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At least 883 records · Page 49Linked to original sources

PubMed1984

Simple and reliable enzyme-linked immunosorbent assay with monoclonal antibodies for detection of Escherichia coli heat-stable enterotoxins.

We have developed a sensitive and specific competitive enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxins consisting of methanol-soluble, suckling mouse active peptides with similar core sequences (STa) by using monoclonal antibodies prepared against STa purified from a human isolate. The assay can detect 3 to 20 pg of purified STa, depending on the monoclonal antibody used in the assay. The assay is rapid, requiring ca. 1 h to complete. With this competitive enzyme-linked immunosorbent assay, we measured STa production by enterotoxigenic E. coli directly in Casamino Acid-yeast extract culture supernatants. The assay was suitable for measuring STa in culture supernatants from human, bovine, and porcine E. coli isolates. No cross-reactivity was observed with heat-labile enterotoxin, cholera toxin, or heat-stable enterotoxin STb, which is a methanol-insoluble peptide(s) active in the ligated pig jejunal loop test. A 100% correlation of toxin production was found by comparing the enzyme-linked immunosorbent assay with the previously established radioimmunoassay for STa and with suckling mouse activity.

Animals↗

PubMed1986

Blinded, two-laboratory comparative analysis of Escherichia coli heat-stable enterotoxin production by using monoclonal antibody enzyme-linked immunosorbent assay, radioimmunoassay, suckling mouse assay, and gene probes.

Heat-stable enterotoxin (ST)-producing enterotoxigenic Escherichia coli (ETEC) can be identified by a variety of assays, including the suckling mouse assay (SMA), radioimmunoassay (RIA), polyclonal or monoclonal antibody enzyme-linked immunosorbent assay (ELISA), and DNA hybridization with STh and STp gene probes. To compare the sensitivity and reliability of these assays, 100 coded ETEC and non-ETEC isolates were blindly tested in two independent laboratories. SMA, RIA, and monoclonal ELISA were performed in Cincinnati, Ohio, while gene probe analysis was performed in Baltimore, Md. The method of storage of organisms had a profound effect on the stability of plasmids in certain strains. Hybridization experiments to determine the presence or absence of the enterotoxin gene showed that strains stored on Dorset egg medium at room temperature better retained their plasmids than strains stored frozen in skim milk. Forty-four of the 100 organisms obtained from the skim milk stock were found to produce STa in liquid culture by the RIA, SMA, and monoclonal ELISA (100% agreement). However, 50 of 54 of the strains stored on Dorset egg medium which were originally classified as STa+ or ST+ LT+ (positive for both heat-stable and heat-labile [LT] enterotoxins) were found to produce STa and retain the plasmid by each of these assays. Three additional strains were found which harbored the plasmid but did not elaborate STa by any of the assays (3% discrepancy). The monoclonal antibody ELISA appears to be highly reliable for determination of STa production by ETEC and can be easily scored visually even by untrained personnel. Furthermore, when this STa assay is coupled with a polyclonal antibody assay, it is possible to predict the genotype of STh- and STp-producing organisms.

Animals↗

PubMed1989

Enterotoxin production and serogroups of Campylobacter jejuni and Campylobacter coli from patients with diarrhea and from healthy laying hens.

Enterotoxin production, a possible virulence factor, was determined in Campylobacter jejuni and Campylobacter coli by two different techniques, the CHO cell test and the GM1 enzyme-linked immunosorbent assay. The frequency of enterotoxigenic Campylobacter strains was 32% in strains from both humans with acute enteritis and healthy laying hens, as measured by the CHO cell test. The CHO cell test was significantly more sensitive than the GM1 enzyme-linked immunosorbent assay in the detection of enterotoxigenic strains. Enterotoxin production was compared with the presence of heat-stable and heat-labile antigens. There was no significant correlation between enterotoxin production and serogroups for C. jejuni or C. coli. The difference in enterotoxigenicity between C. jejuni (34.1%) and C. coli (21.9%) was not significant.

Acute Disease↗

PubMed1989

Simultaneous detection of Escherichia coli heat-stable and heat-labile enterotoxin genes with a single RNA probe.

A single RNA probe was synthesized and used to detect simultaneously the methanol-soluble heat-stable enterotoxin and heat-labile enterotoxin genes in Escherichia coli strains. The results with the biotinylated or radioactive probe correlated 100% with the biological assay results for both toxins. The RNA probe detected the three known heat-stable enterotoxin A alleles.

Alleles↗

PubMed1991

Characterization of Escherichia coli strains producing heat-stable enterotoxin b (STb) isolated from humans with diarrhea.

Two of 49 cytolethal distending toxin-producing strains of Escherichia coli isolated from human stools contained the gene coding for heat-stable enterotoxin b (STb), as detected by a colony hybridization assay. The STb gene was found to be on a 70-kb plasmid also coding for heat-labile enterotoxin (pLT-I). Restriction endonuclease analysis showed the STb gene from human isolates to be similar to the STb gene found in porcine strains. Moreover, by enzymatic amplification based on oligonucleotide primers designed from a porcine STb sequence, the expected portion of the STb gene was amplified for the human E. coli strains. The STb enterotoxin from these strains was bioactive in rat jejunal loops and was detected with an enzyme-linked immunosorbent assay by using polyclonal antiserum raised against purified porcine STb toxin.

Animals↗

PubMed1991

Detection of genes for fimbrial antigens and enterotoxins associated with Escherichia coli serogroups isolated from pigs with diarrhea.

A total of 1,226 Escherichia coli strains isolated from 1979 to 1989 from pigs with diarrhea were examined for serogroup and fimbrial antigen F4 (K88) production. Four main patterns of isolation of the various serogroups were observed, depending on the ages of the pigs from which isolates were obtained and the production of F4. In pattern I, serogroups O8:K"S16", O9:K35, O9/O101:K30, O9/O101:K103, O9 (group), O20:K101, and O64:K"V142" were predominant in pigs aged 0 to 6 days (41.9% of isolates) and were less frequent in pigs aged 7 to 27 days (24.6% of isolates) but were rarely found in pigs aged 28 to 60 days (4.0% of isolates). In pattern II, the F4-associated serogroups O8:K"4627", O157:K"V17", O149:K91, and O147:K89 were predominant in pigs aged 7 to 27 days (29.8% of isolates) and in pigs aged 28 to 60 days (35.0% of isolates). In pattern III, serogroups O8 (group), O115:K"V165", and O147:K89 were rarely isolated from pigs aged 0 to 6 days but were equally distributed in pigs aged 7 to 27 days (10.1% of isolates) and in pigs aged 28 to 60 days (10.9% of isolates). In pattern IV, serogroups O138:K81, O139:K82, O141:K85ac, O45:K"E65", and O26:K60 were most frequently isolated in pigs aged 28 to 60 days (19.3% isolates). Over the period from 1979 to 1989, the proportion of isolates belonging to serogroups of pattern II and the proportion of F4 isolates within the serogroup O157:K"V17" declined, whereas the proportion of isolates of serogroups O147:K89, O8:K"S16", and O9:K35 increased. For 228 isolates selected from the most important serogroups, good agreement was observed between the results of gene probes and immunofluorescence for the detection of fimbrial antigens F4 (K88), F5 (K99), F6 (987P), and F41 and between the results of gene probes and biological assays for the detection of heat-labile enterotoxin (LT) and heat-stable enterotoxins a and b (STa and STb). The STa gene was mostly associated with isolates of pattern I serogroups, which had the F5, F6, and F41 genes alone or in various combinations. The LT and/or STb genes, with the F4 gene, mostly were observed in isolates of pattern II serogroups. The STb gene alone was observed mostly in isolates of pattern III serogroups, although isolates were negative for all fimbrial antigen genes. Similarly, isolates of pattern IV serogroups were negative for all fimbrial antigen genes and rarely positive for the enterotoxin genes. However, verotoxin production was associated with isolates of serogroups O138:K81 and O139:K82. The most important pathotypes among enterotoxigenic isolates in this study were F4:LT:STb, F5:STa, STb, F5:F41:STa, F4:STb, F6, STa, and LT.

Age Factors↗

PubMed1993

Rapid screening test for enterotoxin-producing Bacillus cereus.

Culture supernatants of 30 enterotoxin-producing Bacillus cereus isolates produced a characteristic progressive destruction of McCoy cell monolayers. Enterotoxin-negative B. cereus and other group 1 Bacillus spp. caused no monolayer disruption. The McCoy cell tissue culture system appears to provide a rapid screening assay for detection of enterotoxin-producing B. cereus.

Animals↗

PubMed1999

Prevalence of Shigella enterotoxins 1 and 2 among Shigella strains isolated from patients with traveler's diarrhea.

Shigella spp. are known primarily as a cause of bacillary dysentery. However, in an initial phase, numerous patients exhibit watery diarrhea that may or may not be followed by dysentery. New virulence factors associated with the species of Shigella have recently been described. These are enterotoxins 1 and 2 of Shigella (ShET-1 and ShET-2, respectively). The aim of the present study was to determine the prevalence of ShET-1 and ShET-2 in species of Shigella isolated from patients with traveler's diarrhea. During the period from 1993 to 1998, stool samples from 500 travelers with diarrhea were cultured for the isolation of Shigella spp. and other enteropathogens. The detection of ShET-1 and ShET-2 was performed by a PCR technique with specific primers. Among a total of 51 strains of Shigella isolated during this period (22 S. flexneri, 26 S. sonnei, and 3 S. dysenteriae strains), at least one enterotoxin was detected in 31 (60.78%) strains; 2 (9.09%; both of which were S. flexneri strains) produced only ShET-1, while 21 (41.17%; 3 S. flexneri, 15 S. sonnei, and 3 S. dysenteriae strains) produced ShET-2. Furthermore, 8 (15.69%) of 22 S. flexneri strains presented both enterotoxins. Our results show that the prevalence of ShET-2 was high in all the Shigella species studied and confirm that ShET-1 is detected only in S. flexneri.

Bacterial Toxins↗

PubMed2004

Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli.

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.

Bacterial Toxins↗

PubMed1977

Antigenic similarity of heat-labile enterotoxins from diverse strains of Escherichia coli.

With use of the rabbit intestinal loop model, heat-labile enterotoxins from 21 Escherichia coli strains isolated from a wide spectrum of patients with diarrheal diseases were all neutralized to high titer by two antisera prepared against enterotoxins of either E. coli or Vibrio cholerae. These findings suggest marked immunological similarity among heat-labile enterotoxins from a heterogenous group of E. coli.

Adult↗

PubMed1977

Enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin.

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of heat-labile Escherichia coli enterotoxin is described. The assay, which is based on the immunological similarity between Vibrio cholerae toxin and heat-labile E. coli enterotoxin, is similar in design to a radioimmunoassay but utilizes enzyme-labeled rather than radioactive isotope-labeled reagents. The ELISA system is as sensitive as both radioimmunoassay and the y-1 adrenal cell assay for the detection of heat-labile E. coli enterotoxin but requires neither radioactive reagents nor tissue culture techniques. The ELISA is easy to perform and is adaptable for use in small laboratories.

Drug Stability↗

PubMed1978

Simple assay for staphylococcal enterotoxins A, B, and C: modification of enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay (ELISA) introduced for the detection of staphylococcal enterotoxins by Saunders et al., Simon and Terplan, and ourselves has proved to be a simple, reliable, and sensitive test. A new modification is described that uses polystyrene balls (diameter, 6 mm) coated individually with antibody against one of the toxins A, B, or C. In a single tube, 20 ml of the food extract was incubated with the three balls differently stained, which were then each tested for the uptake of enterotoxin by a competitive ELISA. A concentration of 0.1 ng or less of enterotoxin per ml can be measured, making tedious concentration procedures of the extracts superfluous. Culture supernatants and extracts from foods artificially or naturally contaminated with toxin were successfully examined. Cross-reactions did not occur, and nonspecific interfering substances did not create serious problems.

Enterotoxins↗

PubMed1979

Adaptation of the staphylococcal coagglutination technique for detection of heat-labile enterotoxin of Escherichia coli.

Protein A-containing staphylococci coated with specific antiserum were tested for heat-labile enterotoxin of Escherichia coli. The immunological cross-reactivity of E. coli heat-labile enterotoxin with Vibrio cholerae toxin (choleragen) was the basis for sensitizing stabilized suspensions of the Cowan I strain of Staphylococcus aureus with anticholeragen. Unconcentrated culture supernatant fluid containing E. coli heat-labile enterotoxin produced macroscopic agglutination when mixed with sensitized staphylococci in capillary tubes. A total of 15 toxigenic and 61 nontoxigenic isolates were tested by the staphylococcal coagglutination technique in a coded fashion and found to be in agreement with previous results of the Chinese hamster ovary cell assay and the passive immune hemolysis test. The staphylococcal coagglutination technique is simple, relatively inexpensive to perform, and requires the immunoglobulin fraction of anticholeragen as the only specific reagent. The staphylococcal coagglutination technique appears to have potential for routine use in diagnostic microbiology laboratories.

Agglutination Tests↗

PubMed1976

Influence of staphylococcal enterotoxin on water and electrolyte transport in the small intestine.

The direct and indirect effects of staphylococcal enterotoxins A and C on the flux of water, sodium, and potassium have been studied in paired Thiry Vella fistulae in dogs. Administration of toxin resulted in a significant decrease in absorption, both in the loop to which the toxin had been administered and its pair. This decrease in absorption was associated with a decrease in movement out of the lumen, movement into the lumen remaining relatively unchanged. The mechanism of action of staphylococcal enterotoxins is discussed, and comparisons made with other enterotoxins.

Animals↗

PubMed1989

Comparison of Y1 mouse adrenal cell and coagglutination assays for detection of Escherichia coli heat labile enterotoxin.

A commercial coagglutination assay (COA; Phadebact LT-ETEC) was compared with a Y1 mouse adrenal cell assay for detecting the heat labile enterotoxin of Escherichia coli. Of four different media evaluated for use with the COA, only one (modified blood agar) gave a positive result with all strains known to produce heat labile enterotoxin. With modified blood agar, the COA detected 74 (85%) of 87 such strains. Eighty six strains negative by cell culture assay were also negative by COA, and one strain positive by COA could not be confirmed by cell culture. The Phadebact LT-ETEC kit provides a simple, sensitive, and economical method for detecting E coli heat labile enterotoxin.

Adrenal Glands↗

PubMed1992

Detection of Clostridium perfringens and its enterotoxin in cases of sporadic diarrhoea.

AIMS: To determine the incidence of sporadic and apparently non-food related diarrhoea associated with Clostridium perfringens enterotoxin. METHODS: Enzyme linked immunosorbent assay (ELISA) and reversed phase latex agglutination (RPLA) were used to detect C perfringens enterotoxin in faecal specimens from 818 sporadic cases of diarrhoea. RESULTS: C perfringens enterotoxin was identified as a cause of sporadic diarrhoea in 56 of 818 (6.8%) cases. Diarrhoea was prolonged (three days or more) in most cases. Ages ranged from 3 months to 89 years, although most patients were over 60 years of age. CONCLUSIONS: These results suggest that C perfringens may be a cause of sporadic cases of diarrhoea when causes such as food consumption or cross-infection are absent, particularly in the elderly.

Adolescent↗

PubMed1978

The use of polyethylene glycol in radioimmunoassay of staphylococcal enterotoxins.

The double-antibody radioimmunoassay of enterotoxins A, B, and C was modified by the addition of aqueous polyethylene glycol which precipitated double-antibody bound staphylococcal enterotoxins with little or no precipitation of free enterotoxin. The precipitate formed appeared as a thin coating at the bottom of the test tube and was not removed by aspiration. The procedure obviates the need for normal rabbit serum and for an additional wash of the precipitate.

Enterotoxins↗

PubMed1980

The effect of NaCl on the upper temperature limit for growth of and enterotoxin synthesis by Staphylococcus aureus.

The upper temperature limit for growth of an enterotoxin A producing strain (MF31) and of an enterotoxin B producing strain (S6) was about 44-45 degrees C in either a peptone-based medium or heart infusion broth. In the presence of NaCl, growth and production of enterotoxin, coagulase, and thermostable nuclease occurred up to 2 degrees C higher. The optimal concentration of NaCl for the temperature elevation effect was 1 M.

Bacterial Toxins↗