Properties of Monoclonal Antibodies to the Genus-specific Antigen of Chlamydia and Their Use for Antigen Detection by Reverse Passive Haemagglutination

Thornley, Margaret J.; Zamze, Susanne; Byrne, Marie D.; Lusher, Meryl E.; Evans, R.T.

doi:10.1099/00221287-131-1-7

Cited by 14 publications

(6 citation statements)

References 13 publications

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“…Chlamydial LPS is structurally reminiscent of the deep rough LPS of Re strains of Salmonella spp., including a trisaccharide of ketodeoxyoctonate but lacking additional core polysaccharides and 0 side chains (5,22,23). The novel 2--8 linkage of the second and third ketodeoxyoctonate moieties is the major contributing structure to the antigenic epitope recognized by various "group-specific" serological reagents (4,6,7,9,10,30), including monoclonal antibody 47A2 used in these studies.…”

Section: Discussionmentioning

confidence: 99%

Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells

1989

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The presence of a chlamydia-specified antigen associated with the plasma membrane of infected cell lines was demonstrated by indirect immunofluorescence staining with a monoclonal antibody, designated 47A2, specific for the chlamydial genus-specific lipopolysaccharide (LPS) antigen. Staining of HeLa, L-929, and McCoy cells infected with the L2 or F serovar of Chlamydia trachomatis was observed either without fixation or following aldehyde fixation and brief drying. The 47A2-reactive antigen appeared to be present on the plasma membrane, on bleb-like structures on the host cell surface, and on proximal processes of neighboring uninfected cells. Antibodies to chlamydial protein antigens such as the major outer membrane protein produced no surface staining under similar conditions. Membrane vesicles elaborated from infected cells were enriched for the 47A2-reactive antigen. Superinfection of chlamydia-infected cells with vesicular stomatitis virus, an enveloped virus which buds from the plasma membrane, allowed purification of progeny virions that were enriched with chlamydial LPS. These results are consistent with the presence of chlamydial LPS in the plasma membranes of infected host cells.

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Section: Discussionmentioning

confidence: 99%

Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells

1989

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“…IMS uses antibodies associated with magnetic beads to bind intact Chlamydia, with wash steps designed to remove contaminating material, enabling enrichment of the target species. We used a commercially available anti-Chlamydia mouse IgG primary antibody (IMAGEN Chlamydia, Oxoid) against C. trachomatis lipopolysaccharide (LPS), which binds to all serovars of C. trachomatis and has been tested for cross-reactivity against many other microbial species including Lactobacillus lactis, Mycoplasma spp., Neisseria gonorrhoeae, and Gardnerella vaginalis (IMAGEN Chlamydia booklet; Thornley et al 1983Thornley et al , 1985. LPS is present at ;34,000 molecules per EB (Su et al 1990), creating a high density target for antibody binding.…”

Section: Developing Ims-mda For C Trachomatismentioning

confidence: 99%

Whole-genome sequences ofChlamydia trachomatisdirectly from clinical samples without culture

et al. 2013

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The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C. trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C. trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C. trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.

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“…Two anti-LPS antibodies were used in this study. Monoclonal antibody 512 (Thornley e t al., 1985) was supplied by Boots-Celltech. The other, designated 512F, was locally produced by immunization of mice with C. tracbomatis serotype L2 strain 434 (Wang & Grayston, 1971).…”

Section: S C a M P B E L L A N D O T H E R Smentioning

confidence: 99%

Lipopolysaccharide in cells infected by Chlamydia trachomatis

Campbell¹,

Richmond

Yates

et al. 1994

Microbiology

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In view of the controversy concerning the expression of chlamydial lipopolysaccharide (LPS) in the eukaryotic host cell, the presence of this molecule was examined in three cell types which were experimentally infected with Chlamydia trachomatis serotype E. LPS was detected in the McCoy cell line, human endometrial epithelium and human amniotic epithelium with two monoclonal antibodies. The appearance and distribution of LPS a t the host cell surface during the chlamydial developmental cycle and its transmission to neighbouring cells were examined by immunofluorescence microscopy after air drying of the host cells. LPS distribution was not uniform; it was first observed on regions of the cell surface in close proximity to the chlamydial endosome (inclusion). Soon after, the antigen was also detected a t points of contact with neighbouring uninfected cells. lmmunofluorescent plaques of host cells contaminated with LPS were thus formed in the vicinity of infected cells. These plaques increased in size over 2 d before becoming smaller as host cell lysis occurred. The major outer-membrane protein (MOMP) was not visualized on the host cell surface after air drying. No cell-surface LPS antigen was observed in live cells or those fixed in formaldehyde without air drying. Conventional methanol fixation and immunolocalization of LPS and MOMP in parallel infected cultures stained these antigens within inclusions in the expected fashion. Radio-immunoassays were used to quantify LPS in confluent McCoy cell monolayers during the chlamydial developmental cycle. Cellsurface-associated and inclusion-associated LPS, measured by direct binding of 1251-labelled anti-LPS monoclonal antibodies to air-dried or methanol-fixed monolayers respectively, increased for up to 3 d then declined. Cell-surfaceassociated LPS is not directly accessible to antibodies in the hydrated cell. This apparent masking of the antigen may have a significant advantage for persistence of the parasite in vivo, since such host-cell-associated antigen is unlikely to be a target for immune attack.

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Properties of Monoclonal Antibodies to the Genus-specific Antigen of Chlamydia and Their Use for Antigen Detection by Reverse Passive Haemagglutination

Cited by 14 publications

References 13 publications

Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells

Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells

Whole-genome sequences ofChlamydia trachomatisdirectly from clinical samples without culture

Lipopolysaccharide in cells infected by Chlamydia trachomatis

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