36)(37) and HAT toxicities (13,38) in our protocol.…”

Section: Discussionmentioning

confidence: 86%

An Extreme Strategy for the Production of Hybridoma

2009

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The ethical issues surrounding human immunization hamper the production of human monoclonal antibody through scarcity of immunized B cells in peripheral blood. This defect can be compensated in part by improvement of hybridoma production techniques. We have developed a new strategy to bypass the toxic effects of polyethylene glycol (PEG) as fusogenic reagent and hypoxanthine aminoptrin thymidine (HAT) as selective medium on newly fused cells. The Epstein-Barr virus (EBV) transformed peripheral blood mononuclear cells (PBMC) of accidentally Rh antigen sensitized persons were fused using cephalin as fugenic reagent, with emetine and actinomycin D pretreated heteromyeloma cells. Our results showed that 19-34% of EBV-transformed B cells were grown as hybridoma clones following selection. This extreme improvement in hybridoma production rate may end the fusion efficiency problem and make hybridoma production a plug-and-play technology.

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Rapid isolation of monoclonal hybridoma cultures by a ‘fusion-cloning’ method: the requirement for aminopterin

Cited by 15 publications

References 10 publications

Induction of apoptosis in bone marrow neutrophil-lineage cells by classical swine fever virus

Induction of apoptosis in bone marrow neutrophil-lineage cells by classical swine fever virus

Identification of neutralizing epitopes on a European strain of swine vesicular disease virus.

An Extreme Strategy for the Production of Hybridoma

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