Immunoblotting with Monoclonal Antibodies: A Highly Specific System for Detection and Identification of Bacterial Outer Membrane Proteins

Jürs, M; Peters, Helmut; Timmis, Kenneth N.; Bitter‐Suermann, D.

doi:10.1007/978-3-642-69943-6_13

Cited by 4 publications

(8 citation statements)

References 16 publications

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“…This is consistent with the known narrow host range property of IncF plasmids. Interestingly, studies with an anti-TraT protein monoclonal antibody (Bitter-Suermann et al, 1984;Jurs et al, 1985) revealed not only the presence of TraT protein in nearly all isolates of E. coli and S. typhimurium that were shown by colony hybridization to carry traT, but also the presence of anti-TraT protein antibody-reacting proteins in 91% of isolates of Enterobacter cloacae and in 3 1 % of isolates of non-typhimurium serotypes of Salmonella, neither group of which showed homology to the traTgene probe. The TraT-related proteins of these two latter groups of bacteria have, however, higher molecular weights than that of the TraT protein itself and may, except for the antibody-reacting epitope, be structurally distinct.…”

Section: Discussionmentioning

confidence: 99%

traT Gene Sequences, Serum Resistance and Pathogenicity-related Factors in Clinical Isolates of Escherichia coli and Other Gram-negative Bacteria

et al. 1985

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The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gramnegative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (5670%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.

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Section: Discussionmentioning

confidence: 99%

traT Gene Sequences, Serum Resistance and Pathogenicity-related Factors in Clinical Isolates of Escherichia coli and Other Gram-negative Bacteria

et al. 1985

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“…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 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.…”

mentioning

confidence: 99%

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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“…MAb 900 was generated as described previously (19). Briefly, BALB/c mice were immunized with 2.5 ϫ 10 8 bacteria of E. coli K-12 strain C600 Rif r (pKT146) with five intraperitoneal booster injections.…”

Section: Methodsmentioning

confidence: 99%

“…Alignment of the amino acid sequences deduced from the bacterial EF-Tu gene sequences revealed a high degree of similarity. Previously we described a monoclonal antibody (MAb), MAb 900, which we obtained by immunization of BALB/c mice with E. coli K-12 C600 Rif r (pKT146) bacteria (3,19). This antibody recognized a 43-kDa band from E. coli and other members of the family Enterobacteriaceae on Western blots (immunoblots).…”

mentioning

confidence: 99%

An epitope of elongation factor Tu is widely distributed within the bacterial and archaeal domains

1995

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A monoclonal antibody (MAb), MAb 900, which detects a 43-kDa protein present on Escherichia coli was found. Subsequently, more than 90 organisms, belonging to either the bacterial, archaeal, or eucaryal domain, were tested for reactivity to this MAb. Of the bacterial and archaeal domains, almost all species proved to be positive, whereas all organisms from the eucaryal domain gave negative results. The 43-kDa protein was purified by affinity chromatography and subsequently analyzed by microsequencing methods. Two peptide sequences which showed a high degree of homology (>99%) to the prokaryotic elongation factor Tu (EF-Tu) were obtained. Western blot (immunoblot) analysis using both purified EF-Tu and EF-Tu domains confirmed that the unknown protein was EF-Tu. The panbacterial distribution of EF-Tu, which is present in large amounts in every prokaryotic cell, renders this protein a good candidate for a diagnostic approach. In consequence, we have used the anti-EF-Tu MAb 900 to design both a dot blot assay and an enzyme-linked immunosorbent assay. From either blood culture, urine, or gall-bladder fluid, bacterial contamination could be detected. The sensitivity of these tests is currently 10 4 bacteria per ml.

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Immunoblotting with Monoclonal Antibodies: A Highly Specific System for Detection and Identification of Bacterial Outer Membrane Proteins

Cited by 4 publications

References 16 publications

traT Gene Sequences, Serum Resistance and Pathogenicity-related Factors in Clinical Isolates of Escherichia coli and Other Gram-negative Bacteria

traT Gene Sequences, Serum Resistance and Pathogenicity-related Factors in Clinical Isolates of Escherichia coli and Other Gram-negative Bacteria

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

An epitope of elongation factor Tu is widely distributed within the bacterial and archaeal domains

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