Monoclonal antibody detection of IncF group plasmid-encoded TraT protein in clinical isolates of Escherichia coli

Bitter‐Suermann, D.; Peters, Helmut; Jürs, M; Nehrbass, R; Montenegro, Maria Augusta; Timmis, Kenneth N.

doi:10.1128/iai.46.2.308-313.1984

Cited by 21 publications

(7 citation statements)

References 24 publications

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“…Mice were treated intraperitoneally with whole formalinized E. coli C600 Rif(pKT146) cells. The immunization-and-fusion protocol was exactly as described previously (1,2). After fusion of the immune spleen lymphocytes with the hybridoma line X63-Ag 8.653 (16), the culture supernatant fluids of growing clones were screened by ELISA with whole glutaraldehyde-fixed bacteria or outer membranes as antigens (1).…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, 100 pI of a 0.01% solution of glutaraldehyde was added and incubated for a further 10 min. Plates were washed twice with buffer A, and gelatin (1,15). The reactivity of mAbs with capsular polysaccharides K5, K7, and K56 (1 mg/ml in buffer A) was tested in an ELISA after untreated microtiter plates were coated with 20 ,ul of dilutions of the polysaccharides, air dried overnight at 37°C, and subsequently washed with buffer A containing 0.05% Tween 20.…”

Section: Methodsmentioning

confidence: 99%

“…In every case in which a Western blot was performed, a second slab gel was run in parallel, and proteins and lipopolysaccharides were revealed by silver staining. Marker proteins (Sigma) having molecular weights of 92,000, 66,000, 45,000, 31,000, 21,000, and 14,000 were always run on the same slab gel (1,15). Electrophoretic transfer of LPS, ECA, and proteins to nitrocellulose sheets (BA85; Scheicher & Schull, Dassel, Federal Republic of Germany) was performed with a Transblot apparatus (Bio-Rad, Munich, Federal Republic of Germany) for 2 h at 25 V and 0.2 A, followed by 12 h at 50 V and 0.4 A.…”

Section: Methodsmentioning

confidence: 99%

“…In continuation of our previous studies to evaluate E. coli outer membrane antigens as potential factors of bacterial pathogenicity or as potential immunogens for vaccines (1,5,15), we have produced monoclonal antibodies (mAbs) against the enterobacterial common antigen (ECA), an antigen present in the outer membrane of all members of the family Enterobacteriaceae (21), and against the hexose region of LPS outer cores that occur in a large number of E. coli strains. One of the anti-ECA mAbs was found to cross-react with K5 capsular polysaccharide, indicating the presence of a shared epitope between ECA and K5.…”

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confidence: 99%

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Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide

Peters¹,

Jürs²,

Jann³

et al. 1985

Infect Immun

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We established hybridoma cell lines producing monoclonal antibodies against enterobacterial common antigen (ECA) and a substructure of the outer core of different Escherichia coli lipopolysaccharides (LPSs). Anti-ECA antibodies 865 and 898 reacted with ECA in extracts of heated E. coli and with ECA-bound Rl and R4 core-containing LPS preparations, as well as with a purified sample of ECA from Salmonella montevideo. Antibody 865, but not antibody 898, cross-reacted with K5 capsular polysaccharide, suggesting that 4-linked ce-N-acetylglucosamine is part of an antigenic determinant shared by both K5 polysaccharide and ECA. Anti-LPS antibody 786 recognized an outer core structure common to E. coli K-12, B, R2, and R4 core type LPS, but not to Rl and R3 core type LPS. Its most probable target is the trisaccharide sequence Hexp(1->2)-a-D -Glcp(1-*3)a-D-Glcp->(Hepp) (where Hex is hexose, p is phosphate, Glc is glucose, and Hep is heptose), the first glucose being the immunodominant moiety. These monoclonal antibodies may be used not only for the detection of ECA, K5, and LPS core structures but also for analysis of the molecular forms resolved on polyacrylamide gels (banding patterns) of both ECA and LPS, independently of one another.on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide

Peters¹,

Jürs²,

Jann³

et al. 1985

Infect Immun

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“…Isolation and cloning of hybridoma cell lines. The anti-ECA monoclonal antibody was raised in BALB/c mice by immu-nization with whole cells of E. coli K-12 as previously described (1). Positive hybridoma cultures were identified by screening culture supernatants for reactivity against purified ECA (H. Peters, M. Jurs, B. Jann, K. Jann, and D. Bitter-Suermann, manuscript in preparation).…”

Section: Paper Employing Solvent System D (See Below)mentioning

confidence: 99%

Biosynthesis of enterobacterial common antigen

Rick¹,

Mayer²,

Neumeyer³

et al. 1985

J Bacteriol

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Cultures of Salmonella typhimurium pulse-labeled with N-acetyl-D-[3H]glucosamine ([3H]GlcNAc) incorporated isotope into a GIcNAc-linkced lipid that was tentatively identified as GlcNAc-pyrophosphorylundecaprenol. The incorporation of [3H]GlcNAc into this compound was abolished when cells were pulse-labeled in the presence of the antibiotic tunicamycin. Tunicamycin also abolished the in vivo synthesis of the haptenic form of enterobacterial common antigen (ECA) in S. typhimurium as determined by the passive hemagglutination test. These data indicated that the synthesis of the GlcNAc-linked lipid is related to ECA synthesis. Support for this conclusion was provided by the following observations. Cultures of Escherichia coli and S. typhimurium incorporated [3H]GlcNAc into cell envelope components that migrated as a homologous series of polymers when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The [3H]GIcNAc-labeled polymers were not detected in mutants of E. coli and S. typhimurium defective in ECA synthesis due to lesions in either the rfe or rff gene clusters. These polymers were identified as ECA based on Western blot analyses employing anti-ECA monoclonal antibody. The incorporation of [3H]GIcNAc into ECA polymers was abolished by tunicamycin when the drug was added to cultures to give a minimum concentration of 3 ,ug/ml. In addition, pulse-chase experiments provided evidence for a prvcursor-product relationship between the GlcNAc-linked lipid and ECA. These results strongly suggest that the GlcNAc-linked lipid is involved in the biosynthesis of ECA in a manner analogous to the role of carrier lipid in the biosynthesis of 0-antigen and peptidoglycan.

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An inherited deficiency of the third component of complement, C3, in guinea pigs

et al. 1986

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Hereditary deficiency of the third component of complement, C3, is found very seldom in the human. C3 deficiency is associated with severe bacterial infections revealing the central role of C3 in complement activation via the classical or alternative pathway. We describe a new hereditary C3 deficiency in strain 2 guinea pigs. Serum from these animals had a markedly reduced lytic activity in a standard assay for complement-dependent, antibody-mediated cytotoxicity. In functional assays of individual components, the hemolytic activity of the components C4, C2, C5 and of factors B, D and H was in the normal range. The functional C3 titer, and similarly C3 antigenic activity in the serum of these C3-deficient animals (C3D) was on average only 5.7% of normal activity. Typing the animals with alloantisera or monoclonal antibodies to guinea pig Ia-antigens revealed that the C3D animals had the major histocompatibility complex-haplotype of inbred strain 2 guinea pigs (B.1, Ia.2,4). The C3 defect is not linked to the major histocompatibility complex and, in addition, is not linked to a C3a receptor deficiency. Macrophages and hepatocytes of the C3D animals have an unimpaired capacity for synthesis and secretion of C3 as measured by enzyme-linked immunosorbent assay. There was no indication for hypercatabolism of normal C3 by the animals as shown by plasma clearance of 125I-radiolabeled C3. Thrombocytes of the C3D animals responded normally to stimulation with purified C3a in an ATP-release assay without an indication for a desensitization in vivo. Possibly the fault resides in an enhanced susceptibility of their own C3 to proteolysis. However, C3 partially purified from the plasma of the C3D animals or secreted by hepatocytes exhibited no obvious structural differences to purified normal C3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis or in immunoblotting. The C3D serum had a reduced bactericidal activity compared to normal or to C4-deficient serum. Nevertheless, the animals are apparently healthy without an indication for increased frequency of bacterial infections. These guinea pigs provide an unique model for analysis of the biological functions of C3 in vivo and in vitro without the need for artificial C3-depletion procedures with all their known and unknown side-effects.

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Monoclonal antibody detection of IncF group plasmid-encoded TraT protein in clinical isolates of Escherichia coli

Cited by 21 publications

References 24 publications

Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide

Monoclonal antibodies to enterobacterial common antigen and to Escherichia coli lipopolysaccharide outer core: demonstration of an antigenic determinant shared by enterobacterial common antigen and E. coli K5 capsular polysaccharide

Biosynthesis of enterobacterial common antigen

An inherited deficiency of the third component of complement, C3, in guinea pigs

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