Fast and simple procedure for the detection of cell culture mycoplasmas using a single monoclonal antibody

Blazek, Roman; Schmitt, Karl; Krafft, Ute; Hadding, U.

doi:10.1016/0022-1759(90)90191-w

Cited by 28 publications

(20 citation statements)

References 14 publications

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“…The immunobinding procedure was performed by the method of Kotani and McGarrity [23] with modifications as described previously [24]. As a peroxidase substrate, 4-chloro-1-naphthol (Sigma, Deisenhofen, Germany) or ECLLuminol (Amersham Buchler, Braunschweig, Germany) was used following the suppliers' protocol.…”

Section: Sds-page and Immunostaining Of Recombinant Peptidesmentioning

confidence: 99%

Cyto-adherence studies of the adhesin P50 of Mycoplasma hominis

Kitzerow¹,

Hadding²,

Henrich³

1999

Journal of Medical Microbiology

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Recombinant peptides of Mycoplasma hominis adhesin P50 were expressed in Escherichia coli to investigate which regions of the P50 molecule are responsible for cyto-adherence. The respective DNA fragments were obtained by PCR amplification of the p50 gene with the use of mutating oligonucleotides to change the TGA codons of mycoplasma to TGG codons, which are translated in E. coli as tryptophan. The resulting three clones (I, I + I1 and I+III) contained regions of P50 which closely represent the repeat regions A, A + B and A + C. After expression in E. coli, the polyhistidine-tagged recombinant peptides were purified by metal chelation chromatography. The three recombinant peptides were detected in Western blot analysis by a polyclonal antiserum directed against M. hominis FBG and two P5O-specific monoclonal antibodies, BAlO and BG2. Each of the three recombinant peptides I, I + I1 and I + 111 was able to adhere to immobilised HeLa cells in an adhesion assay. The cyto-adhesion of the peptides could be inhibited by pre-incubation with the appropriate antibody. Therefore, it is suggested that adherence may be mediated by all regions of the P50 molecule. Attachment of the recombinant peptides to immobilised HeLa cells was inhibited by high mol. wt dextran sulphate (MW 500 000), indicating that the respective P50 regions bind to sulphatides on the host cell membrane.

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Section: Sds-page and Immunostaining Of Recombinant Peptidesmentioning

confidence: 99%

Cyto-adherence studies of the adhesin P50 of Mycoplasma hominis

Kitzerow¹,

Hadding²,

Henrich³

1999

Journal of Medical Microbiology

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“…Recently, a MAb against mycoplasmal EF-Tu which was used to detect and differentiate members of the class Mollicutes has been described (8,20). A similar MAb specific for a 43-kDa membrane protein of Mycoplasma fermentans detected 14 other mycoplasma species and some gram-negative and grampositive bacteria (32).…”

Section: Discussionmentioning

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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“…Between 1970 and until the late 1990ies, Germany was struck by several severe and less severe epidemics of CSF (Fritzemeier et al, 2000;Moennig and Plagemann, 1992;Wachendörfer et al, 1978 , 1990;Paton et al, 1995;Wensvoort et al, 1989). In addition, monoclonal antibodies (mabs) were successfully used for typing CSF virus isolates and other Pestiviruses (Kosmidou et al, 1995;Paton et al, 1995).…”

Section: Genetic Typing and The Virus And Sequence Database At The Eumentioning

confidence: 99%