Establishment of Stable Cell Lines Producing Anti‐<i>Pseudomonas aeruginosa</i> Monoclonal Antibodies and Their Protective Effects for the Infection in Mice

O'oka, Hisayoshi; Chonan, E; Mizutani, Kazunori; Fukuda, Tamotsu; Kuroiwa, Yasuyuki; Ono, Yasushi; Shigeta, Shirô

doi:10.1111/j.1348-0421.1992.tb02132.x

Cited by 7 publications

(2 citation statements)

References 21 publications

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“…12 is an Epstein-Barr virus-transformed human lymphoblastoid cell line producing human IgM against Pseudomonas aeruginosa Homma serotype B, and clone 3-4 is a hybridoma established by fusion of clone No. 12 and an established human myeloma cell line P109 mutated so as not to produce IgM -chain (25 Cell Culture-Cells producing human monoclonal IgM were routinely maintained by static culture at 37°C (5% CO 2 /air) in tissue culture flasks containing NYSF-404 medium (26) and 20% fetal bovine serum (FBS). G418 (800 g/ml) was added to cultures of ␤-1,4-GalT-I or GnT-III transfectant cells.…”

Section: Methodsmentioning

confidence: 99%

Control of Bisecting GlcNAc Addition to N-Linked Sugar Chains

Fukuta

Abe

Yokomatsu

et al. 2000

Journal of Biological Chemistry

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In the present study, experimental control of the formation of bisecting GlcNAc was investigated, and the competition between ␤-1,4-GalT (UDP-galactose:N-acetylglucosamine ␤-1,4-galactosyltransferase) and GnT-III (UDP-N-acetylglucosamine:␤-D-mannoside ␤-1,4-N-acetylglucosaminyltransferase) was examined. We isolated a ␤-1,4-GalT-I single knockout human B cell clone producing monoclonal IgM and several transfectant clones that overexpressed ␤-1,4-GalT-I or GnT-III. In the ␤-1,4-GalT-Isingle knockout cells, the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased, where ␤-1,4-GalT activity was reduced to about half that in the parental cells, and GnT-III activity was unaltered. In the ␤-1,4-GalT-I transfectants, the extent of bisecting GlcNAc addition was reduced although GnT-III activity was not altered significantly. In the GnT-III transfectants, the extent of bisecting GlcNAc addition increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular ␤-1,4-GalT activity relative to that of GnT-III. These results were interpreted as indicating that ␤-1,4-GalT competes with GnT-III for substrate in the cells.

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Section: Methodsmentioning

confidence: 99%

Control of Bisecting GlcNAc Addition to N-Linked Sugar Chains

Fukuta

Abe

Yokomatsu

et al. 2000

Journal of Biological Chemistry

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“…However, the use of a murine myeloma cell line results in MAbs contaminated with murine cell products. Alternative methods for the production of human MAbs have also been developed by using Epstein-Barr virus-transformed human lymphocytes previously sensitized to P. aeruginosa (29,36,50,55). In most cases, however, these cell lines are unstable and do not remain an immortal source for Ab production.…”

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confidence: 99%

Phage display and bacterial expression of a recombinant Fab specific for Pseudomonas aeruginosa serotype O6 lipopolysaccharide

Tout

Lam

1997

Clin Diagn Lab Immunol

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Immunotherapy with antibodies (Abs) against the lipopolysaccharide (LPS) of Pseudomonas aeruginosa remains an alternative to serotype-specific LPS-based vaccines due to their limited use and to antibiotics due to the intrinsic resistance to antimicrobials observed in P. aeruginosa. We have chosen a monoclonal Ab (MAb), MF23-1, that binds to the O antigen of the most clinically relevant serotype, IATS O6, for producing a recombinant antibody. Heavy (H) and light (L) chain genes were isolated from MF23-1 to form a functional Fab molecule in the periplasm of Escherichia coli and on the surface of phage by using phagemid vector pComb3. The entire L chain gene was used, but the H chain gene was amplified to 2 amino acids past cysteine 128 which is involved in interchain disulfide bond formation with the L chain. The truncated H chain associated with the L chain in the periplasm of E. coli to form a functional Fab molecule that bound in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay to O6 LPS. Therefore, the remainder of the C H 1 past cysteine 128 is not essential for stable formation of the Fab portion of MF23-1. This recombinant Fab (r-Fab) was shown to be specific for the LPS of the most predominant clinical isolate, serotype O6, while no cross-reactivity was detected to the LPS of the other 19 remaining serotypes. This r-Fab was also expressed on the surface of filamentous phage upon addition of helper phage to recombinant E. coli containing phagemid. Recombinant phage from clones MT13 and MT24 bound specifically to O6 LPS in ELISA. These results represent an important step toward the design of therapeutic Abs to be used against P. aeruginosa infections.

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