Alkaline phosphatase release assay to determine cytotoxicity for
            <i>Listeria</i>
            species

Bhunia, Arun K.; Westbrook,

doi:10.1046/j.1472-765x.1998.00336.x

Cited by 28 publications

(23 citation statements)

References 15 publications

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“…The cytotoxicity effects of bacterial cells or the toxin preparations on collagen-encapsulated Ped-2E9 cells were measured by an ALP release assay as described previously. 40 Briefly, 50-100 ml of bacteria cell suspension (1 Â 10 9 CFU/ ml) or crude toxin preparations (B20 mg/ml, 50-100 ml) were added to each well of the 96-or 48-well microtiter plate, respectively, and held at 371C in a humidified incubator with B cells in gel as biosensors P Banerjee et al 7% CO 2 for a period of 3-6 h. The supernatant from the 48-well plate was collected, centrifuged (1800 g for 5 min) and placed in a mini-cuvette containing a ready-to-go ALP liquid substrate (Sigma). The color development was recorded at 3-5 min intervals at absorbance of 405/595 nm using a handheld USB2000 mini-spectrophotometer (Ocean Optics, Dunedin, FL, USA).…”

Section: Measurement Of Cytotoxicitymentioning

confidence: 99%

“…16,37,38 This cytotoxicity assay can also differentiate the presence of viable pathogenic Listeria species from nonpathogenic ones. 16,[39][40][41][42] Adherent and nonadherent mammalian cell lines of various tissue origins were used previously to distinguish differential cytotoxic response of virulent Listeria species from avirulent ones. 16,[43][44][45] The cytotoxic properties were determined by monitoring the changes in cell morphology, 42 formation of plaques in a monolayer, 43,45 or dye uptake due to loss of membrane permeability.…”

mentioning

confidence: 99%

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A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins

Banerjee

Lenz

Robinson

et al. 2008

Laboratory Investigation

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Cell-based biosensors (CBBs) are becoming important tools for biosecurity applications and rapid diagnostics in food microbiology for their unique capability of detecting physiologically hazardous materials. A multi-well plate-based biosensor containing B-cell hybridoma, Ped-2E9, encapsulated in type I collagen matrix, was developed for rapid detection of viable cells of pathogenic Listeria, the toxin listeriolysin O, and the enterotoxin from Bacillus species. This sensor measures the alkaline phosphatase release from infected Ped-2E9 cells colorimetrically. Pathogenic L. monocytogenes cells and toxin preparations from L. monocytogenes or B. cereus showed cytotoxicity ranging from 24 to 98% at 3-6 h postinfection. In contrast, nonpathogenic L. innocua (F4247) and B. subtilis induced minimal cytotoxicity, ranging only 0.4-7.6%. Laser scanning cytometry and cryo-nano scanning electron microscopy confirmed the live or dead status of the infected Ped-2E9 cells in gel matrix. This paper presents the first example of a cell-based sensing system using collagen-encapsulated mammalian cells for rapid detection of pathogenic bacteria or toxin, and demonstrates a potential for onsite use as a portable detection system.

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Section: Measurement Of Cytotoxicitymentioning

confidence: 99%

mentioning

confidence: 99%

A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins

Banerjee

Lenz

Robinson

et al. 2008

Laboratory Investigation

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“…3). Similarly, in the AP assay, strains AAMU530 and AAMU572 induced AP releases of 16 and 15% respectively, which was significantly lower than the ) release from Ped-2E9 cells [32]. Percentage cytotoxicity was determined based on the total alkaline phosphatase (AP) release from Triton-Xtreated Ped-2E9 cells.…”

Section: Cytotoxicity Projile Of Mutant Strainsmentioning

confidence: 99%

Surface protein p104 is involved in adhesion of Listeria monocytogenes to human intestinal cell line, Caco-2

Pandiripally¹,

Westbrook²,

Sunki³

et al. 1999

Journal of Medical Microbiology

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Adhesion of Listeria monocytogenes to intestinal endothelial cells is an important initial event in the pathogenesis of infection which is not well understood. The suggestion has been made that some proteins, including internalin and actin polymerisation protein (ActA), and carbohydrate molecules mediate, at least in part, the adhesion of listeria to certain cultured mammalian cells. This study investigated the role of a L. monocytogenes cell-surface protein of 104 kDa (p104) in adhesion to human intestinal enterocyte-like Caco-2 cell lines by transposon (Tn916) mutagenesis and a pl04-specific monoclonal antibody (MAb-H7). Genotypic and phenotypic characteristics of Tn916-transformed L. monocytogenes strains, AAMU530 and AAMU572, revealed that these strains did not express p104, and the transposon had been inserted at a single locus in the structural gene. Strains AAMU530 and AAMU572 yielded only 10 and 6.3% adhesion to Caco-2 cells. Coating of L. monocytogenes and L. innocua wild-type strains with MAb-H7 reduced adhesion to Caco-2 cells from 100% to 50 and 45%, respectively, whereas on isotype control MAb EM-7G1 had no effect. Western blot analysis with MAb-H7 indicated that p104 is present in all Listeria spp. except in L. grayi. Furthermore, p104 is also present in internalin (BUGS) and ActA (LUT12) deficient strains, suggesting that p104 is indeed different from internalin or ActA proteins. Cytotoxicity analysis of strains AAMU530 and AAMU572 demonstrated that these strains, although haemolytic and phospholipase-positive, were avirulent when tested with a hybridoma B-lymphocyte cell line. Loss of virulence could be attributed to the interruption of adhesion of mutant strains to the hybridoma cell line. These results strongly suggest that p104 is an adhesion factor in L. monocytogenes and involved in adhesion to intestinal cells.

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“…In order to make the Ped-2E9 assay a rapid and sensitive one, reducing agent DTT in the cell suspensions were added to enhance L. monocytogenes mediated cell cytotoxicity. Data presented here showed that DTT added to the listerial cells or its culture supernatants can augment the cytopathogenic activities, thus lowering the assay time to half or less than what was previously reported (3,5). In this study, we also examined the effect of cysteine HC1…”

Section: Discussionmentioning

confidence: 83%

“…The murine hybridoma Ped-2E9 cell line was produced by the fusion of murine myeloma NS 1 (TIB 18, ATCC, Rockville, Md., U.S.A.) and splenocytes from mice that were immunized with Pediococcus acidilacti- (3,5). Briefly, the hybridoma cells (2 X 106/m1) were inoculated with bacteria at a multiplicity of infection of 100 bacteria to 1 hybridoma cell, and incubated at 37 C. Ped-2E9 cells (0.1 ml) were withdrawn at specified times, stained with trypan blue (0.4%) and counted under a microscope.…”

Section: Methodsmentioning

confidence: 99%

Dithiothreitol Enhances Listeria monocytogenes Mediated Cell Cytotoxicity

Westbrook

Bhunia

2000

Microbiology and Immunology

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The hybridoma Ped-2E9 based cytotoxicity assay was developed to distinguish virulent from avirulent Listeria species in 6 hr. The cytotoxicity effect on Ped-2E9 was reported to be primarily due the cytolytic action of listeriolysin O (LLO), produced by L. monocytogenes. In this study, the effect of a reducing agent, dithiothreitol (DTT, 0-2 mm) that is known to activate LLO was investigated to make the Ped-2E9 based cytotoxicity assay an even more sensitive and rapid. Also, we examined the effect of fetal bovine serum (FBS, 0-50%), a common ingredient of tissue culture media on cytotoxicity. A DTT concentration of 0.5 mm gave an optimum cytotoxicity effect, which could be measured by both alkaline phosphatase (AP) and lactate dehydrogenase (LDH) assays in just 1.5-2 hr. FBS, at levels between 10 to 50%, significantly inhibited Listeria-mediated cytotoxicity. Concentrated culture filtrates from L. monocytogenes or LLO producing recombinant L. innocua (prfA±hlyA±) strain also caused cytotoxicity effects, which were observed by scanning electron microscopy or a cytotoxicity assay in 2-3 hr. Interestingly, addition of DTT to culture filtrates produced 100% cell cytotoxicity in just 15 min. This indicated that LLO activity, which is responsible for Ped-2E9 cytotoxicity, was augmented several folds with the addition of a reducing agent. Examination of Listeria isolates belonging to different serogroups from clinical sources or naturally contaminated meat products with DTT gave cytotoxicity results in 2 hr, which were comparable to the 5-hr assay analyzed concurrently without DTT. These results indicated that DTT, which activated the LLO, could be used in the cytotoxicity assay to enhance Listeria-mediated Ped-2E9 cell cytotoxicity. This knowledge will greatly assist us to develop a user-friendly rapid assay to screen cytopathogenic properties of Listeria species.

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Alkaline phosphatase release assay to determine cytotoxicity for Listeria species

Cited by 28 publications

References 15 publications

A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins

A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins

Surface protein p104 is involved in adhesion of Listeria monocytogenes to human intestinal cell line, Caco-2

Dithiothreitol Enhances Listeria monocytogenes Mediated Cell Cytotoxicity

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